Plasmodium falciparum. In this study, we bioinformatically predicted a total of 63 TA proteins in the P. falciparum proteome and revealed the association of a subset with the P. falciparum homolog of Get3 (PfGet3). In addition, our proximity labelling studies either definitively identified or shortlisted the other eligible GET constituents, and our in vitro association studies validated associations between PfGet3 and the corresponding homologs of Get4 and Get2 in P. falciparum. Collectively, this study reveals the presence of proteins with hallmark TA signatures and the involvement of evolutionary conserved GET trafficking pathway for their targeted delivery within the parasite.Tail-anchored (TA) proteins are defined by the absence of N-terminus signal sequence and the presence of a single transmembrane domain (TMD) proximal to their C-terminus. They play fundamental roles in cellular processes including vesicular trafficking, protein translocation and quality control. Some of the TA proteins are post-translationally integrated by the Guided Entry of TA (GET) pathway to the cellular membranes; with their N-terminus oriented towards the cytosol and C-terminus facing the organellar lumen. The TA repertoire and the GET machinery have been extensively characterized in the yeast and mammalian systems, however, they remain elusive in the human malaria parasite  Plasmodium falciparum. We have used bioinformatic prediction algorithms in conjunction with functional validation studies to identify the candidate TA repertoire and some of the homologs of the trafficking machinery in P. falciparum. Initially, we predicted the presence of 63 putative TA proteins localized to distinct compartments within this parasite, including a few confirmed TA homologs in other eukaryotic systems. We then identified and characterized PfGet3 as a central component in the Guided-Entry of TA (GET) translocation machinery, and our bacterial co-expression and pulldown assays with two selected recombinant TA proteins, PfBOS1 and PfUSE1, showed co-association with PfGet3. We also identified PfGet2 and PfGet4 as the other two components of the GET machinery in P. falciparum using proximity biotinylation followed by mass spectrometry. Interestingly, we also found six TA proteins in the parasite enriched in this fraction. We further validated the direct interactions between a few TA candidates, PfGet4 and PfGet2 with PfGet3 using recombinant-based pulldown studies. In conclusion, this study classified a subset of membrane proteins with the TA nomenclature and implicated a previously unidentified GET pathway for their translocation in this apicomplexan parasite.Tail-anchored (TA) membrane proteins are known to play essential cellular functions in the eukaryotes. These proteins are trafficked to their respective destinations by post-translational translocation pathways that are evolutionarily conserved from yeast to human. However, they remain unidentified in the malaria parasite  The Seciewed in ). TA proa gondii –18. The iewed in , 20). Muiewed in , 22, insiewed in , non-assiewed in  and the iewed in , 26. Theiewed in , 28. Thuiewed in .In the yeast and mammalian cells, a subset of the TA protein repertoire destined for the ER (or post-ER compartments) rely on a proteinaceous machinery known as the Guided Entry of TA (GET) or transmembrane domain recognition complex (TRC), respectively . HoT. gondii [Plasmodium falciparum. P. falciparum causes the most severe form of human malaria [P. falciparum develops a complex network of membranes and vesicles for efficient protein trafficking [in vivo survival and ensure that the virulence determinants are trafficked correctly to their destined organelles. Protein trafficking in malaria-infected erythrocytes requires an additional level of refinement since the parasite resides within a parasitophorous vacuole (PV) of its host and can target proteins not only to the intra-parasite organelles but further beyond, to the parasitophorous vacuolar membrane (PVM) and the infection-induced modified host erythrocytic structures  [P. falciparum necessarily recruits the function of the SS similar to the higher eukaryotes [Plasmodium export element (PEXEL) or host-targeting (HT) motif with a consensus RxLxE/D/Q (where x is any amino acid) [P. falciparum, the parasite ER functions as an obligatory destination for proteins during the initial stages of the secretory pathway. At the ER, important sorting decisions are made and exported proteins reportedly bind phosphatidylinositol 3-phosphate-enriched domains through their HT/PEXEL motif [via ER-recruitment) need not necessarily involve SS since PNEPs without any N-terminus signal peptide are efficiently recruited and exported to the Maurer’s clefts and to the host erythrocyte [In a recent study, 59 novel TA proteins were identified in a related apicomplexan parasite . gondii . Domain  malaria . During fficking . These ee knobs)  (reviewee knobs) ). The eakaryotes . Howeverno acid) , 45, andno acid) . Nonetheno acid)  and PEXEno acid) . In P. fEL motif  prior toEL motif , 50. Howthrocyte .P. falciparum  based on the sequence, predicted structural, and functional similarity to the Get3s of prokaryotic and eukaryotic origins. Secondly, we showed that PfGet3 associates with a subset of the predicted total 63 putative TA proteins in the P. falciparum proteome. Our proximity labelling experiments further revealed the identities of the two other homologs of the GET machinery in P. falciparum, such as PfGet4 and PfGet2, and we validated their association with PfGet3 by in vitro binding studies. We were, however, unable to definitively identify the plasmodial homologs of other GET components, such SGTA/Sgt2, Get5/UBL4A and Bag6 from our shortlisted proteins since all of them featured either the TPR motif (in the case of SGTA/Sgt2 homologs) or the characteristic ubiquitin-like domain , both of which are ubiquitously present across multiple proteins enriched from our proximity labelling studies and LC-MS/MS analyses. We were also unable to identify the plasmodial homolog of Get1/WRB due to extreme sequence divergence. Nonetheless, we believe this is the first study that provides bioinformatic and biochemical evidence for the presence of TA proteins and the partners of the GET machinery in the human malaria parasite P. falciparum and has the potential to serve as a leading resource for future investigations elucidating the dynamics of this pathway and its development for therapeutic interventions.Irrespective of the route of trafficking or the final locale within an infected erythrocyte, the roles of vesicular transport intermediates as carriers of virulence determinants are undisputed in iewed in ). Model P. falciparum database. We thus used sequential selection criteria in the PlasmoDB (www.plasmodb.org) to identify putative TA proteins in the P. falciparum 3D7 strain, based on the following classical TA features: (i) the absence of an ER-type secretory SS as predicted by SignalP , (ii) the presence of a single TMD within 50 amino acids from the C-terminal end of the protein, and (iii) the orientation of the protein with respect to the TMD, i.e., we favoured N-terminuscytosolic-TMD-C-terminuslumenal orientation (using the TMHMM server at http://www.cbs.dtu.dk/services/TMHMM) . The output revealed a total of 130 proteins with these signatures , guanine-nucleotide exchange factor Sec12 (PF3D7_1116400), SNARE proteins like BOS1 (PF3D7_1111300), VAMP7 (PF3D7_0826600), VAMP8 (PF3D7_1303200), mitochondrial Fission1 protein FIS1 (PF3D7_1325600) . We manually removed the 67 misrepresented members from the list of outputs based on results from the earlier studies. These included 64 members of the repeated interspersed multigene family (RIFINs), mainly the subgroup RIFIN-A implying them as putative TA proteins. RIFINs constitute the largest known family of variant antigens in P. falciparum (~150 copies per haploid 3D7 genome) and encode for 27-45-kDa proteins [via the parasite’s induced structures called Maurer’s clefts [i.e., PF3D7_0410000 (erythrocyte vesicle protein-1) and PF3D7_0936000 (ring-exported protein-2) were already confirmed to be exported to the host erythrocytes [S1 Table for the full list of eliminated proteins). The final outcome from our predictions enlisted a total of 63 TA proteins in P. falciparum 3D7 .To date, no annotation as designated ‘TA protein’ exist in the proteins , 54. Preproteins . MembersPF3D7_13200 , with an average length of 22 amino acids, correlating well within the helical length of 18–21 amino acids necessary to span the usual width of a lipid bilayer . A bias towards the enrichment of amino acids with hydrophobic side chains was also observed across these TMDs . In contrast, the length of the CTS was diverse, varying from 1–5 amino acid (23 putative TA proteins) to as high as 45 amino acids (in PF3D7_1422000) . Also, there was no clear preference for any particular type of amino acids in the CTS . In the GRAVY scale (described in theS1 Text), the TMD hydrophobicity values ranged from the lowest 0.74 in PF3D7_1422000 to the highest 3.45 in PF3D7_1341700. PF3D7_1422000 is annotated as single pass putative cytochrome c oxidase assembly protein (COX14) that localizes to the mitochondria [The TA proteins enter multiple cellular membranes including the endomembranous ER, Golgi, plasma membrane, mitochondria, and peroxisomes  chondria . PF3D7_1chondria , and PF3chondria . Similarchondria ). The ERchondria .P. falciparum TA proteins into three broadly predicted sub-cellular localizations; namely the ER (and Golgi), the mitochondria and a collective of other destinations , based on the Gene Ontology (GO) annotations in the Uniprot database (http://uniprot.org) (seeS1 Textfor the predicted distribution criteria details). Our analyses revealed a total of 14 ER-specific TA proteins, 7 mitochondrial-specific TAs and 42 TAs with diverse predicted cellular destinations  and PF3D7_1111300/BOS1/GS27), primarily localized to the ER-derived vesicles and cis-Golgi in P. falciparum and T. gondii [P. berghei homolog of Sec61b localized to the parasite ER and exhibited high degree of co-localization with the ER resident P. falciparum Binding Protein (PfBiP) [H. sapiens RNF5 , selectively identified PfRNF5 at ~50-kDa  in the infected erythrocytic fraction by western blot . In IFAs, PfRNF5 also showed distribution at the perinuclear ER compartments and some degree of co-localization with PfBiP , consistent with the human RNF5 [ bottom blot). HA-Sec61b also exhibited ER distribution with good colocalization with PfBiP . Both PfRNF5 and PfSec61b have GRAVY scores of 1.87 and 1.94, respectively and with short CTS (2 or 3 amino acids) and 0/1 net charge. These have been predicted as ER/Golgi localized proteins (S1 Table).We also manually grouped the 63 putative . gondii , 71; the (PfBiP) . These wman RNF5  SimilarlP. falciparum and the outcome of our cellular localization assays for a few candidate TAs were consistent with the characteristics exhibited by TA protein in the other systems, thereby validating our bioinformatic selection procedures (a few representative TA proteins are shown in Table 1).Thus, the diverse features exhibited by the TMD and CTS of the predicted TA proteins in H. sapiens SGTA and S. cerevisiae Sgt2, as designated in [P. falciparum and subsequently use it as a handle to reveal the identities of the homologous effectors of the GET machinery and the bound TA substrates in this parasite. Although there are >2,000 putative Get3s reported in KEGG and OrthoDB databases [P. falciparum. Thus, we retrieved the amino acid sequences of the Get3 from yeasts , plant  and mammalian  systems from Uniprot (www.uniprot.org) and queried against the P. falciparum 3D7 database by BLAST in PlasmoDB. Results retrieved PF3D7_0415000 