The MC1R/cAMP/MITF pathway is a key determinant for growth, differentiation, and survival of melanocytes and melanoma. MITF-M is the melanocyte-specific isoform of Microphthalmia-associated Transcription Factor (MITF) in human melanoma. Here we use two melanocyte cell lines to show that forced expression of hemagglutinin (HA) -tagged MITF-M through lentiviral transduction represents an oncogenic insult leading to consistent cell transformation of the immortalized melanocyte cell line Hermes 4C, being a melanocortin-1 receptor (MC1R) compound heterozygote, while not causing transformation of the MC1R wild type cell line Hermes 3C. The transformed HA-tagged MITF-M transduced Hermes 4C cells form colonies in soft agar and tumors in mice. Further, Hermes 4C cells display increased MITF chromatin binding, and transcriptional reprogramming consistent with an invasive melanoma phenotype. Mechanistically, forced expression of MITF-M drives the upregulation of the AXL tyrosine receptor kinase (AXL), with concomitant downregulation of phosphatase and tensin homolog (PTEN), leading to increased activation of the PI3K/AKT pathway. Treatment with AXL inhibitors reduces growth of the transformed cells by reverting AKT activation. In conclusion, we present a model system of melanoma development, driven by MITF-M in the context of MC1R loss of function, and independent of UV exposure. This model provides a basis for further studies of critical changes in the melanocyte transformation process. MC1R) has been found to be a low-risk melanoma susceptibility gene [MC1R variants have furthermore been shown to increase the melanoma risk in families possessing cyclin-dependent kinase inhibitor 2A (CDKN2A) mutations [CDKN2A was identified as the first melanoma susceptibility gene more than 20 years ago, and germline CDKN2A mutations have been found in up to 20–40% of the melanoma-prone families worldwide [CDKN2A-encoded p16INK4a forms a functional protein complex with CDK4, and cyclin D1, leading to phosphorylation of the RB1 protein, with subsequent impact on the G1-S control of the cell cycle. This signaling is altered in 90–100% of melanomas [The melanocortin-1 receptor  requires acquisition of somatic mutations, facilitated by the MC1R genotype or aberrant microenvironment due to CDKN2A mutation status [MC1R gene locus is highly polymorphic in populations of European ancestry, and more than 200 coding region variants have been identified to date, with a combined prevalence of any MC1R variant being present in ~60% of the population. Among these variants are the red hair color (RHC) variants associated with red hair, light skin, poor tanning ability, and heavy freckling [MC1R variants, protecting PTEN from degradation, leading to AKT inactivation [It has been proposed that a malignant transformation of melanocytes expressing n status . The MC1reckling . Carrierreckling . The relreckling ,10,11. Itivation . Functiotivation . Solar rtivation . Howevertivation ,16,17, aTERT) or telomere maintenance play a role in familial melanoma predisposition [BAP1, POT1, ACD, and TERF2IP [In recent years, new melanoma susceptibility pathways have emerged , and a gposition , as well TERF2IP .MC1R alleles (R160W/D294H) [MC1R, R160W/D294H, which results in complete loss of function of the MC1R protein, while Hermes 3C is WT for MC1R [MC1R mutant Hermes 4C cell line transforms upon forced MITF expression, whilst the WT Hermes 3C does not. We present a useful model system for studying the development of melanoma in the context of defective MC1R. Here we use the immortalized melanocyte cell lines Hermes 3C and 4C to study the non-UV-related mechanisms of melanoma initiation in the context of familial melanoma. The Hermes 3 and 4 series are immortalized melanocyte cell lines with WT and RHC W/D294H) ,26, respW/D294H) . The Herfor MC1R ,26. We sSOX10 is under MITF control.In order to investigate the role of MITF with respect to melanoma development in varying RHC background, HA-tagged MITF was introduced via lentiviral transduction in both the WT MC1R Hermes 3C and in the compound heterozygote MC1R R160W/D294 Hermes 4C melanocytes. The resulting stably transduced cells are hereafter referred to as 3C-HA-MITF and 4C-HA-MITF, respectively. To confirm successful transduction, we measured exogenous and endogenous MITF levels by both western blotting and RT-PCR A,B. We fBoth SOX10 and MITF are important factors of melanocyte biology, including mediating pigmentation of melanocytes , and theFurther, after lentiviral transduction of HA-tagged MITF, we also observed a transformed morphology in 4C-HA-MITF, in terms of loss of dendricity, flattening and more spread morphology, and greater clustering of cell distribution. These changes in morphology were not observed in 3C-HA-MITF cells D. To valCDK4, CDKN2A, MITF and MC1R, in all cell lines is reported in n = 10; TYR by RT-PCR  differentially expressed genes (DEGs) returned a set of 235 putative direct gene targets of MITF in the Hermes 3C line, and a set of 543 putative direct gene targets in the Hermes 4C line  using siRNA against MITF . Analysi 4C line , of whic 4C line . The 144 4C line . Similar 4C line . In Herm 4C line . Other g 4C line . Overallp = 6.05 × 10−37), and the gene set up-regulated in fibroblasts after KD of RB1 ), respectively, suggesting a possible implication of p53 function, and pRB in the process of transformation of 4C-HA-MITF. Interestingly, we could not observe a marked (log10 (p)< −5.0 = p < 0.00001) enrichment of biological processes among downregulated genes in 3C-HA-MITF vs. 3C-control, while upregulated genes showed enrichment for unfolded protein response and ER stress-related genes  of genes encoding proteins implicated in epithelial to mesenchymal transition . The reason for utilizing an HA-tag in our construct was that high-quality antibodies specific for the MITF-M protein isoform are still lacking. We therefore utilized the HA-tag to ensure antibody-specificity to the MITF-M isoform.We performed two biological ChIP-seq experiments in each cell line and identified 42,075 high confidence (common to both experiments) MITF-M binding sites (MITFBSs) in 3C-HA-MITF cells, and almost twice as many  in 4C-HA-MITF cells. The MITFBSs overlapped to a significant extent between the two cell lines, as approximately 70% of the MITFBSs in 3C-HA-MITF were also found in 4C-HA-MITF A, and thAXL, IL6, TGFBI, and EGFR,) compared to HA-MITF binding in 3C-HA-MITF  of open genomic regions enriched for H3K27Ac and H3K27me3 marks, indicating active or repressed chromatin, respectively, from twelve melanoma cell lines from published studies  and perfA motif-enrichment analysis was undertaken for both 3C and 4C -HA-MITF cells consensus MITFBSs . The maiIn a de novo motif discovery analysis looking for motifs of length between 6 bp and 18 bp, we again found that in 3C-HA-MITF cells, the E-box motifs were the top enriched motifs F. In 4C-AXL was one of the top upregulated genes in 4C-HA-MITF cells C. PrevioNext, we targeted AXL by an AXL siRNA and an AXL inhibitor (R428) to assess whether this could counter the increased proliferation observed in 4C-HA-MITF cells. Depletion of AXL by the siRNA and inhibitor abolished the phosphorylation of AKT, while only the siRNA reduced the AXL level F. SurpriMC1R-variant Hermes 4C cell line, but not in the WT Hermes 3C cell line, we examined TCGA containing a cohort of 470 melanoma cases of Skin Cutaneous Melanoma (SKCM), for the expression status of MITF in MC1R variant carriers. We found that 10–15% of the 470 tumors had relatively high (Z-score > 1) expression of MITF  background . Later, In the model presented here, HA-MITF transduction alone is sufficient to induce transformation of 4C cells. The transformed RHC MC1R melanocytes display increased MITF-binding to the chromatin compared to MC1R WT-carrying melanocytic cells. Moreover, MITF-binding reflects aberrant chromatin structure observed in melanoma . The binThe role of HA-MITF behind the transformation process needs further clarification. However, in agreement with previous reports , our stuPhosphorylation of PRS6 through mTOR activation has been shown to be a marker for malignant transformation in melanocytic lesions downstream of PI3K/AKT . It may It has previously been reported that pharmacological downregulation of MC1R in MC1R-proficient melanocytes is associated with increased PI3K/AKT activation, possibly via MITF . Our finMITF is amplified in circa 20% of melanoma tumors, and this is thought to contribute to tumor progression . It is a2. SKMEL28 was purchased from ATCC and grown in RPMI  with 8% FBS (PAA), 5% CO2, at 37 °C. U2OS cells were obtained from ATCC and grown in RPMI (Sigma Aldrich) with 8% FBS (PAA), 5% CO2, at 37 °C. Further details about cloning, constructs, and lentiviral production, culture conditions, growth factor and anchorage independence assays, as well as siRNA experiments, isolation of RNA, and establishment of tumor xenografts are provided in the The immortalized melanocyte cell lines Hermes 3C and 4C were purchased from the Wellcome Trust Functional Genomics Cell Bank . To ensure melanocyte proliferation, cholera toxin was added to the media as recommended by Wellcome Trust. CT stimulates cyclic adenosine 3′,5′-monophosphate (cAMP) production within the cells. All Hermes lines and their derivatives were cultured as previously described , with thHermes 3C and Hermes 4C melanocytes were subjected to lenti-particle transduction of pLX 3xHAvar4mCh (vector expressing 3xHA tagged MITF-M (variant 4)) or control vector pLVX IRES mCh at a MOI of <1. After 5 passages, the cells were sorted for the expression of bicistronic mCherry, and allowed to expand further. Stable cell lines were subsequently re-sorted based on the presence of mCherry and used further. Cell sorting was performed by flow cytometry at the Oslo University Hospital Core Facility. This process resulted in four cells lines which we called 3C and 4C, for control vector transduced cells, and 3C-HA-MITF and 4C-HA-MITF, for cells transduced with the vector expressing 3xHA tagged MITF-M.Western Blot analysis was performed as previously described . The folAnimal experiments were approved and performed as stated by the Norwegian Animal Authority (Permit number 12080), and conducted according to the regulations of the Federation of European Laboratory Animal Science.NSG mice were anesthetized with 3% Sevoflurane (Baxter) and subcutaneously injected on both flanks with five million Hermes 4C , Hermes 4C transduced with pLVXIRESmCh (vector control transduced 4C cells) and Hermes 4C cells transduced with pLX3xHAvar4mCh vector (4C-HA-MITF) in 200 µL RPMI1640. Tumor volume and body weight was registered once a week. Further details are provided in the 3C-HA-MITF and 4C-HA-MITF cells were used for the Chromatin immuno-precipitation with sequencing (ChIP-seq) experiments in two replicates, essentially as described in Alver et al., , using aMITF-M, SOX10, TBP, RPLP0, AXL, SPOCK1, GREM1 and IRES) were purchased from Integrated DNA Technologies . Primers for PTEN, TGFBI and IL6 were purchased from Bio-Rad Laboratories, Inc. . All sequences are given in Quantitative real time PCR assays were performed as previously described . PrimersChIP-seq analysis was performed as previously described  with minHigh-confidence maps of genomic regions enriched for H3K27Ac , H3K27me3 , and open chromatin 11,065 sites) were compiled with Bedtools  sites we. A separRNA-seq data on MITF knockdown in 3C and 4C cells were analyzed using STAR (v. 20201) with default parameters for alignment to hg19. The resulting bam files were then fed into cuffdiff (v 2.2.1) using all conditions to build a base for transcript analysis.q < 0.05. Mutation analysis from whole-exome sequencing and microarray analyses are described in Gene ontology and pathway analysis were performed using geneMANIA , MetaScaRNA-seq expression data from the TCGA-SKCM cohort , measuren = 471) were obtained from the Genomic Data Common Data Portal using manifest download verification [Alignment files (BAM) for the RNA-sequenced samples  . Pooled Here we propose an HA-MITF-induced model of melanomagenesis based upon the innate oncogenic properties of MC1R RHC, representing enhanced risk for PI3K/AKT pathway activation and oncogenic transformation in families possessing CDKN2A loss of function. Our study opens the path for further investigations on the potential oncogenic role of MITF-M as a molecular switch in MC1R RHC backgrounds.


Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor β superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage mAb that prevents extracellular proteolytic activation of pro- and latent myostatin. Here we used integrated structural and biochemical approaches to elucidate the molecular mechanism of antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. This epitope is highly sequence-divergent, having only limited similarity to other closely related members of the transforming growth factor β superfamily. Hydrogen/deuterium exchange MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen–antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 1:1 (antibody:myostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms. MSTN, gene ID 2260) is a member of the transforming growth factor β (TGF-β)5–Myostatin (growth/differentiation factor 8 (GDF8); gene name A). Upon cleavage of the N-terminal peptide, the secreted pro-myostatin containing the prodomain and growth factor remains intact. The activation mechanism of pro-myostatin involves two distinct proteolytic cleavage events. Furin-like proprotein convertase cleaves an RXXR sequence motif between the prodomain and growth factor, yielding a latent, noncovalent complex of the prodomain and growth factor (latent myostatin) that remains unable to bind its receptor  revealed key insights into the specificity of SRK-015 to the precursor forms of myostatin. The results identified a conformational epitope in the arm region of the prodomain. In addition, changes in conformational dynamics to regions distant from the epitope were observed. Binding of SRK-015 results in decreased solvent accessibility in regions including the latency-associated structural elements and the loops contiguous to the protease cleavage sites. Furthermore, negative-stain EM revealed the precise binding orientation of SRK-015 to both pro- and latent myostatin, demonstrating a stable ring-like conformation of the antibody–antigen complex. Taken together, these findings elucidate the molecular details of a novel strategy for specific inhibition of growth factor activation by targeting the precursor forms.A phage-based discovery campaign aimed at identification of inhibitory antibodies targeting myostatin precursors yielded a number of specific antibodies capable of preventing proteolytic activation . One of TM cells. The secreted proteins were purified using immobilized nickel (II) affinity and gel filtration chromatography. For crystallographic constructs, mutations in the tolloid (D99A) and furin (R263A and R266A) protease cleavage sites were introduced to generate an uncleavable precursor protein, pro-myostatin. Pro-myostatin runs as single bands at approximate sizes of 83 and 41 kDa under nonreducing and reducing SDS-PAGE conditions, respectively (B). The 83-kDa band represents the homodimeric, disulfide-linked form of pro-myostatin that dissociates to a 41-kDa monomeric form under reducing conditions. The latent myostatin is devoid of mutations in the tolloid/furin protease cleavage sites; thus, under nonreducing and reducing conditions, several cleavage products were visible, consistent with the presence of unprocessed precursor forms of myostatin, the prodomain, and the growth factor, which are noncovalently associated.To understand the structural basis of the binding mechanism of SRK-015 to pro- and latent myostatin, biochemical and structural approaches were employed. Human pro- and latent myostatin constructs were generated and expressed in mammalian Expi293Fectively B. The 83C). The antibody variants were tested as full-length IgG4 and as Fab fragments. Full-length SRK-015 showed an approximately nanomolar binding affinity to both pro- and latent myostatin and an approximate 7-fold decrease in binding affinity in its Fab version. 29H4-16 hIgG4 demonstrated high-affinity binding to both pro- and latent myostatin. In fact, the dissociation constant (DK) is not reliably quantifiable, as the off rate of the antibody was observed to be below the limit of sensitivity of the biolayer interferometry Octet instrument. Monomeric 29H4-16 Fab showed a decrease in binding affinity compared with its IgG4 form, a similar trend as observed for SRK-015.Using the recombinantly expressed and purified proteins, the binding affinities of 29H4 and derivative antibodies to pro- and latent myostatin were tested using biolayer interferometry C. The anD). The observed average molar masses for latent myostatin and SRK-015 were 94 and 151 kDa, respectively, which are close to their calculated molar masses . Moreover, the latent myostatin–SRK-015 complex showed a homogenous mass distribution with an average molar mass of 235 kDa, suggesting that one molecule of SRK-015 binds one latent myostatin homodimer, consistent with a 1:1 binding stoichiometry.Further, the stoichiometry of binding of SRK-015 to latent myostatin was investigated. Size-exclusion chromatography (SEC) coupled to multi-angle light scattering (MALS) was used to determine the absolute molar mass of the latent myostatin, SRK-015, and the latent myostatin–SRK-015 complex. The latent myostatin and SRK-015 were each eluted as a single peak with homogenous mass distribution D. The obe.g. type 1 (ALK4/5) and type II (ActRIIa/b) receptors (E), suggesting that both antibodies effectively block mTLL2 tolloid–mediated proteolytic activation and that 29H4-16 is a suitable surrogate for biochemical and structural studies. Monomeric 29H4-GL Fab had no effect on blocking mTLL2-mediated proteolytic activation of latent myostatin, which can be attributed to its weaker binding affinity to latent myostatin (C) and suggests that avidity may play a role in the potent inhibition observed for the bivalent antibody.Similar to other members of the TGF-β superfamily of growth factors, pro-myostatin is activated by extracellular proteases, including proprotein convertase (Furin or PCSK5) , 45, 46 45–eceptors E, suggesyostatin C and sug5GGU and 5F3H), myostatin (PDB code 3HH2), and pro-TGFβ1 (PDB code 3RJR). Subsequently, publication of the pro-myostatin structure (PDB code 5NTU) allowed further improvement in many regions of the structure. The structure was completed after successive rounds of manual building and refinement using Refmac5 .To gain further structural insights into the binding mechanism of SRK-015 to pro- and latent myostatin, the X-ray cocrystal structure of pro-myostatin–29H4-16 Fab was obtained. The structure was solved by molecular replacement using search models derived from crystal structures of Fab (PDB codes  Refmac5  to a finA). Overall, 29H4-16 Fab binds to the arm region in the prodomain, with one Fab occupying each arm region of the pro-myostatin homodimer. The 29H4-16 Fab-binding region in the arm is relatively distant from the loops containing the furin and tolloid protease cleavage sites. This was unexpected, given that the antibody inhibits tolloid protease–mediated activation of the myostatin precursor (E).In the observed crystal form, pro-myostatin exists as a homodimer that sits on the crystallographic two-fold axis so that the asymmetric unit contains one pro-myostatin monomer and one Fab, whereas the entire complex maintained a 2:2 (pro-myostatin monomer:Fab) binding stoichiometry A. Overalrecursor E.24-Ala41), the loops containing the tolloid  and furin (Thr260-Gly269) cleavage sites, a region in the prodomain (Ser125-Pro135), and a region in the growth factor (Gly311-Pro338). As the electron density for the loops that contain the protease cleavage sites are among the missing regions in both structures, the relative conformations of these loops between the unbound and antibody-bound states of pro-myostatin are unresolved.The pro-myostatin structure reported in this study contains several regions with unresolved electron densities, similar to the previously published structure of unbound pro-myostatin . The regA) seen in other TGF-β superfamily members but is structurally different from the ring-shaped, cross-armed structure of pro-TGF-β1  (B) showed a root mean square deviation of 3.03 Å, indicating that the Fab binding elicits conformational changes in the pro-myostatin structure. Particularly the loops and β strands in the arm region of pro-myostatin shift to accommodate the loops of the CDRs of 29H4-16 Fab. Additionally, as part of the global adaptation to 29H4-16 binding, structural elements within the prodomain  and part of the growth factor displayed changes in conformation between the unbound and 29H4-16 Fab–bound states of pro-myostatin.Structural alignment of the pro-myostatin–29H4-16 Fab complex with the previously published unbound pro-myostatin (PDB code 5NTU) B showed C). The pro-myostatin–29H4-16 Fab binding interface buries 4.2% (1650 Å2) of the total solvent-accessible surface area , which is within the typical range for antibody–antigen interactions , CDRH1 , and CDRL2 (Asp51 and Asn53) interact via H-bonding with the residues  in the arm region of pro-myostatin. Additionally, Asp114 from CDRH3 and Asp51 from CDRL2 form salt bridges with residues Arg190 and Lys193 in the β5 strand of pro-myostatin. The nonlinear distribution of the contact residues in pro-myostatin shows that 29H4-16 binds a conformational epitope. The amino acid residues comprising the epitope–paratope of the pro-myostatin–29H4-16 binding interaction are summarized in Fig. 2D.The binding interface of pro-myostatin and 29H4-16 