as the top hit in all queries. PF3D7_0415000 is a 379 amino acid protein annotated as a putative arsenical-pump driving ATPase, owing to its apparent ~17% identity and 32% similarity to the bacterial arsenite transporter ArsA which provides resistance to arsenite [Figs 2A and S1). Within eukaryotes, PF3D7_0415000 shared 46.9% similarity and 35.9% identity with ScGet3; 54.4% similarity and 41.8% identity with SpGet3; 51% similarity and 34.1% identity with AtGet3a and 59.7% similarity and 40.8% identity with HsGet3, respectively  the A-loop for adenosine recognition (QLKNEIR), and (iv) Switch I (DPAHN) and Switch II (DTAPTGHT) loops reported to undergo conformational rearrangements in the presence of γ-phosphate of ATP to form a ‘loaded spring’ that uncoils upon ATP hydrolysis [Fig 2A). Instead PF3D7_0415000 alignment displayed LxxC residues in which the first cysteine was replaced with leucine. Nonetheless, PF3D7_0415000 possessed a ~20 residue insertion called the Get3 motif or TRC40 insert that is deemed essential for the substrate TA binding. Thus, these conserved features prompted us to confidently annotate PF3D7_0415000 as PfGet3 throughout the course of this study. PfGet3 also showed a high level of conservation across the other members of Plasmodium species, including those that infect humans (P. vivax and P. knowlesi), as well as the other rodent-specific malaria parasites  . Phylogenetic analyses suggested that Get3 orthologs in the rodent malaria parasites diverge early in evolution and form a clade separate from the human counterparts. Get3 homologs has also been identified in other apicomplexan parasites, including T. gondii (TGVEG_231190), T. cruzi (Tc00.1047053507763.30), L. donovani (LDBPK_110710) and C. parvum (CGD7_4070) and they share varied similarity and identity to PfGet3 .In eukaryotes, the conserved GET/TRC pathway has been implicated in targeting TA proteins through a concerted relay of events. Therein, the cytosolic ATPase Get3/TRC40 is located at the junctional interface and communicates with the pool of soluble components such as Sgt2/SGTA (hereafter referred to as Sgt2/A to incorporate features of both nated in ), Get4/5nated in –77. We tatabases , no Get3arsenite  www.sbg.bio.ic.ac.uk/phyre2; [in silico prediction of its secondary structure and disordered region. Phyre2 output revealed the presence of 51% α-helices, 10% β-strands and 21% disordered regions . Submission of PfGet3 sequence to the TMHMM server (www.cbs.dtu.dk/services/TMHMM-2.0) predicted no transmembrane helix in PfGet3 consistent with PfGet3 being a soluble cytosolic protein . The Phyre2 server also generated a 3D atomic model of PfGet3 based on remote homology and template-based assembly simulations to the existing Get3 crystal structures in the RCSB Protein Data Bank . Although the predicted PfGet3 structure revealed 100% confidence with the Get3 orthologs from both prokaryotic and eukaryotic origins, it showed highest identity (59%) to the Get3 from the yeast Debaryomyces hansenii  followed by 51% identity to both nucleotide-free SpGet3  and ADP-AlF4 complexed ScGet3 , respectively [4-ScGet3/2WOJ has 99.3% sequence coverage compared to 91.4% for DhGet3/3IO3 (the finger domain of DhGet3 was missing in the electron density map); and more importantly, (ii) the crystal structure of multi-subunit GET translocation complex (Get3-Get4-Get5) from S. cerevisiae was also available in the PDB at 6 Å resolution (PDB ID: 5BWK) [P. falciparum in future studies.Since there is no reported crystal structure of PfGet3, we submitted the amino acid sequence of PfGet3 to the Phyre2 server  for in ectively , 86. We D: 5BWK) , which wS. cerevisiae Get3 (Δget3) has been reported to exhibit no phenotypic growth defects in synthetic complete media (or rich media), or changes in the cellular morphology, colony size, mating, sporulation, or germination [S. cerevisiae Δget3 strains are found to be more sensitive to As3+-, As5+-, Co2+-, Cr3+- and Cu2+-induced stress and high temperature-mediated stress than the parental wild-type strain [S. cerevisiae Δget3 strain with either empty vector or those containing sequences encoding for PfGet3 or ScGet3 and assessed their growth in the absence or presence of 1.5 mM CuSO4. The expression of PfGet3 at ~43-kDa in transformed Δget3 strain was confirmed by western blotting using custom-generated antibodies against PfGet3 . Growth kinetics experiments revealed no apparent differences in growth between the wild-type BY4741 and Δget3 strains in the absence of CuSO4. However, the Δget3 strain transformed with the empty vector displayed a salt-sensitive growth phenotype consistently seen both across the liquid culture-based and the plate-based growth assay. This phenotype was successfully rescued by the in trans expression of PfGet3 to a similar level as seen in the wild type BY4741 cells or in the transformed Δget3 knockout strain expressing ScGet3 . These results indicated that PfGet3 possessed characteristics like the ScGet3 in rescuing the salt-sensitive phenotype in Δget3 knockouts.A null mutant in mination . Insteade strain . To invepfget3 transcriptions in P. falciparum 3D7 (refer to www.plasmodb.org for the transcriptomic data): an initial high wave in ring stages within the first 10 hours post-invasion (hpi) and followed by a short shoulder at 20–36 hpi trophozoite stages, thus suggesting a requirement of PfGet3 function in the intraerythrocytic developmental cycle. To further investigate the expression of PfGet3 during the intraerythrocytic stages, the custom generated polyclonal anti-PfGet3 antibodies  were validated in western blots using P. falciparum 3D7 parasites or with the purified recombinant PfGet3. Results indicated specific recognition at ~43-kDa corresponding to the predicted molecular weight of PfGet3 as 43.3-kDa in P. falciparum parasites but not in uninfected erythrocytes . These anti-PfGet3 antibodies also recognized the recombinant 6×his tagged PfGet3 expressed in E. coli cells .Transcriptomic data in PlasmoDB indicates two waves of Fig 3C, top panel). However, in trophozoites and schizonts additional punctate/reticulate PfGet3 fluorescence were also detected in the perinuclear region characteristic of the parasite ER compartments . We were unable to visualize these concentrated PfGet3 patterns in the young rings owing to their small size and limitations in image resolution. In trophozoites and schizonts, the concentrated puncta of PfGet3 showed good colocalization with the ER chaperone PfBiP and the ER-resident parasite protease Plasmepsin V  thereby suggesting that a fraction of PfGet3 was at the vicinity of the parasite ER. These observations were consistent with the previously reported localization profiles of ScGet3 and the mammalian TRC40 in the perinuclear/ER compartments [P. falciparum to date. Similarly, we also generated transgenic parasites expressing PfGet3-GFP, and western blots confirmed the expression of a fusion protein at ~70-kDa . Live cell microscopy of PfGet3-GFP transgenic parasites further showed the presence of the diffused cytosolic fluorescence as well as punctate PfGet3-GFP spots in the perinuclear region, thus confirming the distribution profiles seen in the IFA images . Overall, the subcellular distribution of PfGet3 and its ability to reverse the CuSO4-sensitivity in the Δget3 yeast knockout strain, encouraged us to further investigate and identify the other homologs of the GET machinery in this parasite.Anti-PfGet3 antibodies were also used in indirect IFA to assess PfGet3 localization within 3D7 parasites. IFAs predominantly showed a diffused PfGet3 fluorescence within the confines of the parasite boundary (demarcated by antibodies to the parasitophorous vacuolar membrane protein Exp-2), thus indicative of the parasite cytosolic staining of PfGet3 in the ring stage parasites . Since the interactions of TA proteins with Get3 are deemed transient; to experimentally validate our bioinformatic predictions of TA proteins in P. falciparum 3D7 and their association with PfGet3, we generated transgenic parasites expressing PfGet3 fused to BirA*-HA at the C-terminus . Expression was confirmed in western blots using antibodies to the HA tag, which detected PfGet3- BirA*-HA fusion protein of ~76-kDa . IFA of the PfGet3-BirA*-HA-expressing parasites showed diffused distribution within the parasite cytosol . We were unable to find any concentrated fluorescent puncta of PfGet3-BirA*-HA similar to those seen in the native PfGet3 images , likely either due to the increased intensity of cytosolic PfGet3-BirA*-HA fluorescence or due to the use of antibodies to the HA-tag. However, in magnified images we noticed relative enrichment areas of PfGet3-BirA*-HA near the perinuclear region . Equal volumes of total extracts from PfGet3-BirA*-HA cells grown either in the absence (control fraction) or in the presence of D-Biotin (named as the BioID fraction hereafter)  were then used to affinity purify the proximal biotinylated interactors using StrepTactin XT beads and identified either by western blotting or LC-MS/MS. In the western blots using antibodies to both HA tag and PfGet3, the PfGet3-BirA*-HA was enriched only in the BioID fraction  although it could be detected in the crude lysates of both the control and BioID fractions . Interestingly, antibodies to PfGet3 also detected the native untagged PfGet3 on longer exposure of the membrane thereby implying the formation of a ‘heteromer’  between the untagged PfGet3 and PfGet3-BirA*-HA and thus the ensuing biotinylation. Self-biotinylated PfGet3-BirA*-HA was also detected in western blots. Mass spectrometry results yielded a list of proteins enriched in the BioID fraction of PfGet3-BirA*-HA as compared to the control sample (seeS2 Tablefor the complete list). PfGet3 was one of the top hits in the BioID fraction with 24 different peptides and ~10-fold excess over the control. Since the other homologs of Get3 are confirmed to form oligomers, we speculate that this low fold change might have been influenced either by the sample size, the overall abundance of PfGet3 (even when the transgenic parasites express PfGet3-BirA*-HA under the constitutive cam promoter), the ability of PfGet3-BirA*-HA to self-biotinylate or due to the low resolution LC separation from complex protein mixtures  [trans-biotinylated native PfGet3 . These also included two biotinylated peptides GTLNVLKNFTNNEMEFDSLYEK and KEMFDNILPELLHSFPGIDEALCFAELMQSIK (K indicates the site of biotinylation), thus confirming successful recruitment of the biotinylizer.BioID harnesses the potential of biotin ligase BirA* (BirA with a R118G mutation) to promote promiscuous biotinylation. When fused to a ‘bait’ protein, the ligase catalyses a two-step reaction in the presence of biotin to generate reactive bioAMP that covalently biotinylates adjacent primary amines of the proximate ‘prey’ proteins . These b lysate) . The ideFig 4F). These included PF3D7_0806300 , PF3D7_1111300 , PF3D7_1116400 (Guanine nucleotide-exchange factor SEC12), PF3D7_1227800 , PF3D7_1228800  and PF3D7_1412600 (Deoxyhypusine synthase), and accounted for approximately 43% and 10% of the 14 ER-predicted and the total 63 predicted TA proteome, respectively in P. falciparum. We also observed that the sum of peptides derived from each TA protein was limiting and we were unable to find biotinylation in any of the peptides corresponding to these proteins. This could either be due to the sample size or due to their low abundance or transient interactions with PfGet3-BirA*-HA that limits the scope of biotinylation at all the possible sites within these TA proteins. Nonetheless, we strongly believe that these TA proteins were biotinylated since they were affinity-purified with the StrepTactin-resin and enriched in the BioID fraction as compared to the control. Among the