involves two CDRs from the heavy chain (CDRH1 and CDRH3) and one CDR from the light chain (CDRL2) of the 29H4-16 Fab and the arm region within the prodomain of pro-myostatin spanning the β4, β5, and β7 strands C. The prs 4.2% 16 Å2 of thE). The low sequence conservation comprising the epitope supports previously reported data showing the specificity of SRK-015 over the other relevant members in the TGF-β family, including pro-GDF11, proactivin A, BMP-9, BMP-10, and pro-TGFβ1  are relevant. To gain additional structural insights into the binding of SRK-015 and the variant 29H4-16 antibody to the pro-myostatin and latent myostatin forms, the in-solution conformational changes accompanied by these binding interactions were measured using H/DX-MS. This method relies on exchange of the protein backbone amide hydrogens with deuterium in solution, in which the rate of H/D exchange depends on solvent accessibility for a given region. Thus, the structural and regional dynamics of the protein play a large role in the rate of exchange (e.g. antigen–antibody) can be generated.The activation pathway of pro-myostatin involves cleavage by furin and tolloid proteases, which releases the growth factor for downstream signaling. Importantly, a pool of extracellular, uncleaved pro-myostatin has been observed in skeletal muscle in addition to furin-cleaved, latent myostatin, which predominates in serum , 45, 47.exchange . For exaFig. S1). The regions with no peptide coverage are assigned to regions within the growth factor that contain disulfide-bonded cysteines, suggesting a very stable region recalcitrant to reduction and protease digestion. Heatmaps representing the relative differential deuteration profiles of pro- and latent myostatin in the unbound and Fab-bound states over the course of H/D exchange are illustrated  to provide the overall conformational flexibility profile across regions of the protein. As expected, in both pro- and latent myostatin, the buried regions in the core of the protein structure showed lower H/D exchange levels than the solvent-exposed or flexible regions of the protein. The peptic peptides with higher H/D exchange are localized within regions in the prodomain, including the N-terminal association region, latency lasso, α2 helix, and the top of the arm region comprising the loop region adjoining the β5 strand. Relatively higher H/D exchange events have also been observed in loop regions contiguous to the furin and tolloid protease cleavage sites. These data suggest that the higher conformational flexibility of these regions could be essential for the structural and dynamic regulation of pro- and latent myostatin as well as for its accessibility to proteolytic cleavage for activation.H/DX-MS experiments were performed on pro- and latent myostatin in its unbound and 29H4-(GL/16) Fab-bound states. The protein samples were incubated in deuterated buffer between 10 s and 1 h for H/D exchange, followed by on-column pepsin digestion and MS for peptide mass analysis. The peptic peptide coverage was found to be 94.2% and 88.0% across the linear amino acid sequence for pro-myostatin and latent myostatin, respectively (E) and latent myostatin . The deuterium uptake plots of all resolved peptic peptides for pro-myostatin and latent myostatin are shown in Figs. S3 and S4. Overall, the binding of 29H4-16 Fab resulted in a higher extent of significant H/D protection across regions of pro- and latent myostatin than 29H4-GL Fab  Fabs. These regions included the top of the arm of the prodomain, including the amino acid residues adjoining the β4 (170–176), β5 (174–195), and β7 (234–238) strands . The repFig. 3A, C, and E). The observed decrease suggests that the peptic peptide regions containing the proteolytic cleavage sites become less flexible and less solvent-exposed when bound to 29H4-(16/GL) Fabs. In contrast, the corresponding peptides containing the tolloid/furin protease cleavage sites in latent myostatin displayed no significant changes in H/D exchange profile upon binding to 29H4-(16/GL) Fabs . This suggests that the overall flexibility of these regions is not affected in the presence of the antibody fragments. The differences in H/D exchange pattern in the region contiguous to the tolloid and furin cleavage sites between the pro- and latent myostatin indicate that these two forms are in distinct conformational states before and after furin cleavage.The H/DX-MS dynamic studies of the Fab bound complexes revealed multiple protected regions in the arm of the prodomain, which is in agreement with the result of the conformational epitope identified in the cocrystal structure of pro-myostatin–29H4-16 Fab . AdditioA), suggesting that these regions become less flexible in both complexes. On the other hand, latent myostatin showed increased H/D exchange in the latency lasso region, part of the β6 strand, and α4 helix, indicative of enhanced solvent exposure and conformational flexibility of these regions when bound to 29H4-(16/GL) Fabs (B). The antibody-mediated changes in overall conformational flexibility of regions adjoining the α1 helix, latency lasso, α2 helix, and β1 strand in pro- and latent myostatin are particularly interesting because these regions are proposed to be key in maintaining myostatin latency  Fab. In pro-myostatin, a significant decrease in H/D exchange was observed in the regions of the N-terminal association site, α1 helix, latency lasso, α2 helix, and β1 and β10 strands A, suggesGL) Fabs B. The an latency , 16./GL Fab. Altogether, the dynamics of pro- and latent myostatin suggest that binding of SRK-015 and the antibody variant 29H4-16 induced a decrease in the conformational flexibility in the antibody epitope in the arm region of the prodomain. Binding of the antibody also evoked a decrease in solvent exposure of structural elements that are important for maintaining pro-myostatin latency, stabilizing the prodomain–growth factor interaction. Likewise, binding of the antibody resulted in a decrease in solvent exposure of the tolloid and furin protease cleavage sites, which may suggest limited access of these sites to proteases for conversion of pro-myostatin to latent myostatin.A). The antigen–antibody complexes were then stained with uranyl formate for negative stain EM imaging. Further 2D class averages obtained for pro-myostatin–29H4-16 IgG4  and latent myostatin–SRK-015  showed limited but well-defined orientations of the particles representing the antigen–antibody complex. In fact, only two rounds of 2D class averaging were necessary for both antigen–antibody samples because of the observed sample homogeneity.To visualize the overall molecular organization and precise orientation of SRK-015 binding to pro- and latent myostatin, negative-stain EM was conducted using pro-myostatin and latent myostatin incubated with full-length monoclonal antibodies, 29H4-16 hIgG4, and SRK-015, respectively. Prior to conducting negative-stain EM, the samples were subjected to size-exclusion chromatography to ensure that complex formation of full IgGs to pro- and latent myostatin could be observed A. The anB) and latent myostatin–SRK-015 (D) showed particles that represent the antigen–antibody complex as well as the unbound full-length mAb. The samples were combined at a 1:2 (antigen:antibody) molar ratio to achieve a saturating condition; hence, there were no observed class averages that could represent the unbound pro- or latent myostatin homodimers. The class averages representing the antigen–antibody complexes showed lengths of about 21–22 nm with an average width of 10 nm. The electron density displaying the relative orientations of the pro- and latent myostatin dimers and the Fab and Fc arms of the antibody could be readily identified. Visible in the class averages are the two Fab arms of the antibody occupying the two arm regions of the prodomain for the pro- and latent myostatin homodimers, suggesting a 1:1 binding stoichiometry of the antigen–antibody complex. The pro- and latent myostatin homodimers adopted a V-shaped conformation with an angle of ∼80° between the two arms of the prodomain. This angle is conserved across class averages and between the two different antigen–antibody samples, suggesting that there is minimal flexibility around the dimer interface of both myostatin samples when bound to the antibody. In contrast, the Fc region showed a less well-defined density with variable orientations relative to the rest of the structural organization of the complex, suggesting a conformationally elastic domain that is likely a consequence of flexibility around the hinge region of the antibody.Representative 2D class averages for pro-myostatin–29H4-16 hIgG4 B and lat–SRK-015 D showed A) into the representative 2D class averages for pro-myostatin–29H4-16 hIgG4 (C) and latent myostatin–SRK-015 (E) showed a strikingly similar overall structural organization, with the exception of the presence of an additional density assigned to the Fc region of the full length antibody. Taken together, these data suggest the utility of an integrated structural approach to map out the details of the binding mechanism and stoichiometry as well as the mechanism of action of SRK-015, a neutralizing antibody against pro- and latent myostatin.The superposition of the cocrystal structure of pro-myostatin–29H4-16 Fab A into th16 hIgG4 C and lat–SRK-015 E showed The structural characterization data described here reveal the molecular mechanism of a novel strategy to inhibit myostatin signaling by preventing proteolytic activation of the myostatin precursors. This strategy is distinct from other approaches that target the interaction of the mature growth factor with its receptor , 52–55. 