enriched TA proteins, PF3D7_1111300 and PF3D7_1116400 have been predicted in the ER-localized TA family in this study and corresponding homologs already validated in other systems [S1 Table), it is possible that their targeting in P. falciparum intersects with the GET pathway involving PfGet3. Interestingly, our BioID fraction was devoid of any mitochondrial TA proteins, an expected result since mitochondrial TA targeting essentially recruits GET/TRC-independent mechanisms [via the conserved ER membrane protein complex (EMC) rather than the GET pathway [P. falciparum and further experiments are warranted to ascertain their detailed characterization. To further validate the binding of TA proteins with PfGet3, we selected two representative TAs for our co-association studies. While one of the candidates, i.e., PF3D7_1111300 (PfBOS1) was enriched in our BioID fraction , we did not detect any peptides corresponding to PF3D7_0710800 (PfUSE1) by LC-MS/MS. Pulldown from the soluble crude lysates of transformed E. coli co-expressing recombinant 6×his-PfBOS1 or 6×his-PfUSE1 along with the recombinant untagged PfGet3  using Ni2+-NTA resin and detection by western blotting identified PfGet3 in the Ni2+-NTA purified 6×his-PfBOS1 or 6×his-PfUSE1 fractions, thereby confirming a direct binding of PfGet3 to these TA proteins. The recombinant PfGet3 (when singly expressed in the same plasmid vector) did not display any Ni2+-NTA-binding characteristic . These co-association studies further suggested that multiple TA proteins likely interacted with PfGet3 in the proximity biotinylation assay but were not detected in LC-MS/MS due to a paucity in their peptide abundance, limited sample size or the detector sensitivity.Peptides corresponding to six predicted TA proteins were also quantitatively enriched in the BioID fraction . Interestingly, PF3D7_1438600 was also the only significant hit in BLAST searches with the amino acid sequences of the well-characterized Yor164c (the S. cerevisiae homolog of Get4) and the H. sapiens Golgi to ER traffic protein 4 homolog/TRC35 (HsGet4) against P. falciparum 3D7 proteome, with significant e-values of 2e-4 and 9e-11, respectively. PF3D7_1438600 encodes for a 277 amino acid cytosolic protein, without any ER-type secretory signal sequence or TMD and has been annotated as a conserved protein with a domain of unknown function (DUF410) in PlasmoDB. PF3D7_1438600 shares considerable similarity with Yor164c (17.6% identity and 36.3% similarity), HsGet4/TRC35 (15.6% identity and 37.8% similarity) as well as other Get4 homologs , including the residues involved in interactions with Get3 [schematic inFig 5A; ). Evolutionary analyses of the orthologs of Get4 by the Maximum-likelihood method [Plasmodium parasite species that infect humans . Surprisingly, the Get4 ortholog of the rodent malaria parasite P. yoelii (PY00666) also grouped closer to the human malaria parasites  than other rodent malaria species (P. berghei and P. chabaudi). We thus annotated PF3D7_1438600 as PfGet4 hereafter in this study. Prior structural studies have shown that yeast Get4 is an alpha-helical repeat protein with the N-terminus (residues 1–148) involved in Get3 binding and its C-terminus interacting with Get5 [www.sbg.bio.ic.ac.uk/phyre2) for in silico secondary structure prediction. Phyre2 output revealed the presence of 72% α-helices and 14% disordered regions . PfGet4 structure showed 100% confidence with the crystal structure of the S. cerevisiae Get4-Get5 complex (PDB ID: 2WPV) and the human Bag6-NLS & TRC35 complex . Essentially, PfGet4 was also predicted to form 14 right-handed helices arranged pairwise with an α-solenoid like ScGet4 [In trans expression of PfGet4 (and confirmed by western blotting) also rescued the CuSO4-sensitive growth phenotype in Δget4 strain (as compared to those transformed with the vector only) to a similar level as the wild-type BY4741 cells or Δget4 cells transformed with ScGet4 in both the liquid culture-based or the plate-based growth assays . Custom anti-peptide antibodies against PfGet4  detected a parasite protein at ~33-kDa  in infected erythrocytes but not in uninfected RBC . IFA revealed localization of PfGet4 as diffused fluorescence indicative of the parasite cytosolic staining. Immunolocalization studies in transgenic parasites expressing PfGet3-BirA*-HA using mouse-anti-HA antibodies in conjunction with anti-PfGet4  showed diffused cytosolic staining pattern for PfGet4 with some areas of colocalization . In some late stage trophozoites, we also observed a few punctate spots of PfGet4 fluorescence in the parasite cytosol that were devoid of PfGet3 . These likely represent the pre-targeting PfGet4 complex (PfGet4-PfGet5-PfSgt2/A-TA) prior to its association with PfGet3. To assess the interaction between PfGet4 and PfGet3 in vitro, we expressed and purified recombinant MBP-PfGet4 in E. coli . Purified recombinant MBP-PfGet4 or recombinant MBP (as a control) were incubated with recombinant PfGet3-6×his and affinity-purified using Ni2+-NTA beads to assess co-association. Western blots using anti-MBP antibodies confirmed the selective binding of MBP-PfGet4 (but not MBP) to PfGet3 . Based on these observations and our earlier BioID results, we reasonably concluded that the P. falciparum genome encodes for a functional PfGet4 with features similar to the other Get4 homologs, and PfGet4 directly interacts with PfGet3, as expected for the canonical GET pathway.Our BioID fraction also enriched 8 peptides from PF3D7_1438600, while only one was detected in the control  and the scaffolding proteins Get4  and Get5  [Fig 5A; [in vitro translated TA proteins from mammalian lysates and mutations in the residues of the TPR domain which prevent Hsp70 binding impairs the loading of TA substrates onto Sgt2/A [Tables 2 and S2). Neither PfGet3 nor PfGet3-BirA*-HA display any obvious chaperone-binding motif, so we speculated that the proximal presence of the plasmodial homologs of Sgt2/A (PfSgt2/A) with Hsp-recruitment potential to PfGet3-BirA*-HA ensured their biotinylation and enrichment in the BioID fraction. To identify the putative PfSgt2/A, we queried P. falciparum 3D7 proteome with amino acid sequences of ScSgt2 or HsSGTA. Results retrieved multiple hits with significant e-values, such as PF3D7_1434300 (Hsp70/Hsp90 organizing protein/PfHOP), PF3D7_1241900 (Tetratricopeptide repeat protein), PF3D7_0213500 (Tetratricopeptide-repeat protein), PF3D7_1355500 (Serine threonine protein phosphatase 5), PF3D7_0707700 (E3 ubiquitin ligase) and PF3D7_1334200 (Chaperone binding protein), respectively. Unfortunately, our LC-MS/MS analyses did not yield any peptide corresponding to PfHOP, however, we detected of 12, 4, 3 and 2 peptides corresponding to PF3D7_0707700, PF3D7_1334200, PF3D7_1355500 and PF3D7_1241900, respectively in the BioID fraction and none were detected in the control . Interestingly, all these proteins contain TPR domains and could likely recruit the Hs(c/p) cascade to function as a putative PfSgt2/A. Indeed, a subclass of TPR-domain containing proteins such as Protein phosphatase 5, HOP and Sti1 in yeast act as cochaperones in regulating the nucleotide hydrolysis cycles and connecting protein folding pathways with the alternate route to ubiquitination and degradation [P. falciparum.Get3 is a central component of the GET pathway, orchestrating connections between the TA-recognition complex  and the downstream Get1/2 receptors at the ER membrane that functions to insert the TA proteins. Thus, the transgenic PfGet3-BirA*-HA parasites also offered possibilities to biotinylate both upstream and downstream effectors of the GET pathway. We therefore mined the BioID fraction further to identify the other GET machinery homologs in complex) . The bin[Fig 5A; , 77, 105[Fig 5A; . The cen[Fig 5A; , 107. Heo Sgt2/A , 33. We radation . HoweverScythe), TRC35 (HsGet4) and Ubl4A (Ubiquitin-like protein 4A), the Bag6 complex associates with the ribosomes and awaits the emergence of TMDs from the ribosomal tunnel [P. falciparum could possibly be biotinylated during its proximity to the putative PfSgt2/A as it ‘transfers’ the TA substrates to PfGet3-BirA*-HA, and accordingly get affinity-purified. PF3D7_0922100 was the top hit for H. sapiens Bag6 BLAST search against P. falciparum in PlasmoDB with an e-value of 2e-08. However, other hits from the BLAST query, like PF3D7_1113400, PF3D7_1365900, PF3D7_1313000 and PF3D7_1211800 also exhibited significant e-values of 1e-05, 3e-06, 2e-05 and 3e-04, respectively. Our BioID fraction detected 14 peptides corresponding to PF3D7_0922100, while none was detected in the control fraction (Tables ). Surprisingly, BLAST query with the amino acid sequence of the human Ubl4A also yielded plasmodial proteins overlapping with the Bag6 hits, notably PF3D7_1211800, PF3D7_1365900, PF3D7_0922100, PF3D7_1113400 and PF3D7_1313000 with e-values of 8e-12, 3e-12, 3e-08, 1e-07 and 2e-08, respectively. PF3D7_1365900 is 128 amino acids in length and annotated as ubiquitin-60S ribosomal protein L40. It was discarded as a candidate PfBag6/PfUbl4A because firstly, peptides were detected both in the BioID and control fraction (with only 5.03-fold enrichment), and secondly because PF3D7_1365900 has been solved as a part of the P. falciparum 80S ribosome by cryo-EM  [P. falciparum since they exhibit conservation in the critical residues implicated in the binding of Bag6-UBL to the CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) domain or Ubl4A-UBL to SGTA  in P. falciparum. Although, we detected no peptides corresponding to PF3D7_1313000 in the BioID fraction, we still retained PF3D7_1313000 as a plausible parasite homolog of Bag6/Ubl4a/Get5. We reasoned that the overlapping hits between the BLAST output of Bag6, Ubl4A and ScGet5 were due to the shared ubiquitin-like domain feature and thus, we were unable to conclusively designate any one of them. It is also possible that multiple proteins play redundant roles as Get5/Bag6/Ubl4A in P. falciparum and further experiments are necessary to identify and characterize the plasmodial homolog.The mammalian Bag6 complex also functions as an upstream loading factor, delivering TA substrates to the TRC40  , 109. Col tunnel . The tral tunnel , 112. WeD: 2BWE) , 115 and [P. falciparum 3D7 proteome in PlasmoDB to shortlist proteins fulfilling both the criteria, i.e., (i) the presence of the RR8422 (Arg-Arg-Polar-Basic-Aliphatic-Aliphatic) motif . Our results retrieved PFD_0215600 as the only hit. Interestingly, two peptides corresponding to PF3D7_0215600 were detected in the BioID fraction while none in the control  thereby strongly indicating it as a potential homolog of Get2/CAML. In the PlasmoDB, PF3D7_0215600 is a 403 amino acid protein, without the ER-type SS and annotated as a conserved plasmodial protein of unknown function. It has three transmembrane regions spanning between 247–267, 299–322 and 348–368 amino acids and shared only 27% similarity and 14.4% identity with the H. sapiens CAML and 22.9% similarity and 12.4% identity with ScGet2 . We have henceforth designated PF3D7_0215600 as PfGet2 in this study. PfGet2 also rescued the CuSO4-sensitive phenotype exhibited by the Δget2 knockout stain of S. cerevisiae as compared to the vector-only transformed Δget2 cells both in the liquid culture and plate-based growth assay , thereby confirming its role as a putative homolog of ScGet2 in P. falciparum. The transmembrane topology prediction for PfGet2 by MEMSAT 3 [CD; schematic shown in Fig 6D) fused to the MBP at the N-terminus . Accordingly, purified MBP-PfGet2CD or recombinant MBP (as a control) was incubated with the purified PfGet3-6×his, followed by enrichment with Ni-NTA beads and western blotting using anti- MBP or anti-PfGet3 antibodies . Results revealed co-purification of MBP-PfGet2CD (but not MBP) with recombinant PfGet3-6×his bound to Ni-NTA, thereby confirming a direct interaction. Thus, based on our bioinformatics selection parameters, enrichment in the BioID fraction and in vitro interactions results, we concluded that the P. falciparum genome encodes for a functional PfGet2 which exhibits properties similar to the other Get2 homologs.The terminal step in TA translocation involves interaction of the TA-loaded Get3 with Get1/2  receptors at the ER membrane to achieve membrane insertion, a process coupled with the ATP hydrolysis –121. DurLuminal) . We thern CAMLs; ) within MEMSAT 3  revealedP. falciparum, namely the existence of TA proteins in the proteome, and the active role of GET machinery for the protein translocation onto destined organelles . A recent study identified 59 putative TA proteins encoded by the Toxoplasma genome [P. falciparum. We further validated these predictions in cellular assays using the BioID approach with PfGet3-mediated proximal labelling and showed that a subset of the TA repertoire is indeed associated with PfGet3, thus directly implicating the role of PfGet3 in their translocation. In addition, our in-depth mining, and analyses of mass spectrometric data from the PfGet3-BioID pool either precisely identified or shortlisted a few other homologs of the GET machinery in P. falciparum. These included PfGet4 and PfGet2. We were, however, only able to shortlist (and not conclusively identify) the candidate plasmodial homologs of some of the other GET components due to the omnipresence of the following conserved domains/sequence motifs across multiple hits in the BioID fraction: (i) TPR in Sgt2/A and, (ii) ubiquitin-like domain in Get5/UBL4A/Bag6. We were also unable to identify Get1/WRB in P. falciparum, possibly due to the extreme sequence divergence from the canonical Get1/WRB. Identification and validation of PfGet1 along with homologs of the plasmodial Get5/UBL4A/Bag6 forms the next challenge in our understanding of GET pathway in this parasite. Until then, we offer our results to the community to investigate further alongside us.In conclusion, the outcome from this study revealed two novel aspects of protein trafficking in the human malaria parasite a genome . SimilarG, parameter = 4.1388 for PfGet3 and 66.6765 for PfGet4). The rate variation model allowed for some sites to be evolutionarily invariable  for PfGet4. The analysis involved 13 and 12 amino acid sequences for homologs of PfGet3 and PfGet4, respectively. There was a total of 409 (for PfGet3) or 327 (for PfGet4) positions in the final datasets. Evolutionary analyses were conducted in MEGA X [The evolutionary history of PfGet3 (PF3D7_0415000) and PfGet4 (PF3D7_1438600) were inferred by using the Maximum Likelihood method plus frequency model . The boon MEGA X .P. falciparum 3D7 genomic DNA was used as a template. Briefly, pET42b (PfGet3-6×his) was generated by PCR amplification of pfget3 using the forward PfGet3-NdeIF (5’-AGATCTCATATGAGTGAG GATGAATCGAATTCCGTTTCTTGTTCATTAAGC-3’) and reverse PfGet3-XhoIR (5’-AGATCTCTCGAGCAGATTATCTTTATATATAGGTATATCTTTCGATTGTAAGAGCATCTCCG-3’) primers, digestion of the PCR product with NdeI-XhoI and cloning at corresponding sites within similarly digested pET42b. The yeast complementation plasmid pYES260 was purchased from Euroscarf (http://www.euroscarf.de/) and contained ura3 selectable marker [pfget3 was PCR amplified using PfGet3-NcoIF forward (5’-AGATCTCCATGGATAGTGAGGATGAATCGAATTCCG-3’) and PfGet3-XhoI reverse (5’-AGATCTGTCGACCTCGAGTTACAGATTATCTTTATATATAGGTATATCTTTCGATTG-3’) primers. The PCR product was digested with NcoI-XhoI and cloned at the corresponding sites in digested pYES260. Similar strategy was also used to generate pYES260 (PfGet4/PF3D7_1438600) and pYES260 (PfGet2/PF3D7_0215600) using the following primer pairs: PfGet4-HindIIIF (5’-CCCATAAAGCTTATGTATCCATATGATGTTCCAGATTATGCTGCAGCTGCTAAAAAGTTCAAATTTAGTAAAGAAAAGCTAGCC-3’)/ PfGet4-XhoISalIR (5’-AGATCTGTCGACCTCGAGTTATGCAAATATGTTTTGGAACATACTGAACAAGTTATG-3’) or PfGet2-HindIIIF (5’- CCCATAAAGCTTATGTATCCATATGATGTTCCAGATTATGCTGCAGCTGCTGATAAAAATACATTAAAAAGAAGAAG-3’)/ PfGet2FL-XhoIR (5’-AGA CCGCTCGAGTCATAATAATACTACCTTCTGG -3’). Cloning within these sites also ensured incorporation of sequences encoding for the HA-tag at the N-terminus of the recombinant PfGet4 in the S. cerevisiae transfectants.All plasmid constructs used for this study were generated using molecular biology grade reagents and verified by Sanger sequencing. For all the PCRs involving the amplification of plasmodial homologs of the GET pathway and sub-cloning into different plasmids, e marker . For genS. cerevisiae homologs of Get3 (ScGet3) and Get4 (ScGet4). Briefly, scget3 and scget4 were amplified by PCR from S. cerevisiae genomic DNA using the following primer pairs: ScGet3NcoIF (5’- CATGCCATGGATGATTTAACCGTGGAACCTAATTTG-3’)/ScGet3XhoIR (5’-AGATCTCTCGAGCTATTCCTTATCTTCTAACTCATAAATGACTTTG -3’) or ScGet4-NcoIF (5’-CATGCCATGGATTTCCTGCTGAATCTAATGCTGTACAAGC-3’)/ScGet4-XhoIR (5’-AGATCT CTCGAGTCACTTCGATCCGCCCAGGAAT-3’) and cloned at the NcoI and XhoI sites in pYES260.The strategy was further extended to generate pYES260 constructs with genes encoding for the P. falciparum expression plasmid construct pA150 (PfGet3-GFP) was generated by PCR amplifying pfget3 using PfGet3-AvrIIF (5’-GTACCGCCTAGGATGAGTGAGGATGAATCGAATTC-3’) and PfGet3-AAA-BglIIR (5’-TCCTTTAGATCTAGCTGCTGCCAGATTATCTTTATATATAGGTATATCTTTCGATTG-3’), restriction digestion and cloning into similarly digested pA150. For the BioID experiments, the plasmid pcDNA3.1 MCS-BirA(R118G)-HA was purchased from Addgene (USA) and used to construct pA150 (PfGet3- BirA*-HA) through a series of subcloning steps. Briefly, pfget3 was PCR amplified using PfGet3-AvrIIF and PfGet3-AAA-BglIIR primers, first cloned at NheI-BamHI sites of pcDNA3.1 MCS-BirA(R118G)-HA. Subsequently, this plasmid used as template to PCR amplify out the entire pfget3-birA (R118G)-ha fragment using PfGet3-AvrIIF and HA-XhoIR (5’-TCTAGACTCGAGCTATGCGTAATCCGGTACATCGTAAG-3’) primers, digested with AvrII-XhoI and subcloned into similarly digested pA150. The construct pA150 (HA-PfSec61b) was generated by amplifying pfsec61b/pf3d7_0821800 from P. falciparum genomic DNA using PCR with the forward HA-PfSec61b-AvrIIF (5’-CCCATAAAGCTTCCTAGGATGTATCCATATGATGTTCCAGATTATGCTGCAGCTGCTAACGCCGCACCAGTAATAG-3’) and PfSec61b-XhoIR (5’-AGATCTCTCGAGTATTTTACTAATGATATGTAAAATAACTACACTTGCCATAAATATTAATG-3’) primers, digestion with AvrII-XhoI and cloning into similarly digested pA150.The CD, i.e., pMALc2X (PfGet4) or pMALc2x (PfGet2CD) were generated by amplification of either full-length pfget4 or nucleotides encoding for the first 246 amino acids of PfGet2CD using PCR with the PfGet4-EcoRIF (5’-GTACCGGAATTCATGAAAAAGTTCAAATTTAGTAAAGAAAAGCTAGCC-3’)/PfGet4-XhoISalIR or PfGet2CD-BamHIF (5’-GTACGCGGATCCATGGATAAAAATACATTAAAAAGAA-3’)/PfGet2CD-XhoISalIR (5’-AGACCGGTCGACCTCGAGTTATTCATGTTTCGTAATAATAAATTG-3’) primer pairs and subsequently cloning at corresponding sites in pMALc2x plasmid (New England Biolabs). For the co-expression and co-association studies in E. coli, pf3d7_1111300/bos1 and pf3d70710800/use1 were amplified by PCR using P. falciparum 3D7 genomic DNA and the respective primer pairs: BOS1-BamHIF (5’-GATCGGATCCTATGAATAAAAAGTATGAACCAATATTAGATAATGAATTG-3’)/ BOS1-AgeISalXhoIR (5’-CTCGAGGTCGACACCGGTTCATCTCTTAAAGTAAGAATATATGACATAAAAAAATATC-3’) and USE1-BamHIF (5’-GATCGGATCCTATG ATTGTGTTCGACGAACTTTTTG-3’)/ USE1-AgeISaIlXhoIR (5’-CTCGAGGTCGACACCGGTTTATAAAAGTATTATTACAAAAAAGGTAAATATAAAAAGTACTACTGAAG-3’). The PCR products were digested with BamHI and SalI and cloned in the MCS-1 of pCDFDuet-1 . This resulted in in-frame fusion at the 5’ end with sequences in the vector encoding for the 6×his tag at the N-terminus of the BOS1 and USE1 fusion proteins. Subsequently, pfget3 was subcloned from pET42b (PfGet3) construct at the NdeI-XhoI sites at the MCS-2, which generated in-frame fusion with at the 3’ end with nucleotides encoding for the S-tag at the C-terminus of the PfGet3 fusion protein.The constructs for the expression of N-terminus MBP tagged recombinant PfGet4 or PfGet2E. coli BL21 arabinose-inducible (AI) cells (Invitrogen) were transformed with the plasmids pET42b (PfGet3-6×his), pMALc2x (PfGet4) or pMALc2x (PfGet2CD) and transformants were selected on 100 μg/ml ampicillin or 25 μg/ml kanamycin containing LB plates. Primary cultures of transformed E. coli cells were grown overnight at 37°C under shaking conditions and then used to seed secondary cultures. Cultures were induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 0.2% L-Arabinose at OD600 ≈ 0.6 for 16 h at 25°C. For the purification of PfGet3-6×his, induced cultures were harvested at 6,000 rpm for 20 mins and resuspended in lysis buffer  containing 100 mM Phenylmethylsulphonylfluoride (PMSF) and EDTA-free Protease inhibitor cocktail tablets (Roche). Resuspended cells were first treated with 1 mg/ml lysozyme to digest the cell wall and subsequently frozen at -80°C overnight. The thawed cell suspension was sonicated at 30% amplitude with repetitive pulses of 30 s and off for 30 s till clarity. The sonicated cells were centrifuged at 14,000 rpm for 1 h at 4°C to remove the cell debris and the supernatant was incubated to Ni2+-NTA resin (Thermo Fisher) pre equilibrated with lysis buffer. After binding 4°C for 4 h under rotating conditions, the supernatant-resin suspension was packed onto a column and washed with at least 20 column volumes of wash buffer (lysis buffer containing 40 mM imidazole). PfGet3-6×his was eluted by elution buffer (lysis buffer containing 300 mM imidazole) and the eluted fractions were analyzed by running SDS-PAGE. Fractions containing PfGet3-6×his were pooled, dialyzed against 10 mM Tris. HCl pH 8.0, 150 mM NaCl, 1 mM MgCl2, 5 mM β-mercaptoethanol and 5% glycerol with minimum of three changes in buffer and concentrated using membrane concentrators . The purified recombinant PfGet3-6×his was subsequently used for the custom generation of anti-PfGet3 antibodies or for in vitro binding experiments.CD, the respective secondary cultures (OD600 ≈ 0.6) of transformed AI cells were induced with 0.3 mM IPTG at 25°C for 16 h, harvested at 6,000 rpm for 20 min and resuspended in column buffer . Soluble supernatant fractions were generated following similar protocol as described above. MBP, MBP-PfGet4 and MBP-PfGet2CD were affinity purified using amylose-resin  and eluted with elution buffer  as according to manufacturer’s instructions. The corresponding proteins were dialysed against 10 mM Tris. HCl pH 8.0, 150 mM NaCl, 1 mM MgCl2, 5 mM β-mercaptoethanol and concentrated. The purity of each protein was assessed by SDS-PAGE and subsequently used for in vitro binding studies.For the purification of recombinant MBP, MBP-PfGet4 and MBP-PfGet2Antibodies were generated against PfGet3 and PfGet4. Anti-PfGet3 antibodies were custom-generated in rabbits by Biobharati LifeScience (India) using the purified recombinant PfGet3-6×his as immunogen. Following booster, the anti-PfGet3 antibodies were affinity purified from harvested sera using column bound recombinant PfGet3-6×his. For anti-PfGet4 antibodies, the KLH-conjugated peptide CPPNSTEEKYKFMN, corresponding to the amino acids 97–110 in PfGet4 (PF3D7_1438600), was used as an immunogen in New Zealand rabbits and custom-generated by Genscript (USA). Antibodies were also affinity purified against the same peptide.S. cerevisiae parental BY4741 , Δget3 ,Δget4  or Δget2  strains were purchased from Euroscarf (http://www.euroscarf.de). The constructs pYES260 (PfGet3), pYES260 (PfGet4), pYES260 (PfGet2) or empty plasmid pYES260 were used to transform the respective strains. All the yeast transformations were performed following the previously published protocol [S. cerevisiae, cells were inoculated in YNB Sc-Ura media and grown. The