52–To date, the pro-myostatin–29H4-16 Fab cocrystal structure represents the first crystal structure of an antibody bound to the proform of myostatin. More importantly, SRK-015 and related analogs represent first-in-class antibodies that recognize both prodomains in the symmetrical V-shaped structure of the precursor myostatin homodimer. This precise binding orientation was confirmed by negative-stain EM showing that both Fab arms of a single antibody occupied the prodomains of one pro- or latent myostatin dimer forming a stable ring-like antigen–antibody complex. This unique anchoring mechanism of SRK-015 could be attributed to the distinct set of complementary residues in the binding interface as well as the avidity observed because of the bivalency of the IgG4 framework.Observed in the cocrystal structure and by H/DX-MS, binding of SRK-015 and the high-affinity 29H4-16 analog elicits changes in conformation and solvent accessibility to regions distant from the epitope, including the latency-associated structural elements that are crucial for the overall activation mechanism of myostatin. One distinguishing feature in the prodomains of TGF-β superfamily members is the N-terminal helix–loop–helix motif in the forearm region comprised of residues 42–115, which covers regions in the α1 helix, latency lasso, and α2 helix. This peptide region was previously identified as an inhibitory fragment of pro-myostatin . In the in vitro proteolytic inhibition assay shown in this work (E) and further validated by previous in vitro and in vivo assays showing that SRK-015 and analogs prevent tolloid-mediated cleavage of myostatin precursors , suggesting that this region becomes less flexible upon antibody binding, which could impact deposition of myostatin into the extracellular matrix.Additionally, the H/DX-MS dynamics studies of myostatin precursors were able to resolve representative peptides covering the N-terminal association region, which is typically not resolved in the crystal structures of pro-myostatin. This region in the prodomain is an important structural feature of the TGF-β family, as it serves as an anchor point for various presenting molecules for storage in the extracellular matrix or for cell surface expression . The myoFig. 3The discovery of SRK-015 and related antibodies opens opportunities for specific therapeutic intervention for myostatin signaling. Previous clinical approaches that inhibit myostatin signaling were directed to block the growth factor–receptor interaction and included binders of the growth factor  or recep37–The approach of targeting the precursor forms to modulate growth factor activation could be useful in discovery of specific modulators for other members of the TGF-β superfamily or, more broadly, other signaling proteins requiring precursor processing for activation. Practically, this approach takes advantage of the sequence diversity in the prodomain relative to the growth factor domain, reducing the challenge of identifying antibodies selective for one growth factor over others that share a high level of sequence and structural identity.en route to its receptor. Such a mechanism requires a high circulating concentration of inhibitory antibodies and an antibody with a very fast on-rate to achieve sustained functional inhibition. Preventing activation altogether substantially alleviates such requirements, reducing the bar for achieving efficacy.Perhaps more importantly, targeting (inactive) growth factor precursors may also offer a biological advantage. Activation and signaling of the TGF-β superfamily of growth factors are local events. Growth factor activation occurs proximally to the cellular location of the growth factor receptors. In the case of myostatin, pro- and latent myostatin reside predominantly in the extracellular space within muscle fibers, in close proximity to its cognate receptors on the cell surface . SimilarIn summary, use of an integrated structural approach facilitated elucidation of the molecular mechanism of a novel therapeutic strategy of targeting the precursor forms of myostatin to prevent activation and signaling for treatment of muscle-related disorders. Extracellular proteolytic processing is a common theme in the activation mechanism of members in the TGF-β superfamily. Thus, the approach of specifically targeting the precursor protein to block activation can be broadly applied to other members of the TGF-β superfamily, which may facilitate discovery of its specific modulators.6 tag, tobacco etch virus protease cleavage site, and myostatin amino acid residues 24–375 (Uniprot ID 014793) with furin (D99A) and tolloid (R263A and R266A) sites mutated to render it uncleavable and inactive. Latent myostatin was generated similarly with the same signal sequence, His6 tag, and the WT myostatin sequence (residues 24–375).The constructs for pro- and latent myostatin were cloned into the pTT5 vector using EcoRI and NotI restriction sites via Gibson assembly . Pro-myostatin was designed with an N-terminal signal peptide derived from the mouse IgG heavy chain, followed by a HisH and CH1 regions into the EcoRI and BamHI restriction sites of pTT5. The full-length IgGs of each were cloned similarly into pTT5, using identical variable domains for each of the two variants with a human IgG4 constant region with a stabilized hinge to reduce heavy-chain swapping. The 29H4-GL and 29H4-16 human λ chains were cloned via Gibson assembly by inserting DNA encoding the mouse IgG heavy chain V region 102 signal peptide followed by the 29H4-GL and 29H4-16 VL and CL regions into the EcoRI and BamHI restriction sites of pTT5.The constructs for both 29H4-GL and the 29H4-16 human IgG1 Fabs were cloned via Gibson assembly by inserting the DNA encoding the mouse Ig heavy chain V region 102 signal peptide followed by the 29H4 VTM cells (Invitrogen) using Expi293 growth medium . For pro-myostatin used in crystallographic experiments, 2 days after initial seed culture of 0.6 × 106 cells/ml, kifunensine  was added to the cell culture medium at a final concentration of 1 mg/liter. The transfection method described below was followed for all antigens, full-length antibodies, and Fabs. Briefly, transfection was done using polyethylenimine hydrochloride  and a total DNA amount of 1 mg/liter of culture with viable cell densities of 2–3 × 106 cells/ml. The DNA:PEI solution was then added to the cell culture medium. Twenty-four hours post-transfection, the cells were supplemented with 0.5% (w/v) tryptone N1  and allowed to grow for 4–5 days under optimal culture conditions . The cells were harvested by centrifugation at 6.9 × g for 20 min at 4 °C. The supernatant containing the secreted proteins was sterile-filtered using a 0.22-μm filter.All antigens, full-length antibodies, and Fabs in this study were transiently expressed in Expi293Fm NaCl and pH 8.0, respectively. The pH and salt-adjusted conditioned medium was loaded onto a 5-ml HiTrap Excel column  pre-equilibrated, and washed with 20 mm phosphate and 0.5 m NaCl (pH 8.0). Protein was eluted using 20 mm phosphate, 0.5 m NaCl, and 0.25 m imidazole (pH 7.8). The protein was further purified by SEC using a HiLoad 26/600 Superdex® 200 pg column  with 20 mm HEPES and 150 mm NaCl (pH 7.5) as eluent buffer. The protein was concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C until use.Pro- and latent myostatin were subsequently purified using immobilized nickel (II) ion affinity chromatography and SEC. Briefly, the salt and pH levels of the supernatant were adjusted to 0.45 m H3PO4 (pH 2.7) and neutralized with 10% (v/v) 1.6 m HEPES (pH 8.0). Protein fractions were pooled and adjusted to 150 mm NaCl and sterile-filtered using a 0.22-μm filter. Pooled protein was dialyzed against 20 mm HEPES and 150 mm NaCl (pH 7.5) at 4 °C, concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C until use.The antibody fragments, 29H4-16 Fab and 29H4-GL Fab, were purified by affinity binding to a 5-ml HiTrap λ Fab Select Column  in tandem with a 5-ml HiTrap Protein L Column  equilibrated and washed with 1× PBS (pH 7.4). The protein was eluted using 0.1 m phosphoric acid (pH 3.0) and neutralized with 10% (v/v) using 1.6 m HEPES (pH 8.0). The protein was dialyzed against 20 mm citrate and 150 mm NaCl (pH 5.5) at 4 °C, concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C until use.The full-length antibodies, 29H4-16 human IgG4 and 29H4-GL human IgG4 (SRK-015), and ActRIIb-mouse Fc fusion protein were purified using a 5-ml HiTrap rProtein A FF Column  pre-equilibrated and washed with 1× PBS (pH 7.4). The protein was eluted using 100 mm HEPES, 200 mm NaCl, and 5% glycerol (pH 7.4) as eluent buffer.For protein crystallization, pro-myostatin and 29H4-16 Fab were mixed at a 1:1 molar ratio and incubated at room temperature for 1 h. The pro-myostatin–29H4-16 Fab complex was purified from the constituent components by SEC using HiLoad 16/600 Superdex® 200 pg Column  using 30 mm Na citrate (pH 5.5), 20% PEG 3350, 15% 2-propanol, and 3% trimethylamine N-oxide and equilibrating at room temperature. Crystals were harvested with a cryo-loop and cryoprotected by soaking in well solution with 25% ethylene glycol before plunge-freezing in liquid nitrogen.The pro-myostatin–29H4-16 Fab complex was crystallized using sitting drop vapor diffusion by mixing 200 nl of 6.8 mg/ml protein complex with 200 nL of well solution containing 0.1 Diffraction data were collected from a single crystal at the Advanced Photon Source of Argonne National Laboratory on Industrial Macromolecular Crystallography Association Collaborative Access Team beamline 17-ID-B with a Dectris Pilatus 6 M and a wavelength of 1.00000 Å. The crystal was rotated 180°, and images were collected with 0.5° oscillation and 0.5-s exposure time. The diffraction data were indexed, scaled, and merged using the XIA2 pipeline (CCP4) .5GGU and 5F3H), myostatin (PDB code 3HH2), and pro-TGFβ1 (PDB code 3RJR). Buccaneer . Finally, a 15-min dissociation step was performed.The human IgG4 and Fab fragments of 29H4-GL and 29H4-16 were measured for their binding affinities to human pro- and latent myostatin by biolayer interferometry using Octet Red384 (FortéBio). Polystyrene 96-well black half-area plates were utilized for this experiment (Greiner Bio-One). Briefly, pre-equilibrated anti-human IgG Fc capture biosensor tips (FortéBio) were baselined for 1 min in 1× kinetics buffer , and antibodies were loaded onto the anti-human IgG Fc capture tips at a concentration of 1 μg/ml for 5 min. An additional 1-min baseline was performed in 1× kinetics buffer followed by a 5-min association step using varying concentrations of human pro- or latent myostatin . The Fab was then loaded onto the FAB2G tips at a concentration of 1 μg/ml for 5 min. An additional 1-min baseline was performed in 1× enhanced kinetics buffer followed by a 5-min association step using varying concentrations of human pro- or latent myostatin (0–300 nm). Finally, a 15-min dissociation step was performed.To determine the affinities of the Fabs to human pro- and latent myostatin, anti-human Fab-CH1 second-generation (FAB2G) biosensor tips (FortéBio) were used. Briefly, pre-equilibrated FAB2G tips were baselined for 5 min in 1× enhanced kinetics buffer  for each antigen/antibody pair, a global fit was performed using all concentrations of antigen for which the association response was greater than 0.1 and the dissociation resulted in a measurable off-rate. For samples where off-rates were not detectable for any of the concentrations, the off-rate and DK were assigned to be below the limit of detection on the Octet instrument. A 1:1 model was utilized for all curve fitting. The data were analyzed using Data Analysis 11.0 software (FortéBio).For analysis and determination of the dissociation constant (m phosphate buffer and 200 mm NaCl (pH 6.8) as eluent. SEC-MALS was performed using an HPLC  connected to a series of detectors, including a UV-visible multi-angle scattering detector  and Optilab T-rREX refractometer (Wyatt Technologies). SEC separation was accomplished using a silica-based SRT SEC-500 column . Prior to loading onto the SEC column at 0.3 ml/min, samples of pro- and latent myostatin alone and in complex with SRK-015 were incubated overnight at room temperature. The absolute molar masses were then determined from the light scattering data using ASTRA 6.1 software (Wyatt Technologies).The size and binding stoichiometry of pro- and latent myostatin with SRK-015 were determined by SEC-MALS using 20 mm HEPES, 1 mm CaCl2, 1 μm ZnCl2, and 0.01% Brij-35). Antibodies and latent myostatin were added at a 1:1 volume ratio and incubated for 30 min at 37 °C. Conditioned medium containing mTLL2 protease, prepared and validated as described previously (m phosphoric acid. The absorbance readings at 450 nm were measured using a Synergy H1 microplate reader (BioTek) with a background correction at 630 nm. The limit of detection for this assay was 1 ng/ml based on detection of a recombinant myostatin growth factor standard.The ActRIIb–mouse Fc fusion protein was coated at 2 μg/ml in 1× PBS buffer onto a plate  and stored overnight at 2 °C-8 °C. The next day, plates were washed and blocked with assay buffer  at room temperature. Anti-myostatin antibodies and Fabs were diluted to 200 μg/ml in 1× PBS and titrated 1:2 in 1× PBS. Human latent myostatin was diluted to 4 μg/ml in activity assay buffer  in sample buffer (20 mm HEPES and 150 mm NaCl (pH 7.5)). A 15 μm pro- or latent myostatin sample was also prepared for the unbound antigen control. The protein samples were incubated at room temperature for 1 h and then introduced into the nanoACQUITY UPLCTM system equipped with H/D exchange technology (Waters Corp.), which was used to perform all subsequent manipulations for the H/D exchange experiments. H/D exchange was initiated by dilution of each sample 10-fold with H/D exchange buffer  and the mixture was incubated at predetermined H/D exchange time points  at 25 °C. At the indicated time point, an aliquot from the exchange reaction was removed and quenched by adding an equal volume of quench buffer phosphine (pH 2.5)) at 3 °C. For the nondeuterated samples, the same procedure was performed in sample buffer. Quenched samples were introduced into the 5 μm ethylene bridged hybrid 2.1 × 30 mm EnzymateTM immobilized pepsin column (Waters Corp.) at 100 μl/min using 0.1% formic acid as eluent at 10 °C and incubated for an additional 9.5 min to allow on-column digestion. Peptide fragments were collected at 0 °C on a C18 VanGuard trap column  for desalting using 0.1% formic acid (in water). The digested peptides were introduced onto the 1.8-μm high strength silica T3 C18 2.1 × 30 mm nanoACQUITY UPLC® column  at 40 μl/min (0 °C) using a 10-min gradient from 0.1% formic acid to acetonitrile . Fragments were subsequently mass-analyzed using the Synapt G2Si electrospray ionization quadrupole time-of-flight mass spectrometer (Waters Corp.). Between each sample run, a clean blank was run by injecting pepsin wash buffer  into the H/DX-MS system to minimize peptide carryover.H/D exchange experiments were performed using a 1:3 molar ratio of pro- and latent myostatin–29H4-(16/GL) Fab (15 μE) and ProteinLynx Global Server 3.0 software (Waters Corp.) were used to determine the peptic peptides in the undeuterated protein samples analyzed on the same UPLC-ESI-QTOF system used for H/DX-MS experiments. Peptic peptides generated from the ProteinLynx Global Server were imported into DynamX 3.0 (Waters Corp.) with peptide quality thresholds of MS1 signal intensity of 1000 or higher and a maximum mass error of 1 ppm. Automated results were manually inspected to ensure that the corresponding m/z and isotopic distributions at various charge states were properly assigned to the appropriate peptic peptide. DynamX 3.0 was used to generate the relative deuterium incorporation plot and H/D exchange heatmap for each peptic peptide. The relative deuterium incorporation of each peptide was determined by subtracting the weight-averaged centroid mass of the isotopic distribution of the undeuterated control sample from that of the weight-averaged centroid mass of the isotopic distribution of deuterium-labeled samples at each labeling time point. All comparisons were performed under identical experimental conditions, negating the need for back-exchange correction in determination of the deuterium incorporation. Thus, H/D exchange levels are reported as relative (Accurate mass and collision-induced dissociation in data-independent acquisition mode (MSrelative . The frarelative . Differem phosphate buffer and 200 mm NaCl (pH 6.8 as eluent). The samples were combined at a 1:2 molar ratio (antigen:antibody), incubated on ice for 1 h, and diluted 150-fold in 20 mm HEPES and 150 mm NaCl (pH 7.5) prior to imaging. Each sample was loaded onto a carbon-coated, 400-mesh copper grid; the buffer was wicked off, and the sample was immediately stained with uranyl formate. Negative-stain EM imaging was performed using an FEI Eagle 4k × 4k charge coupled device camera. After identifying suitable target areas for imaging at lower magnifications, high-magnification images were acquired at nominal magnifications of ×110,000 (0.10 nm/pixel) and ×67,000 (0.16 nm/pixel) with a nominal underfocus of −1.6 μm to −0.8 μm and electron dose of 25 e−/Å. For 2D class averaging, particles were identified at high magnification  images prior to alignment and classification. Particles were selected and boxed out, and individual subimages were combined into a stack to be processed using reference-free classification based on the XMIPP (The samples for negative-stain EM include the antigen–antibody complexes of pro-myostatin–29H4-16 hIgG4 and latent myostatin–SRK-015. Prior to negative-stain EM analysis, formation of an antigen–antibody complex was confirmed by size-exclusion chromatography using Superdex 200 Increase 5/150 GL with 20 mhe XMIPP  and manuK. B. D., E. T., J. W. J., R. R. F., A. D. C., and G. J. C. conceptualization; K. B. D., E. T., F. C. S., J. W. J., R. R. F., S. C. L., S. B. N., A. D. C., and G. J. C. formal analysis; K. B. D., E. T., F. C. S., J. W. J., A. N., S. C. L., C. J. B., S. B. N., and A. D. C. investigation; K. B. D., E. T., F. C. S., J. W. J., R. R. F., A. N., S. C. L., C. J. B., A. D. C., and G. J. C. methodology; K. B. D., F. C. S., J. W. J., R. R. F., and S. C. L. writing-original draft; K. B. D., S. B. N., and G. J. C. writing-review and editing; C. J. B., S. B. N., A. D. C., and G. J. C. supervision.