cell density was subsequently adjusted to OD600 nm ≈ 0.01 and induced with 2.5% galactose and 1% raffinose for 8 h following which cells were harvested at 6,000 rpm for 5 min at 25°C and washed with autoclaved milliQ. Cells were then resuspended in lysis buffer  containing 0.05% PMSF and EDTA-free Complete protease inhibitor cocktail (Roche). Equal volume of acid washed 0.45 mm glass beads (Sigma) were added to the cells and homogenized using BeadBeater (BioSpec) at 3,000 rpm for 30 s followed by 30 s on ice for a total of 8 cycles. Cell suspensions were decanted away from the glass beads and centrifuged twice at 10,000 rpm at 4°C to remove unlysed cells and cellular debris. Supernatants were quantified and analyzed by western blotting using anti-PfGet3 or anti-PfGet4 antibodies.protocol  and seleS. cerevisiae parental BY4741 strain or the Δget2/Δget3/Δget4 knockouts transformed with either the empty pYES260 plasmids or expressing the plasmodial homologs were grown overnight in YNB Sc-Ura at 30°C under shaking conditions at 200 rpm. After growth, the respective cell density was adjusted to OD600 nm ≈ 0.01, cells transferred to 50 ml falcon tube containing fresh 15 ml YNB SC-Ura and induced with 2.5% galactose and 1% raffinose in the presence or absence of 1.5 mM or 1.75 mM CuSO4. Cells were grown for 32 h at 30°C under shaking conditions during which samples were harvested at every 8 h time interval to measure OD600 nm and the growth curve was plotted accordingly. For the plate-based growth assay, induced cultures were normalized to OD600 nm ≈ 1.0, serially diluted five-fold in sterile autoclaved water and 5 μl of each of the 10−1 to 10−5 dilutions were spotted onto YNB only or YNB with CuSO4 plates. Plates were then incubated at 30°C for 36 h before analysis.The P. falciparum 3D7 parasites were grown in fresh O+ human RBCs (kindly provided by the Rotary Blood Bank) in RPMI 1640 media supplemented with 0.5% Albumax II, 0.2 mM hypoxanthine, 11 mM glucose, 5% NaHCO3 and 50 μg/ml gentamycin in a humidified CO2 incubator as described previously [eviously . Cultureeviously . For liveviously  and imagP. falciparum 3D7 or transgenic parasites following protocol as described previously [4Cl, and slides blocked with 2% BSA. The respective primary antibodies were diluted in blocking buffer and incubated at RT for 2 h. The following antibody concentrations were used: anti-PfGet3, 10 μg/ml; anti-Exp2 ([Indirect immunofluorescence assays (IFA) were performed with either non-transfected eviously . Brieflyti-Exp2 , 20 μg/ti-Exp2 , 10 μg/P. falciparum parasites, cultures are 10–12% parasitemia were permeabilized with PBS  containing 0.05% saponin to release the hemoglobin content of the host red blood cell. The parasite fraction was repeatedly washed  with ice cold PBS till no hemoglobin was detected in the supernatant fraction. The parasite pellet was subsequently solubilised in Laemmli’s sample buffer containing 1 mM dithiothreitol (DTT), denatured for 10 min at 95°C and resolved by 12% SDS-PAGE. Resolved proteins were then transferred onto a nitrocellulose membrane and blocked with 5% fat-free skim milk for 2 h. Membranes were incubated with either anti-PfGet3, anti-Hog , anti-GFP (Invitrogen) or anti-β actin antibodies for 3 h on a platform shaker and followed by respective HRP-conjugated secondary antibodies. The blots were finally developed by chemiluminescence detection (Amersham).The preparation of lysates from transformed bacterial and yeast cultures are described previously. For the preparation P. falciparum 3D7 parasites expressing PfGet3-BirA*-HA following previously published protocol [2 flasks at 2% parasitemia and 5% hematocrit. D-Biotin was added at a final concentration of 50 μg/ml to one of the flasks (designated as the BioID sample) and not to the other (representing the control). The parasites were allowed to grow for 48 h at 37°C with replacement of fresh media (± D-Biotin) after 24 h. Cultures were subsequently harvested, washed twice with cold PBS and RBCs were lysed in PBS containing 0.03% saponin for 10 min. The parasite pellet was washed at least five times with 10 volumes of cold PBS to remove all the hemoglobin and stored at -80°C till further use. When planned, the frozen parasites were thawed at 4°C and extracted for 1 h at 4°C with 2 ml lysis buffer  containing 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail (Roche) under rotating conditions. The soluble extract was separated from insoluble cellular debris by centrifugation at 16,000 × g for 30 min at 4°C and immediately diluted two-fold with 50 mM Tris. HCl, pH 7.5. An aliquot each of the BioID and control extracts was stored at -80°C for SDS-PAGE and western blotting. To the remaining extracts, 30 μl of StrepTactin XT was added and allowed to incubate overnight under rotating condition at 4°C. After incubation, the beads were pelleted by centrifugation at 3,000 rpm for 5 min at 4°C and washed sequentially: thrice with lysis buffer, twice with cold double distilled water and twice with 50 mM Tris. HCl, pH 7.5. The beads were finally boiled in SDS-PAGE sample buffer and processed either for western blotting using anti-PfGet3 or anti-HA, or for LC-MS/MS analyses.Proximity biotinylation experiment was performed with the transgenic protocol . BrieflyMass spectrometry of the control and BioID fractions were performed at Wistar Proteomics Facility, Philadelphia, PA, USA. Briefly, the fractions were resolved by SDS-PAGE (only to the extent when the samples entered 0.5 cm within the resolving gel) and stained with colloidal Coomassie. Bands were excised from the gel, de-stained with water, alkylated with iodoacetamide, and digested with trypsin as previously described  see S1 . ProteinE. coli Rosetta™ (DE3) cells  were transformed with pCDFDuet plasmids, cloned with pfget3 along with either pf3d7_1111300/bos1 or pf3d70710800/use1. As a control, pCDFDuet expressing PfGet3 alone was used. Transformants were selected in LB agar plates with spectinomycin and inoculated into terrific broth containing the desired antibiotic. Cultures and grown at 37°C under shaking conditions, induced with 0.5 mM IPTG at OD600 nm of 0.6–0.8 for 3–4 h at 37°C. Cells were harvested prior to and after induction by centrifugation at 6,000 × g for 20 mins at 4°C. The induction of specific proteins was confirmed in western blots prior to processing for pulldown experiments.For co-expression and co-association studies, 2+-NTA resin, the harvested cells pellets (post-induction) were lysed by lysis buffer , 1 mg/ml lysozyme and protease inhibitor tablets cocktail tablets . The cell suspensions were disrupted by sonication with cycles of 30 seconds on and off each at 30% amplitude, for approximately 5 minutes till the turbidity of the solutions were clarified. The sonicated cells were centrifuged at 12,000 rpm for 30 mins at 4°C to remove the insoluble cellular debris and the soluble lysates were incubated to Ni2+-NTA resin  pre-equilibrated with the lysis buffer for 2 h at 4°C under gentle rotating conditions. Following binding, the resin-lysate suspensions were packed onto column and extensively washed with wash buffer . The proteins were stripped off the beads by boiling in 2× SDS PAGE sample buffer for 5 min at 95°C, resolved SDS-PAGE and processed for western blotting using anti-PfGet3 and anti-His  antibodies.For the downstream pulldown protocols using Niin vitro binding studies, purified recombinant MBP-PfGet4 or MBP-PfGet2CD (2–5 μg) were incubated with recombinant PfGet3-6×his in 1 mg/ml BSA containing binding buffer  for 2 h at 4°C. As a control, PfGet3-6×his was also incubated with purified MBP at similar concentration and duration. Following binding, binding buffer-equilibrated Ni2+-NTA beads were added to all protein mixtures and allowed to bind at 4°C for 2 h under shaking conditions. Beads were then washed extensively with binding buffer (without BSA), boiled in the presence of SDS-PAGE sample buffer for 5 min at 95°C and resolved by SDS-PAGE. Nitrocellulose membrane-transferred proteins were incubated with anti-PfGet3 or anti-MBP (NEB), probed with corresponding HRP-conjugated secondary antibodies and developed by chemiluminescence.For S1 FigA. Clustal X sequence alignment [S. cerevisiae, D. hansenii, S. pombe, A. fumigatus, H. sapiens, A. thaliana and the bacterial Arsenite transporter ArsA. Residue colouring is based on the program output (type of amino acid). The shading of the bars from brown to yellow reflects the degree of conservation, quality, and the consensus amino acids of the ordinates. Occupancy at a particular residue position is indicated by increasing intensity from light to dark grey shading. B. Table summarizing the similarity and identity between the amino acid sequences of PfGet3 (PF3D7_0415000) in comparison to the other validated or predicted homologs of Get3.lignment  between (TIFF)Click here for additional data file.S2 FigA. Predicted secondary structure of PfGet3 by the Phyre2 server (www.sbg.bio.ic.ac.uk/phyre2) and revealing the presence of 51% α-helices, 10% β-strands and 21% disordered regions. Residues are colored according to a simple property-based scheme: A, S, T, G and P; small/polar are in yellow, M, I, L and V; hydrophobic are in green, K, R, E, N, D, H and Q; charged are in red, and W, Y, F, C; aromatic + cysteine are in purple. The secondary structure prediction comprises three states: α-helix, β-strand, or coil. Green helices represent α-helices, blue arrows indicate β-strands and faint lines indicate coils. The ‘SS confidence’ line indicates the confidence in the prediction from PSIPRED, with red indicating high confidence and blue showing low confidence. A large amount of blue or green in the confidence line is indicative of few homologous sequences detected and a consequent low probability of modeling success. B. Outcome of the TMD prediction for PfGet3 by the TMHMM server (www.cbs.dtu.dk/services/TMHMM-2.0). No transmembrane helix was predicted in PfGet3.(TIFF)Click here for additional data file.S3 FigE. coli cells only under IPTG induced conditions, as compared to the uninduced control. The induced recombinant PfGet3 is indicated as filled arrowhead in the SDS PAGE (left) or by empty arrowheads in the western blots (middle and right) using custom-generated antibodies to PfGet3 (middle) or commercial anti-6×his antibodies . Molecular weight standards (in kDa) are as indicated.SDS PAGE (left) and western blot (middle and right) showing the expression of recombinant 6×his tagged PfGet3 in (TIF)Click here for additional data file.S4 FigA. Table showing the percentage identity and similarity between the various homologs of Get4 in comparison to PfGet4. B. Result from the secondary structure prediction for PfGet4 by the Phyre2 server (www.sbg.bio.ic.ac.uk/phyre2) and revealing the presence of 72% α-helices and 14% disordered regions. Residues are colored according to a simple property-based scheme: A, S, T, G and P; small/polar are in yellow, M, I, L and V; hydrophobic are in green, K, R, E, N, D, H and Q; charged are in red, and W, Y, F, C; aromatic + cysteine are in purple. The secondary structure prediction comprises three states: α-helix, β-strand, or coil. Green helices represent α-helices, blue arrows indicate β-strands and faint lines indicate coils. The ‘SS confidence’ line indicates the confidence in the prediction from PSIPRED, with red indicating high confidence and blue showing low confidence. A large amount of blue or green in the confidence line is indicative of few homologous sequences detected and a consequent low probability of modeling success. C. Multiple sequence alignment between PfGet4 and a few representative homologs of Get4 using ClustalX [D. Phyre2 predicted 3D structure of PfGet4 (rainbow colored) aligned with the crystal structure of the H. sapiens TRC35  [ClustalX . ResidueA; grey) . The α- (TIFF)Click here for additional data file.S5 FigNMR chemical shift perturbation patterns of Ubl4A-UBL and Bag6-UBL caused by their corresponding interaction partners (N-terminus of SGTA for Ubl4A-UBL and CUE for Bag6-UBL are indicated by asterisks . Black t(TIF)Click here for additional data file.S1 Tablehttp://uniprot.org): ER (shaded light orange), mitochondria (grey) and other destinations (not shaded). Thus, a total of 14 ER-specific TA proteins, 7 mitochondrial-specific TAs and 42 TAs with diverse cellular destinations were predicted.The list of 130 proteins includes RIFINs (64), EVP1, REX-2 and MSP5. The PlasmoDB ID, description and other features are shown for each predicted TA protein. Corresponding GRAVY and Adagir scores are also shown and shaded according to the scale provided. Intracellular localization is also predicted for each TA protein based on three different machine learning tools  and reveals no clear consensus for any particular organelle. Thus, the 63 predicted TA proteins were manually grouped (in this study) into three predicted categories based on their Gene Ontology (GO) annotations in the Uniprot database ((XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Forkhead box protein M1 (FOXM1) is a pivotal regulator of G2/M cell cycle progression in many types of cancer. Previously, our study demonstrated that histone deacetylase inhibition (HDACi) sensitizes hepatocellular carcinoma cells (HCC) to oxidative stress through FOXM1 suppression. However, the mechanism underlying its suppression by HDACi still requires elucidation. We hypothesized that HDACi induce genes responsible for destabilizing and inactivating FOXM1. The transcriptome in the HepG2 was revealed by massive analysis of cDNA end (MACE). Expression of mRNA and proteins were analyzed by quantitative real-time PCR (qPCR) and western blot, respectively. Cell cycle was analyzed by fluorescence-activated cell sorting (FACS). Oxidative stress and HDACi suppressed CDK4/6 levels while enhancing CDK inhibitor 2B and 2D (CDKN2B and CDKN2D) expressions in HCC. Palbociclib, a specific inhibitor of CDK4/6, induced G2/M cell cycle arrest in HCC by down-regulating phosphorylation level of FOXM1, and its downstream target genes such as aurora kinase A (AURKA) and polo-like kinase 1 (PLK1). HDACi treatment increased the ubiquitination level of FOXM1 by suppressing ubiquitin-specific peptidase 21 (USP21), which deubiquitinates FOXM1. Inhibiting FOXM1 degradation with MG132 treatment affected neither palbociclib-induced G2/M cell cycle arrest nor expression of its target genes. Double knockdown of CDKN2B and CDKN2D reduced the oxidative stress and HDACi-induced G/2M cell cycle arrest. In conclusion, oxidative stress and HDACi synergistically cause G2/M cell cycle arrest via CDKN2 induction, which sequentially inhibits CDK4/6, FOXM1, and its downstream target genes AURKA, PLK1, and CCNB1 phosphorylation in HCC. Human hepatocellular carcinoma (HCC) is one of the most common cancers and global mortality associated with it has increased during the past 15 years, primarily due to delayed detection and frequent recurrence that hinders HCC therapy Forkhead box protein M1 (FOXM1) belongs to the forkhead box transcription factor family. Unlike FOXO, another forkhead box transcription factor that is activated in quiescent cells and inhibits cell proliferation, FOXM1 is expressed in proliferating cells and acts as a master regulator of cell cycle progression Histone deacetylase inhibitors (HDACi) emerged as anticancer drugs decades ago Our previous study has demonstrated that treatment with histone deacetylase inhibitor (HDACi) suppressed FOXM1 expression and sensitized HCC cells to oxidative stress, which led to G2/M cell cycle arrest at a low level of oxidative stress 2. Cell viability was analyzed using Cell Counting Kit 8 (CCK8). Cells (5 x 103/well) were seeded in 96 well plate with 100 μl medium. After 24 h, cells were treated with agents. The next day, 10 ul of CCK 8 solution was added to the wells and then incubated for 2 h. Optical density was measured with a microplate reader at 450 nm . For knockdown, siRNA  was transfected using the INTERFERin® transfection reagent  following the manufacturer's instructions.Human hepatocellular carcinoma cell lines (HepG2 and Hep3B) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C with 5% COAntibodies to detect human CDK4, CDK6, CDKN2B, CDKN2D, AURKA, PLK1, CCNB1, and phospho-CCNB1 (pSer147) were purchased from Cusabio . Ubiquitin, FOXM1 (ChIP grade), and p-FOXM1 (pThr600) antibodies were purchased from Santa Cruz Biotechnology, Inc . CDKN2B and CDKN2D siRNA were purchased from Santa Cruz Biotechnology, Inc . MS-275, suberanilohydroxamic acid (SAHA), palbociclib, tertiary-butylhydroperoxide (tBHP), and MG132 were purchased from Tocris Bioscience .https://www.ebi.ac.uk/QuickGO/). Heatmap of differentially expressed genes was generated with Multiple Experiment Viewer (MeV) software. Fastq files from MACE were deposited in the NCBI SRA repository under accession number PRJNA678827 (https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA678827).Total RNA was extracted using RNAeasy mini kit . RNA quality and quantity were determined using NanoDrop One . Massive Analysis of cDNA End (MACE), an RNAseq method was performed as described previously TM First Strand cDNA synthesis kit  was used to synthesize single strand cDNA according to the manufacturer's recommendation. Then qPCR was perform using micPCR  as described previously Gapdh and used to calculate ΔCt values. Data were presented as fold change of 2-ΔCt. All primer sets used in the present study are shown in Total RNA extraction and determining RNA quality and quantity were performed as described above. Two micrograms of total RNA was used to synthesize cDNA. RevertAidFACS analysis for cell cycle arrest was conducted as described previously 5) were seeded in 60 mm dishes and the next day, cells were treated with drugs for 24 h. After washing with PBS, cells were lysed with lysis buffer. For IP, cells were lysed with non-denaturing lysis buffer  containing protease inhibitor cocktail. Cell lysates were incubated with primary antibody (2 ug) at 4 °C overnight (O/N). Target protein-antibody complex was precipitated using protein A/G agarose (60 ul of 50% slurry) at 4 °C for 2 h. The immune complexes were washed with lysis buffer three times. After elution of the immune complex, a western blot was performed to detect precipitated target protein. For western blotting, cell lysates (30 ug) were separated on a polyacrylamide gel and transferred to a nitrocellulose (NC) membrane. Transferred membrane was incubated with 5% skim milk in TBST buffer  for 1 h. The membranes were incubated with primary antibody (0.1~0.5 ug/ml) at 4 °C, O/N. After washing the membrane with TBST three times, secondary antibodies (1:5000) were added and incubated for 1 h. Target protein bands were developed using enhanced chemiluminescence (ECL) agent  in the darkroom. The band density was calculated with ImageJ software.Cells (5 × 103), followed by protein lysis with protease K at 45 °C for 30 min. DNA was eluted with DNA binding columns. FOXM1 binding on the promoter region of target genes was analyzed using qPCR. Primer sequences for ChIP assay are shown in ChIP assay was performed using the EpiTect ChIP OneDay Kit  according to the manufacturer's instructions. Cells were fixed with fresh formaldehyde (1% in PBS) for 5 min. Fixation was quenched with 125 mmol/L glycine for 10 min. Cells were lysed with SDS lysis buffer containing protease inhibitor cocktail. Soluble chromatin was generated by sonication . After pre-clearing with protein A/G agarose, ChIP grade anti-FOXM1 antibody (2 μg) was added and incubated at 4 °C for O/N. The antibody-chromatin complex was precipitated with protein A/G agarose and then sequentially washed with a low-salt solution, high-salt solution, LiCl solution, and Tris-EDTA solution twice. Precipitated chromatin was eluted with elution buffer , suberanilohydroxamic acid (SAHA), or tBHP and SAHA combined (SA+tB) were used to generate a heatmap  and minuscule changes at a relatively low level of tBHP , but drastic reductions became evident from 50 μmol/L onwards. Conversely, dose-dependent increases in the expressions of CDKN2B and CDKN2D were observed  in a dose-dependent manner. Pairing palbociclib with either SAHA (IC50 = 25.0 ± 0.92 μmol/L) or MS-275 (IC50 = 27.5 ± 0.94 μmol/L) enhanced its effect on HepG2 cells viability , and incorporating SAHA (IC50 = 24.3 ± 0.72 μmol/L) or MS-275 (IC50 = 25.0 ± 0.9 μmol/L) incurred further viability reduction in a dose-dependent manner  play a role in the maintenance of FOXM1 protein by preventing its proteasomal degradation p21 Fig. B. Immunop21 Fig. C. Low cop21 Fig. D.Aurka and Plk1 was investigated by ChIP assay. FOXM1 enrichments on the promoters of Aurka . Phosphorylation of FOXM1 in the TAD was closely related to the induction of target genes involved in G2/M regulation such as AURKA and PLK1. Additionally, our findings revealed that HDACi suppressed the expression of FOXM1 deubiquitinating peptidase Cdk4 and Cdk6 were decreased while their endogenous inhibitors Cdkn2b, Cdkn2d were increased  is an alternative variant of RNAseq, which requires a relatively short sequence (50-800 bp) from poly-A-tail of mRNA. In comparison to the standard RNAseq, MACE is a more powerful method for analyzing differential gene expressions and less susceptible to transcript length and degradation bias sed Fig. . The rolWAF1/Cip deleted cancer cells Cdk4 and Cdk6. Double knockdown of CDKN2B and CDKN2D reversed the Cdk4 and Cdk6 suppression incurred upon tBHP and SAHA co-treatment  specifically induced CDKN2B and CDKN2D in the cancer cells, including p21ent Fig. B and 5C.Aurka and Plk1 that play a critical role in G2/M progression were inhibited by palbociclib treatment (Fig. Ccnb1 (Fig. Aurka and Plk1 was investigated by ChIP assay. Palbociclib treatment significantly decreased the enrichment of FOXM1 near the transcription start site of Aurka (Fig. Plk1 (Fig. Usp21 (Fig. Aurka and Plk1 (Fig. FOXM1 is known as a master regulator of proliferation, metastasis, and cell cycle in cancer cells nb1 Fig. E and 4J.rka Fig. D and Plklk1 Fig. E. Interelk1 Fig. . Stabilip21 Fig. A and 7B.p21 Fig. D and 8E The ROS-scavenging capacity of cancer cells is higher than that of normal cells because cancer cells are exposed to a high level of ROS resulting from genetic, metabolic, and microenvironment-associated alterations FOXM1 acts as a master regulator of G2/M cell cycle as well as a critical regulator of oxidative stress during oncogenesis. ROS scavenging enzymes such as MnSOD, catalase, and PRDX3 are induced by FOXM1, which contributes to reducing intracellular ROS levels. Thus, FOXM1 activity is essential to cancer cells expressing ROS-producing oncogenes such as RAS or AKT In conclusion, HDACi induced the expression of CDKN2B and CDKN2D, which resulted in decreased activity of CDK4/6 as well as suppression of Usp21. These results were related to diminished activity and stability of FOXM1, which plays a critical role in cell cycle progression and anti-oxidative activity in HCC. Thus, HDACi combination with other anticancer agents could be a potential treatment option for cancer cells expressing FOXM1.Supplementary table.Click here for additional data file.