N-methyl- D-aspartate (NMDA) receptor encephalitis associated with homozygous C4B deficiency, who did not respond to intravenous immunoglobulin and pulse methylprednisolone but plasmapheresis and rituximab.Complement C4A or C4B deficiency has never been reported in autoantibody-associated encephalitides patient. Here we present a case of anti-C4B gene inherited from his mother.A fourteen-year-old boy presented to our unit with subacute onset of behavioral changes and confusion, and was later confirmed to be anti-NMDA receptor encephalitis. He was initially managed with intravenous immunoglobulin (IVIG) and pulse methylprednisolone but did not achieve any clinical improvement. Seven sessions of plasmapheresis was commenced with remarkable improvement after the second session, and was followed by four doses of rituximab. His neurological and cognitive functioning gradually returned to baseline. Immunological investigations demonstrated persistently low C4 levels below 8 mg/dL. A more in-depth complement analysis of the patient and his family showed that he has homozygous C4B deficiency. Genetic analysis revealed that the index patient has homozygous deficiency in complement C4B and he carries one non-functioning mutant Low levels of serum C4 correlate with reduced functions of the classical and lectin pathways, leading to the impairment of immune-complexes removal. Plasmapheresis ameliorates complement deficiency and removes the offending immune-complexes leading to clinical improvement that was not achieved by IVIG and steroids. We postulate that serum C4 levels may serve as a biomarker for the need of plasmapheresis upfront rather than only after non-response to steroid and IVIG in treating anti-NMDA-receptor encephalitis. N-methyl- D-aspartate (NMDA) receptor encephalitis associated with homozygous C4B deficiency.Complement C4 plays a crucial role in the activation of the classical and lectin complement pathways. It has two isotypes, C4A and C4B. While deficiency of C4A and C4B are detectable at low frequency in healthy populations, they have also been reported to be associated with various autoimmune and inflammatory diseases , 2. Howe2, with normal protein and glucose levels. Oligoclonal protein was detected in the CSF. Microbiological investigations including bacterial cultures and viral polymerase chain reactions for herpes simplex virus, varicella zoster virus and enteroviruses were all negative. Anti-NMDA receptor antibodies based on commercial assay  were detectable (titer < 1:10) in the CSF but not the serum.Our patient is a fourteen-year-old boy with background of mild attention-deficit and hyperactive disorder. He has insignificant family history of neurological, psychological or immunological disorders. Preceded by two weeks of viral illness-like prodromal symptoms, he progressively developed hypersomnolence, confused speech with echolalia, self-muttering, dysarthria, mood fluctuation, bilateral upper limbs tremor, and headache. Physical examination was unremarkable, apart from having confusion, inappropriate speech with foul languages, urine and fecal incontinence. Investigations showed normal complete blood count, liver and renal function tests. Autoimmune markers including anti-nuclear antibodies and anti-double stranded DNA antibody were negative. His serum C3 levels were within normal range between 109 and 115 mg/dL, but his C4 levels were persistently below 8 mg/dL  and monomodular (L) RP-C4-CYP21-TNX (RCCX) haplotypes (panel A). Regular Southern blots of TaqI digested genomic DNA revealed the presence of three long C4 genes in LL/L configurations (panel B), by which two C4 genes belong to the C4A isotype and one C4 gene belongs to the C4B isotype, as shown by PshAI/PvuII Southern blot (panel C). Immunofixation experiment of EDTA-plasma showed that the patient expressed C4A protein but no C4B protein (panel D) [C4B gene that did not produce a C4B protein  and thus assigned a C4B mutant. Concurrent study of the patient’s parents and maternal stepbrother suggested the patient  than female healthy controls (N = 16), the sample sizes (for controls) were relatively small and required validation. Moreover, genetic deficiency of C4B was not examined [Further studies investigating the complement profiles and circulating immune complexes in patients with anti-NMDA receptor and other autoimmune encephalitides would clarify the role of examined . While texamined , inheritAdditional file 1.


Met T4454C, tRNATrp A5568G, tRNACys T5794C, tRNAArg A10438T and T10462C, tRNALeu(CUN) A12308G, tRNAThr A15907G, A15924G and G15928A. However, through the database searches and phylogenetic conservation analysis, we noticed that the tRNAThr A15924G, G15928A and tRNALeu(CUN) A12308G mutations should be classified ‘pathogenic’. Thus, the roles of mt-tRNA mutations in clinical expression of NDM needed to be further experimentally addressed.According a recent report by Heidari et al., a mutational screening for candidate pathogenic mitochondrial tRNA (mt-tRNA) mutations were performed in 45 Iranian patients with non-dystrophic myotonia (NDM) and 70 control subjects. Through PCR amplification and direct sequence analysis, nine mt-tRNA mutations were identified: tRNA However, the pathogenicity of these mt-tRNA mutations remained mysterious. In this study, we examined the genetic susceptibility of these mt-tRNA mutations and further discussed the relationship between these mutations and clinical phenotype.Most recently, Heidari et al.  and collhttps://pubmed.ncbi.nlm.nih.gov) and other public resources (Mitomap database: www.mitomap.org)  A12308G mutation’; ‘mitochondrial tRNAThr A15907G mutation’; ‘mitochondrial tRNAThr A15924G mutation; or ‘mitochondrial tRNAThr G15928A mutation’ to identify the case-control studies published to date on the association between these nine mutations and various clinical diseases.We carried out the literatures searches for the presence of these nine mt-tRNA mutations via Pubmed Central  was compared the human mtDNA variations with other 14 species. Notably, the CI > 75% was believed to have functional potential  A12308G, tRNAThr A15907G, A15924G and G15928A mutations were individually analyzed using the published secondary structures for the mt-tRNAs with the stem and loop structure , A5568G in the T-stem of tRNATrp (position 62), T5794C in the anticodon stem of tRNACys (position 33), T10462C (position 66) in the acceptor arm and A10438G (position 37) in the anticodon stem of tRNAArg, A12308G in the variable region of tRNALeu(CUN) (position 44), A15907G (position 22) in the D-stem; A15924G (position 39) and G15928A (position 43) in the anticodon stem of tRNAThr  A12308G, tRNAThr A15924G and G15928A mutations, other mutations are not conserved, with the CI varied from 21.1% to 82.6%, the lower level of CIs ruled out their roles in clinical expression of mitochondrial diseases.We performed the phylogenetic conservation analysis for these nine mt-tRNA mutations, as seen Leu(CUN) A12308G, mt-tRNAThr A15924G and G15928A mutations were 15, 13 and 10 points, respectively, belonging to ‘definitely pathogenic’ and ‘possibly pathogenic’. Similarly, the total scores of T4454C, A5568G, T5794C, A10438T, T10462C and A15907G mutations were 4,4,2,4,4 and 2 points, respectively, suggesting that they belong to ‘neutral polymorphisms’ at this stage  A12308G created a conserved 25 A-37T base-pairing, whereas the A15924G mutation abolished the 31 T-39A base-pairing, while the G15928A mutation disrupted the 27 C-43G base-pairing (Leu(CUN) A12308G mutation had been reported to be associated with CPEO, stroke and breast cancer risk  and fatal infantile respiratory enzyme deficiency  and Parkinson’s Disease (PD) (Mayr-Wohlfart et al. In the present study, we reassessed the roles of nine mt-tRNA mutations in the phenotypic manifestation of NDM. Among these sequence alternations, the T4454C mutation was located at position 58 in the T-loop of tRNA-pairing . In addiAccording to these observations, we believed that the possible molecular mechanism underlying the A12308G, A15924G and G15928A mutations in clinical expression of NDM may be as follows, first of all, these mutations altered the secondary structure and affected the steady-state levels of corresponding tRNAs, subsequently, these mutations may lead to the failure in tRNA metabolism such as CCA addition, aminoacylation, or defects in tRNA modifications. As a result, mitochondrial protein synthesis may be affected by these events and the respiratory chain functions impaired, thus, the ATP declined and ROS increased, which led to mitochondrial dysfunction that was involved in the pathogenesis of NDM.Phe C628T variant and hearing loss (Zhu et al. Most recently, several studies had been reported on the associations between mt-tRNA mutations and mitochondrial disorders, as in the cases of T4454C variant and hypertension (Wang et al.


Moreover, blocking of PI3K-Akt signaling pathway abolished heterozygous p53-R280T mutation-promoting NPC cell proliferation and survival. Our data indicate that p53 with heterozygous R280T mutation functions as an oncogene, and promotes the oncogenicity of NPC cells by activating PI3K-Akt signaling pathway.A heterozygous point mutation of p53 gene at codon 280 from AGA to ACA (R280T) frequently occurs in nasopharyngeal carcinoma (NPC) cell lines, and about 10% NPC tissues. However, the role of this mutation in the pathogenesis of NPC remains unclear. In this study, we generated p53 knockout (KO) NPC cell lines from CNE2 cells carrying heterozygous p53 R280T (p53-R280T) mutation and C666-1 cells carrying wild-type p53 by CRISPR-Cas9 gene editing system, and found that KO of heterozygous p53-R280T significantly decreased NPC cell proliferation and increased NPC cell apoptosis, whereas KO of wild-type p53 had opposite effects on NPC cell proliferation and apoptosis. Moreover, KO of heterozygous p53-R280T inhibited the anchorage-independent growth and  Nasopharyngeal carcinoma (NPC) arises from the epithelial lining of the nasopharynx . It has P53 is a sequence-specific DNA binding protein, which consists of two N-terminal transactivation domains, a central DNA binding domain (DBD), a C-terminus including nuclear localization signals and an oligomerization domain needed for transcriptional activity –8. P53 mIn this study, we generated p53 knockout NPC cell lines from CNE2 carrying heterozygous p53-R280T mutation  and C666https://zlab.bio/guide-design-resources/). CNE2 and C666-1 cells were transfected with the plasmid pGK1.1 containing p53 gRNA and control vector pGK1.1, respectively. Forty eight hours after transfection, cells were treated with puromycin at a concentration of 3 μg/ml for 2 days. Then, a single cell was seeded into 96-well plates and cultured for 1 month, and knockout of p53 protein was evaluated by western blot, and DNA sequencing was used for confirmation. For DNA sequencing, genomic DNA was extracted from cells, and a 383-bp polymerase chain reaction (PCR) amplicon flanking the CRSPR-Cas9-targeted sites (GCAGTCACAGCACATGACGG) was generated using the primers 5′-TCACTTACCTCTCAGAGAC-3′ (forward) and 5′-ACAGGGCAGGTCTTGGCCGTT-3′ (reverse). The PCR product was purified and ligated into the pMD18-T vector. The recombinant plasmids were introduced into competent DH5α cells. Plasmid DNA was extracted and sequenced across the insert using one of the PCR primers in the Life Technologies Corporation . The sequence of p53 KO CNE2 (KO-41) cells after knockout by CRISPR-Cas9 gene editing system was 5′-GGAGGCTACACGACACTGACGAACATC-3′. The sequence of p53 KO CNE2 (KO-49) cells after knockout by CRISPR-Cas9 gene editing system was 5′-GGAGGCAGGGGTCCGGAGACTAAGTACA-3′ and 5′- GGAGGCACTGACGAAC-3′. The sequence of p53 KO C666-1 (KO-9) cells after knockout by CRISPR-Cas9 gene editing system was 5′-GGAGTACACGACACTGACGAACATCTACCG-3′. The sequence of p53 KO C666-1 (KO-29) cells after knockout by CRISPR-Cas9 gene editing system was 5′-GGAGTACACGACACTGACGAACATCTACCG-3′.Human NPC cell lines  were cultured and maintained in RPMI-1640 medium containing 10% (v/v) fetal bovine serum (FBS)  at 37°C. For p53 knockout, the guide RNA (gRNA) sequence was GCAGTCACAGCACATGACGG, which was designed using the website software from Massachusetts Institute of Technology (USA) . Blots were incubated with antibodies against p53 (DO-1) , phospho-AKT , or AKT  overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody for 1 h at room temperature. The signal was visualized with an enhanced chemiluminescence detection reagent .Sanger sequencing was performed to detect the heterozygous R280T mutation of p53 gene using genomic DNA extracted from NPC CNE2, 5-8F, 6-10B, and C666-1 cell lines. Mutation was confirmed by at least two independent PCR amplifications and a DNA sequencing reaction on both strands. Oligonucleotide primers were designed to amplify exon 8 of p53 gene. The primers used were: 5′-GCTGGGGAGAGGAGCTGGTG-3′ (forward) and 5′-GGTTCATGCCGCCCATGCAG-3′ (reverse). The products were examined by sequencing in the Sangon Biotech, Shanghai, China.EdU incorporation assay was performed to detect cell proliferation as described previously by us . The assCell proliferation was measured using a CCK-8 kit as described previously by us . The assFlow cytometry analysis of cell cycle and apoptosis was performed as described previously by us . All assPlate colony formation assay and soft agar colony formation assay were performed to detect the anchorage dependent or independent growth of cells as described previously by us . All ass6 cells resuspended in 200 μl of serum-free medium were subcutaneously injected into the flanks of mice (n = 3 mice each). The mice were monitored daily for palpable tumor formation, and tumor volume (in mm3) was measured by a vernier caliper every 3 days and calculated by using the modified ellipse formula (volume = length × width2/2). At the end of the experiments, the mice were killed by cervical dislocation, and tumors were excised, and weighted.Nude male mice that were 4 weeks old were obtained from the Laboratory Animal Center of Central South University  and were maintained under specific pathogen-free conditions. 5 × 102 Fold change|≥2, and q < 0.05). The GO enrichment of differentially expressed genes (DEGs) was implemented by the hypergeometric test, in which p-value is calculated and adjusted as q-value, and data background is genes in the whole genome. GO terms with q < 0.05 were considered to be significantly enriched. The KEGG enrichment of DEGs was implemented by the hypergeometric test. KEGG terms with p < 0.05 were considered to be significantly enriched.Total RNA was extracted from NPC cells with Trizol reagent . Two microgram RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® , and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. First strand cDNA was synthesized using random hexamer primer and RNase H. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then terminal repair, A-tailing and adapter added were implemented. The aimed products were retrieved and PCR was performed, then the library was completed. The libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated. Reads count for each gene in each sample was counted by HTSeq v0.6.0, and FPKM (Fragments Per Kilobase Millon Mapped Reads) was then calculated to estimate the expression level of genes in each sample. DESeq (v1.16) was used for differential gene expression analysis between two samples with biological replicates using a model based on the negative binomial distribution. The DEGs standard is  and listed in Total RNA was extracted from NPC cells with Trizol reagent . One microgram of total RNA was reversely transcribed for cDNA using a RT kit according to the manufacturer's protocol and Oligo dT primer  according to the manufacturer's instruction. The RT products were amplified by real-time PCR using SYBR qPCR Master Mix kit  according to the manufacturer's instruction. The products were quantitated using 2t-test was used for comparisons between groups. Differences were considered statistically significant when P < 0.05.All the quantified data represented an average of three times. Data are represented as mean ± SD. One-way analysis of variance or two-tailed Student's Genomic DNA obtained from CNE2, 5-8F, 6-10B, and C666-1 cells was amplified and detected for mutations at codon 280 of p53 gene by Sanger sequencing. Alignment analysis of DNA sequences was performed using the NCBI BLAST. A heterozygous G changed to C point mutation at codon 280, position 2 (AGA coding for arginine changed to ACA coding for threonine) was detected in the CNE2, 5-8F, 6–10B cell lines , which iTo study the roles of heterozygous p53-R280T mutation in NPC cells, we established p53 knockout (KO) CNE2 and C666-1 cell lines, in which p53 was knocked out at the chromosomal level by using CRISPR/Cas9 gene editing system. Single-guide RNA (sgRNA) was designed to delete exon 5 of the p53 gene . Guide RWe evaluated the effect of p53 KO on NPC cell proliferation by plate colony formation assay, EdU incorporation assay and CCK-8 assay. The results showed that p53 KO significantly suppressed cell proliferation in the CNE2 cells with heterozygous p53-R280T mutation, whereas significantly promoted cell proliferation in C666-1 cells with wt p53 . Flow cyin vivo tumorigenicity of NPC cells respectively. The results showed that p53 KO significantly decreased the formation ability of soft agar colony in the CNE2 cells, whereas significantly increased the formation ability of soft agar colony in the C666-1 cells  and control CNE2 cells. As a result, a total of 2,612 differentially expressed genes (DEGs) (fold change≥2) were identified in the KO-41 and control CNE2 cells . Of themTo investigate whether the DEGs in the p53 KO and control CNE2 cells are abnormally expressed in NPC biopsies, we downloaded the gene expression profile of NPC tissues from the GEO database (GSE12452) , and comThe 2612 DEGs identified in the present study were formulated into an XML-based input data set to query the GO database. The results showed that all DEGs were divided into three major groups: cellular component, molecular function and biological process, as well as 55 functional groups . In the The 2612 DEGs were uploaded into the KEGG database for pathway enrichment analysis. The results showed that 37 pathways were found to be statistically enriched , includiin vitro cell proliferation of p53 KO CNE2 and p53 KO C666-1 cells, which was abolished by LY294002  , and obsPI3K plays an important role in cancer development and progression –32. OnceIn summary, our data suggest that heterozygous p53-R280T mutation functions as an oncogene in NPC, and promotes the oncogenicity of NPC cells by activating PI3K-Akt signaling pathway. P53 knockout NPC cell lines and heterozygous p53-R280T mutation-associated DEGs provide a valuable tool to investigate the role and molecular mechanism of heterozygous p53-R280T mutation in NPC.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE130398.The datasets of mRNA-sequencing for this study can be found in the GEO: All animal experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Xiangya Hospital, Central South University, with the approval of the Institutional Animal Ethics Committee.Z-QQ and Z-QX designed experiments. Z-QQ, Q-GL, and HY implemented experiments. Z-QQ, Z-QX, Q-GL, WH, S-SL, Y-YT, and Z-XR analyzed experimental results. ZQ-Q and Z-QX wrote the manuscript.The 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. The reviewer GL declared a shared affiliation, though no other collaboration, with the authors to the handling Editor.