The rare and heterogeneous kidney disorder C3 glomerulopathy (C3G) is characterized by dysregulation of the alternative pathway (AP) of the complement system. C3G is often associated with autoantibodies stabilizing the AP C3 convertase named C3 nephritic factors (C3NeF). The role of classical pathway (CP) convertase stabilization in C3G and related diseases such as immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) remains largely unknown. Here, we investigated the CP convertase activity in patients with C3G and IC-MPGN. Using a refined two-step hemolytic assay, we measured the stability of CP convertases directly in the serum of 52 patients and 17 healthy controls. In four patients, CP convertase activity was prolonged compared to healthy controls, i.e. the enzymatic complex was stabilized. In three patients  the convertase stabilization was caused by immunoglobulins, indicating the presence of autoantibodies named C4 nephritic factors (C4NeFs). Importantly, the assay also enabled detection of non-immunoglobulin-mediated stabilization of the CP convertase in one patient with C3G. Prolonged CP convertase activity coincided with C3NeF activity in all patients and for up to 70 months of observation. Crucially, experiments with C3-depleted serum showed that C4NeFs stabilized the CP C3 convertase (C4bC2a), that does not contain C3NeF epitopes. All patients with prolonged CP convertase activity showed clear signs of complement activation, i.e. lowered C3 and C5 levels and elevated levels of C3d, C3bc, C3bBbP, and C5b-9. In conclusion, this work provides new insights into the diverse aspects and (non-)immunoglobulin nature of factors causing CP convertase overactivity in C3G/IC-MPGN. C3 glomerulopathy (C3G) is an umbrella term for kidney diseases characterized by glomerular inflammation and accumulation of breakdown fragments of complement component C3 in the glomeruli , 2. The Before the definition of C3G was introduced in 2010  and consNeisseria meningitidis infection  and lectin pathway that are activated by specific pattern recognition –14. Althnfection . NonetheIn this work, we investigated whether CP convertase overactivity occurred in our patient cohort consisting of 45 patients with C3G and 7 patients with IC-MPGN. First, we validated a two-step CP convertase activity assay measuring the stability of CP convertases directly in the serum of patients, to screen for factors prolonging the CP convertase activity. Next, we characterized the nature of these factors and investigated their prevalence in the disease course and relation to complement activation markers.Between July 2001 and March 2020, blood samples from 116 patients whose physicians indicated a suspicion of C3G or MPGN were sent to the Radboud university medical center for complement investigation corresponding to their suspected disease. All samples were measured for abnormal CP convertase activity. For 58 patients the final diagnosis could be retrieved  and P3  and P10 The study was performed in accordance with the appropriate version of the Declaration of Helsinki. Whole blood samples were processed within 1 hour after sampling according to the standard protocol . To obtaSheep erythrocytes (ShE) suspended in Alsever’s solution were obtained from Håtunalab AB . For each experiment, freshly sensitized ShE working suspensions were prepared as described previously , by washThe Ig fractions were isolated from 0.2-1.0 mL EDTA-plasma  or serum  samples as previously described , using NActivity of CP C3/C5 convertases was measured using a two-step convertase activity assay that follows the same principles as described previously , 28, 29 ® Incubating Microplate Shaker with 3 mm orbit . Finally, the supernatant was collected by centrifugation and transferred to flat-bottom 96-well plates (Greiner Bio-One). Hemolysis was quantified as percentage of full lysis generated by an equal amount of erythrocytes in water: (A405 test sample − A405 blank)/(A405 full lysis − A405 blank) × 100%.Per experimental time point, 10 µL of sensitized ShE were mixed with 20 µL DGVB++ containing 10 nM of the C5-inhibitor eculizumab . Subsequently, 20 µL of 1.9-2.5% test serum or EDTA-plasma diluted in DGVB++  was added at different time points to allow convertase assembly for 20, 10, 5, 2.5, 1.0, and 0.5 min. Where applicable, NHS or C3-depleted serum  were first mixed with purified Igs in a volume ratio of 1:1, 1:3, or 1:5 before being added as the test sample. Then, 150 µL of ice-cold 40 mM EDTA–gelatin veronal buffer  was added, after which the cells were washed and collected by centrifugation . For the second step, erythrocytes were incubated for 60 min with 50 µl of 2.5% guinea pig serum  in EDTA-GVB as a source of C5b-9 components, supplemented with 50 µl of DGVB++ buffer. All incubation steps above were performed in V-shaped 96-well plates (Greiner Bio-One) at 37°C with 600 rpm agitation in a VWRCFH, CFHR5, CFI, CD46, C3, and CFB were screened for rare genetic variants  according to previously described protocol , was performed to analyze the CFH/CFHR region in all patients.C3NeF activity was measured using a two-step hemolytic AP convertase activity assay . Levels protocol . In addiWhere indicated, data were analyzed using two-way analysis of variance with Bonferroni’s post-test using GraphPad Prism 5.03 for Windows .To establish the normal CP convertase activity profile, 17 healthy control sera were analyzed. These individual controls showed activity profiles that were comparable to each other and to NHS  and CFHR5 (P4) complement genes (We then screened for deviant CP convertase activity in the samples of 52 C3G/IC-MPGN patients whose samples were collected for complement investigation in the context of their disease. The patients had a median (IQR) age at the time of investigation of 15 –18 yearsTo investigate whether Igs, i.e. C4NeFs, were responsible for the increased convertase stability in the sera of patients P1-P4, we purified the Igs from the samples. Purified Ig fractions were concentrated in phosphate buffered saline to the initial sample volume and added to NHS in different volume ratios in the convertase activity assay (In the first step of the CP convertase activity assay both C3 convertases (C4bC2a) and C5 convertases (C4bC2aC3b) are assembled and further complement activation is blocked by eculizumab. C3b is also a component of AP C3 (C3bBbP) and C5 (C3bBbPC3b) convertases. Thus, to specifically analyze the CP C3 convertase-stabilizing activity of C4NeFs, C3-depleted serum was used in the first step of the assay so that only C4b2a could be formed. Addition of the Ig fractions from the patients showing Ig-mediated convertase stabilization in NHS, i.e. P1, P2, and P4, to C3-depleted serum resulted in prolonged CP C3 convertase activity profiles in all cases  of the patients displayed prolonged AP convertase activity, i.e. C3NeF activity  and C5b-9, and decreased levels of C5 , a urinary protein/creatinine ratio that varied between 69-132 mg/mmol, and normal blood pressure while receiving mycophenolate mofetil and ACE inhibition.This male patient presented at the age of 7 years with macroscopic hematuria and nephrotic range proteinuria with normal renal function. C3 serum levels were low (60 mg/L) but with normal serum C4 (260 mg/L). Serology findings for antineutrophil cytoplasmic antibody (ANCA), antinuclear antibody (ANA), and antistreptolysin O (ASO) titer were all negative. The kidney biopsy findings were consistent with DDD. With treatment including intravenous methylprednisolone therapy for 3 days followed by oral steroids, ACE inhibitor, and hydrochlorothiazide he reached partial remission, i.e. normal renal function with still some mild persisting proteinuria. His follow-up described in this study, however, started at the age of 15 years, when he presented again with nephrotic range proteinuria. This was preceded by diarrhea followed by general fatigue 5 weeks before presentation. C3 serum levels were again low (82 mg/L), C3d levels elevated (32.2 mg/L), and serum C4 levels normal (213 mg/L). This time exanthemata-like, non-itchy skin lesions were present at his face, trunk, arms, and legs as well as a staggering painful knee and ankles without redness and swollenness. Skin biopsy showed neutrophilic dermatosis. Fever developed during intravenous methylprednisolone treatment. Blood culture demonstrated a This girl was diagnosed at the age of 16 years with nephrotic syndrome with normal estimated glomerular filtration rate (eGFR) and was treated with a high dose of steroids and mycophenolate mofetil. Kidney biopsy taken on admission showed a mesangial proliferative glomerulonephritis with some global glomerulosclerosis and subepithelial humps. No staining for IgA, IgG, IgM, kappa or lambda light chains on immunofluorescence, marginal staining in capillaries for C1q and C3, but strong staining for C4d along the glomerular basement membrane were observed. Diffuse intramembranous, electron dense deposits were present on electron microscopy. On admission low serum C3 (40 mg/L), low C4 (50 mg/L), high C3d (18.9 mg/L) as well as low AP50 and CH50 activity were observed, and these values did not change during her three year follow-up, except for C4 normalization within one year. Although in follow-up she did not have peripheral and general edema, her serum albumin remained low (21-27 g/L) and her urinary protein/creatinine ratio ranged between 160-450 mg/mmol. Another renal biopsy performed at the age of 18 years demonstrated the pattern of C3GN with still extensive mesangial proliferation with mild endocapillary hypercellularity as well as endothelial, epithelial, intramembranous, endothelial, and mesangial depositions. Immunofluorescence showed this time C3 3+ next to C1q 1+, IgG, and IgM 1+. Unfortunately, no staining for C4d was performed. No glomerulosclerosis or interstitial fibrosis or tubular atrophy were seen. While on low dose of steroids, mycophenolate mofetil, losartan, and hydrochlorothiazide, she was transferred at 18 years to the adult nephrology clinic having a serum creatinine of 80 µmol/L and being normotensive but still with nephrotic range proteinuria.2 (serum creatinine 41 µmol/L) with nephrotic range proteinuria (urinary protein/creatinine ratio 1656 mg/mmol) and microscopic hematuria. She started with oral steroids for the presumed minimal change nephrotic syndrome. After 4 weeks of treatment she had responded partially with decline in proteinuria (urinary protein/creatinine ratio 717 mg/mmol) and no peripheral edema. Serum complement C3 level and C4 were very low (90 mg/L) and low (70 mg/L), respectively, and serum C3d level was elevated (16.9 mg/L). Serology findings for ANCA, ANA, and ASO titer were all negative. Kidney biopsy demonstrated a mesangioproliferative glomerulonephritis with no chronic lesions. C3 was prominently present on immunofluorescence microscopy (3+), and electron microscopy showed no intramembranous dense deposits. The referring center diagnosed the patient as C3GN. Mycophenolate mofetil was added to the steroids therapy followed by ACE inhibition, and later losartan was added. Six months later she is feeling well with no edema and a normal renal function, normotensive but still nephrotic range proteinuria (urinary protein/creatinine ratio 200-400 mg/mmol). The serum C3 level (150 mg/L) and C4 level 130 mg/L) are still low, and C3d remained elevated (22 mg/L). Her treatment consists of daily low steroids, mycophenolate mofetil, ramipril, and losartan.This female patient presented at the age of 10 years at the pediatric nephrology clinic. She had periorbital edema for already 2-3 weeks. At presentation she had a normal renal function with an eGFR of 136 ml/min/1.73 m2. The urinary protein/creatinine ratio on admission was 760 mg/mmol. Serology findings for ANCA, ANA, and ASO titer were all negative. Serum C3 was low (68 g/L), C3d elevated (28.6 mg/L), and C4 normal (178 mg/L). Due to the extreme fluid overload (estimated 18 L), fluid and salt restriction as well as diuretics were immediately started. Differential diagnosis mentioned post-infectious glomerulonephritis, IgA nephropathy, lupus nephritis, and C3G. Several attempts to perform a kidney biopsy under anesthesia in the acute phase of the disease failed due to his persistent refusals. In the light of the negative results for autoimmune diseases (see above) in combination with very low serological C3 level, elevated complement activation markers, and the presence of C3NeF, C3G was suggested. Treatment including high dose of steroids, mycophenolate mofetil, ACE inhibition, and diuretics was started. Fortunately, three weeks after the start of therapy a renal biopsy could be performed and showed an IC-MPGN without extracapillary proliferations and no interstitial-tubular damage. Immunofluorescence demonstrated granular deposits in the capillary wall: C3 3+, IgG 2+, IgM 1/2+, lambda 2+, kappa 1/2+, with no IgA or C1q. No chronic lesions were observed. Electron microscopy showed subendothelial electron dense deposits and presence of neutrophils. While on mycophenolate mofetil and ACE inhibition, three months later his serum albumin showed an increase (22 g/L) and his urinary protein/creatinine ratio (610 mg/mmol) some decrease.This boy presented at the age of 15 years with nephrotic range proteinuria, microscopic hematuria, and general edema at the emergency department. Blood pressure was 126/86 mmHg. One week before admission he had a possible throat infection for which he visited the general practitioner. His medical history mentioned an autism spectrum disorder and mental retardation. His laboratory results showed a low serum albumin (14 g/L) and low-normal eGFR 81 ml/min/1.73 mThe C3G classification was introduced to replace the older term MPGN in order to increase awareness of the AP dysregulation as the central event in disease pathogenesis. Most frequently, AP-directed autoantibodies are recognized as the underlying cause. In most cases C3NeFs are identified, but in fewer cases autoantibodies directed against Factor H, Factor B, or C3b can be detected , 33. C3GC2 gene of patient P3.We validated a reliable and time-effective approach to screen for CP convertase-stabilizing factors in the samples of 52 patients whose samples were sent for C3G/IC-MPGN workup. We found four patients whose sera showed prolonged CP convertase activity: three patients with C3G and one patient with IC-MPGN. In three patients , we could confirm that Igs, i.e. autoantibodies named C4NeFs, were responsible for prolonged CP convertase activity, since the Igs from these patients induced convertase overactivation in NHS. This low prevalence of C4NeF of 6% is in line with reported data in other C3G/(IC-)MPGN patient cohorts, where the C4NeF prevalence was 3-19% , 19, 34.Interestingly, all four patients showing prolonged CP convertase activity also showed prolonged AP convertase activity caused by C3NeFs. Such double positivity has been shown previously in 20% , 41% 1616, and 5C3 [c.691A>C (p.Ser231Arg)] in patient P2, which has not been described previously and is predicted as probably pathogenic by mutation prediction software . In patient P4, a frameshift variant in CFHR5 [c.485_486dup (p.Glu163Lysfs*10)] was found, which leads to a truncated protein. Similar frameshifts (p.Glu163Argfs*35) have been previously described in C3G patients  exaggerate the initial (weak) CP-mediated response, after which the disease evolves to a primarily AP-driven disease process e to C3G , 43.In conclusion, we have shown in-depth analysis of CP convertase activity in our C3G/IC-MPGN patient cohort and found that CP convertase overactivation can be caused by C4NeFs but also by serum factors other than Igs that are still to be identified. Patients with prolonged CP convertase activity additionally showed AP dysregulation as seen by C3NeF activity and clear signs of full complement activation in their blood. We recommend that all patients with C3G and related diseases such as IC-MPGN are tested for CP convertase-stabilizing factors for full characterization of the underlying complement defect, especially in the light of a broad spectrum of upcoming complement-inhibitors targeting the complement pathways at different levels . FurtherThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by ethics committee of the Radboud University Medical Center.MM: Methodology, formal analysis, investigation, writing – original draft, and visualization. NK: Conceptualization, resources, and supervision. SK: Investigation. SS: Investigation. VG: Resources. FE: Resources. ED: Resources. HD: Resources. CD: Resources. JW: Resources and writing – review and editing. BH: Conceptualization, project administration, supervision, and writing – review and editing; EV: Conceptualization, supervision, methodology, and writing – review and editing. All authors contributed to the article and approved the submitted version.This work was supported by the Dutch Kidney Foundation (13OCA27 COMBAT Consortium).JW has received grants from Achillion and Chemocentryx, outside the submitted work.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The OGG1 glycosylase function depletion has been seen to obstruct pathological conditions such as inflammation, A3 T-cell lymphoblastic acute leukemia growth, and neurodegenerative diseases, thus warranting OGG1 as an attractive anti-cancer enzyme. Herein, we employed several drug libraries intending to screen non-toxic inhibitory molecules against the active pocket of the enzyme that achieved stable binding mode in dynamics. Two anti-cancer compounds ([O-]C1=C(CC2=CC=CC=C2)SC(=[N+]1CC(=O)NC3=NC=C(CC4=CC=CC=C4)S3)S and CCCN(CCC)[S]-(=O)(=O)C1=CC=C(C=C1)C(=O)NNC2=NC3=CC=C(Br)C=C3C(=N2)C4=CC=CC=C4) from Selleckchem.com were identified to occupy the active pocket of OGG1 and bind with greater affinity than Control TH5487. The binding affinity of Top-1 is −11.6 kcal/mol while that of Top-2 is −10.7 kcal/mol in contrast to TH5487 Control . During molecular dynamic simulations versus time, the said compounds are tightly held by the enzyme with no minor structural deviations reported except flexible loops in particular those present at the N and C-terminal. Both the compounds produced extensive hydrophobic interactions with the enzyme along with stable hydrogen bonding. The docking and molecular dynamics simulations predictions were further validated by molecular mechanics with generalized Born and surface area solvation (MM/GBSA) and Poisson Boltzmann surface area (MM/PBSA), and WaterSwap binding energies that validated strong binding of the compounds to the enzyme. The MM/GBSA binding free energy for Top-1 complex is −28.10 kcal/mol, Top-2 complex is −50.14 kcal/mol) and Control is −46.91 kcal/mol while MM/PBSA value for Top-1, Top-2 and Control is −23.38 kcal/mol, −35.29 kcal/mol and −38.20 kcal/mol, respectively. Computational pharmacokinetics support good druglike candidacy of the compounds with acceptable profile of pharmacokinetics and very little toxicity. All these findings support the notion that the compounds can be used in experiments to test their anti-cancer activities.The  Reactive oxygen species (ROS) are the product of exogenous and endogenous sources, the latter ROS are generated as a by-product of oxygen metabolism in the endoplasmic reticulum, mitochondria, and peroxisomes . It has https://www.medchemexpress.com/su0268.html (accessed on 8 December 2021)) and O8 (https://www.tocris.com/products/o8-ogg1-inhibitor_6236 (accessed on 8 December 2021)), effectively targets OGG1 enzyme and thus can serve beneficial approach for targeting oxidative DNA repair and alleviating cancer pathologies C1=C(CC2=CC=CC=C2)SC(=[N+]1CC(=O)NC3=NC=C-(CC4=CC=CC=C4)S3)S and CCCN(CCC)[S](=O)(=O)C1=CC=C(C=C1)C(=O)NNC2=NC3-=CC=C(Br)C=C3C(=N2)C4=CC=CC=C4) were selected as the best inhibitors of the enzyme compared to the Control TH5487, SU0268, and O8. The GOLD fitness scores of Top-1 and Top-2 are 79 and 76, respectively, while their AutoDock Vina binding energy is −11.6 kcal/mol and −10.7 kcal/mol, respectively. In case of negative Controls, the binding affinity of TH2840 or TH5411 was −8.61 kcal/mol and −8.01 kcal/mol, respectively. The different docking scores of the Control and compounds are tabulated in To evaluate the binding affinity of the filtered libraries’ compounds for the OGG1 enzyme, structure-based virtual screening was conducted. The active site information was retrieved from crystal structure of the enzyme reported with TH5487 inhibitor . PerformControl TH5487 molecule’s GOLD fitness score is 72 and its binding energy value is −9.0 kcal/mol. Both hydrogen bonding and van der Waals interactions are reported at the active pocket by the Control. Cys243, Val259, and Hie260 are hydrogen bonding residues with distance lengths of 4.02 Å, 3.8 Å, and 4.54 Å, respectively. While Gly32, Gln33, Lys239, Val257, Asp309, Hie263, and Gly32 formed van der Waals interactions with the compound. Besides these, Phe134, Leu246, Pro256, Phe306, and Leu310 were noticed in different alkyl interactions. Interestingly, the docked site of the compounds and the Control remain the same and interact with an almost similar set of active pocket residues. The binding mode of Control and its interactions with the active site residues are shown in The Top-1 interactions at the docked active cavity are dominated by van der Waals. These interactions are formed along the length of the compound where the central oxygen and nitrogen atoms are engaged by hydrogen bonding in particular with Lys239 (distance length is 2.3 Å). The end benzene on one side produced interactions of van der Waals and pi-anion while the on the other side make pi-alkyl and van der Waals. The central sulfur atoms of the compounds interact with Cys243, and Phe134 through sulfur bonding. From all around, the compound is strongly held at the active pocket and favors interactions with majority of the active pocket residues  and  results . The sim results ,29 was pControl was reported with an excellent RMSD plot and is a demonstration of greater intermolecular strength. The mean RMSD of Top-1, Top-2, and Control complexes is 1.68 Å, 2.04 Å, and 2.27 Å, respectively. The Top-1 compound enzyme is seen as not stable until 52 ns, as it experienced deviations touch 3.1 Å. Following this, the system remained in stable RMSD until the end of simulation time. Top-2 compound had a lower RMSD until 60 ns and then behaved inconsistently with maximum RMSD of 3 Å, and after 140 ns the system became stable. These acceptable RMSD variations upon snapshots visualization identified that compounds are trying to stabilize their binding conformation at the active pocket by giving flexibility to some of its chemical moieties, which forces the enzyme flexible loops to lose their original XYZ coordinates  (8 December 2021). The top 10 solutions of GOLD were compared, and the best common binders were selected for downward computational studies. The docking process was done in Genetic Optimization for Ligand Docking (OLD) 5.2  and the OLD) 5.2 . In GOLDControl complex is presented in For the molecular dynamic simulations, we employed AMBER20 software package . Force fTo determine both total binding free energy and per-residue decomposition of selected complexes MM/GBSA and MM/PBSA approaches were used . This waThe entropy energy of each complex was determined using a bash script applied in a previous study . The docked complexes were then subjected to WaterSwap based binding energy predictions ,27. WatebindG and calculated as:Alanine scanning analysis was further performed for OGG1 enzyme residues that are seen consistently important for interactions with the compounds . Key resbindG value and vice versa. More stability of the mutants can be inferred by a lower ΔIn silico predictions about compounds, pharmacokinetics were done using online SWISSADME  and pkCSComputer aided drug designing (CADD) techniques have been useful in discovery, developing and analyzing biological active drugs . Compare