PiD). We have used docking, molecular dynamics simulations, binding-affinity and tunnel calculations to explore TauPiD binding sites, binding modes, and binding energies of PET probes  with TauPiD. The probes bind to TauPiD at multiple surface binding sites as well as in a cavity binding site. The probes show unique surface binding patterns, and, out of them all, PM-PBB3 proves to bind the strongest. The findings suggest that our computational workflow of structural and dynamic details of the tau filaments has potential for the rational design of TauPiD specific PET tracers.In recent years, it has been realized that the tau protein is a key player in multiple neurodegenerative diseases. Positron emission tomography (PET) radiotracers that bind to tau filaments in Alzheimer’s disease (AD) are in common use, but PET tracers binding to tau filaments of rarer, age-related dementias, such as Pick’s disease, have not been widely explored. To design disease-specific and tau-selective PET tracers, it is important to determine where and how PET tracers bind to tau filaments. In this paper, we present the first molecular modelling study on PET probe binding to the structured core of tau filaments from a patient with Pick’s disease (Tau AD filaments are composed of two C-shaped protofilaments arranged either symmetrically base-to-base (PHF) or back-to-base (SF). The cores in both types of filament consist of 73 residues (V306–F378) with eight β-strands (β1–β8). On the other hand, distinct tau filament structures are found in PiD, CBD or CTE subjects PBB3, [18F]PM-PBB3, [18F]AV1451, [18F]THK5351, [18F]MK-6240, [18F]R06958948, [18F]GTP-1, and [18F]PI-2620, were developed and have been applied to human subjects [18F]AV1451 and [18F]THK5351 exhibit off-target effects such as binding to monoamine oxidase (MAO)-A and MAO-B, respectively [18F]THK5351 to MAO-B is strongly suggested in the detection of MAO-B-positive astrogliosis [18F]PM-PBB3, [18F]MK-6240, [18F]R06958948, [18F]GTP-1, and [18F]PI-2620) seem to have less off-target binding PBB3, FPM-PBB3,ectively . In partogliosis ,22. Seco binding ,23,24. M binding . TauPiD C]PBB3, 1FPM-PBB3, with AD ,26. Impo with AD . HoweverPiD, we performed computational modeling to characterize the binding of various PET probes to TauPiD, and obtained insights into their unique binding properties.Of the various neurodegenerative diseases, Pick’s disease is a rare type of age-related dementia that affects the frontal lobes of the brain, resulting in speech problems like aphasia, behavior difficulties and eventually death. Pick’s tau folds quite differently from Alzheimer’s tau filaments ,8, and i18F]AV-1451 ([18F]flortaucipir), [18F]THK5351, [11C]PBB3, [18F]PM-PBB3 and [18F]MK-6240  PBB3 and [18F]PM-PBB3, for MAO-A/B and α-synuclein by in vitro radioligand binding assay [Most recent findings with both clinical and pre-clinical studies of [ disease . To conf TauPiD) A,B, conf similar . On the ng assay ,35,36, iPiD are rather unique. S8 site is highly selective for PM-PBB3 over PBB3  where PET probes bind within a cavity formed between P312-V313-D314 and P332-G333-G334 (−1), MK-6240 , PBB3 , PM-PBB3 , and THK-5351  have comparable docking scores in the C1 site, but PiB  binds comparatively weakly. Of all the docking scores, those of PiB in C1 and at the surface sites are weakest. AV-1451, PBB3, and THK-5351 bind with similar strength to C1, but MK-6240 and PiB show weak affinity. Binding energies from MD simulations followed by MM/GBSA calculations on 1000 frames suggest that binding site C1, which is formed due to folding of the fibril, is a favored binding site. All seven compounds show stable binding to C1, and the binding is stronger than it is to surface binding sites S1 to S11  without any PET probes in the binding sites. We further performed a Tunnel analysis around C1, in a 1-μs MD trajectory to see if the tunnel is realistic and maintained during the MD simulation. The tunnel profile in the cryo-EM structure of TauPiD shows two potential tunnels with some slightly narrow entry paths between filaments to C1 (PiD assembly (Tau49PiD) taken at every 2 ns from 1-μs long MD simulation  of multiple binding sites such as those that exist in the tau fiber. Experimental approaches cannot provide binding constants for each of such multiple binding sites. Although a gap still exists between theoretical predictions and experimental results, a computational approach will ensure advances in elucidating tracer-protein interactions of multiple binding sites.PiD (PDB ID: 6GX5) and TauAD (PDB ID: 5O3L) were taken from the Protein Data Bank [PiD and TauAD structures comprise 94 and 73 amino acids, respectively, from the total 441 residues in the 2N4R tau sequence. The reported TauPiD assembly has three tau fibrils (K254–F378), so we prepared a five-fibril assembly by superimposing structures over each other to have a structure comparable to the one used in the TauAD studies [antechamber of Amber18 [The structures of Tauata Bank . TauPiD fibrils K4–F378, s studies . The Res Amber18  with ESPchi_coeff = 0 and chi_cutoff = 12. The used values of chi_coeff of and chi_cutoff switch Vina-carb to AutoDock Vina [−1 were used. All the binding poses were refined by a rescoring function of X-Score [The tau assembly was centered in a conformational search space of 30 Å × 30 Å × 70 Å. We chose this size to ensure that it was large enough to capture all the surface sites in the fibrils. A total of 100 docking conformations of each PET probe was obtained by software AutoDock Vina-carb using ock Vina  are deno X-Score . The docPiD assembly with bound PET probe in all potential binding sites was prepared. For each PET probe, we performed MD simulation of fully solvated assemblies where the PET probe is simultaneously bound to TauPiD in all the binding sites predicted by docking. For each binding site, the highest-scoring binding pose from each cluster was selected for MD simulation. The protein was treated with Amber ff14SB force field [− ions was added to neutralize the whole system. A multi-step protocol published elsewhere [AD complexed with PM-PBB3 in five binding sites was performed.A Tauce field , whereasce field  with RESlsewhere  was usedPiD cryo-EM structure possesses a core cavity at a site between β-sheets containing residues P312 and G332, which can accommodate PET tracers. To check the validity of the existence of the core cavity , a 230 Å long 49 fibril TauPiD assembly (Tau49PiD) was prepared for MD simulation. The MD of Tau49PiD in apo form was performed on the microsecond timescale. In contrast to the MD of five fibril TauPiD, this Tau49PiD assembly was solvated with a rectangular water box, extending 10 Å from the protein surface, and comprised a total of ~80,000 water molecules and 441 Cl− ions. Prepared systems were quite large . MD of Tau49PiD fibrils was performed for 1 µs in explicit solvent using cuda version of pmemd in Amber18 [cpptraj [The Tau Amber18 . The non[cpptraj .OBC generalized-born model [−1 and 0.0, respectively, for the solvent accessible surface area (SASA) calculation. An offset value of 0.0 was used to correct non-polar solvation free energy contributions. The interior and exterior dielectric constants were set to 1 and 80, respectively. Other parameters were their default values in Amber18.To calculate binding free energy of the PET probe in each site, solvent molecules and tracers present in remaining binding sites were removed from the trajectory. A total of 1000 snapshots were extracted after each 10 ps of a 100-ns MD simulation and used for the PET probe binding energy calculation with the MM/GBSA approach . A similrn model  was usedPiD were calculated using the CAVER Analyst 2.0 program [Tunnels around the core-site in Tau program  with a p


HLA class I molecules bind antigenic peptides and present them on the cell surface to cytotoxic T cells to initiate immune responses. The peptide selection process occurs intracellularly with the aid of a molecule called tapasin. HLA class I molecules are highly variable, which influences their structural characteristics and the level of tapasin involvement in peptide selection. We measured tapasin dependence levels of nearly 100 HLA variants and found that the level of tapasin dependence negatively correlates with the number of peptides that the HLA class I molecule presents to T cells, thereby affecting breadth of the immune response. Analysis of HLA genotypes in HIV cohorts reveals that greater tapasin independence associates with slower disease progression and lower viral load. n = 97), which revealed a broad continuum of values. Ex vivo examination of cytotoxic T cell responses to the entire HIV-1 proteome from infected subjects indicates that tapasin-dependent allotypes present a more limited set of distinct peptides than do tapasin-independent allotypes, data supported by computational predictions. This suggests that variation in tapasin dependence may impact the strength of the immune responses by altering peptide repertoire size. In support of this model, we observed that individuals carrying HLA class I genotypes characterized by greater tapasin independence progress more slowly to AIDS and maintain lower viral loads, presumably due to increased breadth of peptide presentation. Thus, tapasin dependence level, like HLA zygosity, may serve as a means to restrict or expand breadth of the HLA-I peptide repertoire across humans, ultimately influencing immune responses to pathogens and vaccines.Human leukocyte antigen (HLA) class I allotypes vary in their ability to present peptides in the absence of tapasin, an essential component of the peptide loading complex. We quantified tapasin dependence of all allotypes that are common in European and African Americans ( The classical HLA class I molecules, HLA-A, HLA-B, and HLA-C, present antigenic peptides to CD8+ T cells, eliciting an adaptive immune response . They ar3HLA class I allotypes vary in level of cell surface expression in the absence of tapasin. Some allotypes are expressed at very low levels on the surface of tapasin-deficient cells , while others exhibit normal expression on these cells  –17. The Tapasin function can be targeted by viruses as a means of downmodulating HLA class I and evading cytotoxic CD8+ T cell (CTL) responses , 21. SimA panel of 97 distinct lentiviral HLA class I expression constructs were generated, in which HLA heavy chains were fused with a FLAG tag at the N terminus and linked to a ZsGreen reporter via a self-cleaving peptide for transduction control. The constructs were transduced individually into the human B cell line .220, which does not express tapasin , and intSI Appendix, Fig. S1). This antibody specifically detects transduced allotypes, in contrast to W6/32 mAb, a broadly used pan-HLA class I antibody that can bind endogenous HLA class I, which may be expressed by .220 cells at low levels. Nevertheless, we observed a strong correlation between HLA expression levels measured using anti-FLAG mAb and those determined by staining with W6/32 . This indicated that the FLAG-tagged heavy chains remained properly folded on the cell surface, since W6/32 only binds β2-microgloblin–associated HLA class I complexes. Further, allotype-specific HLA class I expression levels in .220tpn cells were similar to the expression levels in isogenic .221 cells bearing endogenous tapasin, indicating that V5-tagged tapasin was functioning similarly to the native protein . Comparison of the flow cytometric data obtained from cells expressing the R240 vs. T240 tapasin variants did not reveal a differential effect of this polymorphism on HLA expression levels.HLA class I cell surface expression levels were quantified in tapasin-deficient and tapasin-sufficient cell lines by flow cytometry using an anti-FLAG mAb . For example, the highly tapasin-dependent HLA-A*01:02 and HLA-B*44:02 have a ratio above 100, whereas the highly tapasin-independent HLA-A*68:01 and HLA-B*35:01 have a ratio of ∼1. TD values across each HLA class I locus represent a continuum, demonstrating some level of dependence on tapasin for most allotypes.TD values for each allele were generated by calculating the ratio of expression level in .220tpn cells over expression level in .220 cells, such that higher scores for a given allotype indicate its greater dependence on tapasin for cell surface expression .Our dataset included 26 of the 27 ed cells . DespiteHLA class I , HLA-A , and HLA-A/B/C allotypes combined  were observed . A locus-specific TD score for each individual was calculated as the sum of the TD values for the two alleles at a given locus, or a “global” TD score was generated by summing TD values for the four alleles of their HLA-A/B or six alleles of their HLA-A/B/C genotypes. The global TD score demonstrated a significant negative correlation with the total number of HIV-1 peptides to which CD8+ T cell responses were detected  ≤ 500 nM are expected to be presented by the corresponding HLA class I. Similar to the HIV-1 ELISpot data, the predicted dengue repertoire size showed a negative correlation with TD of HLA class I allotypes , HLA-A , and HLA-A/B allotypes combined . The prediction algorithms NetMHCpan EL 4.0 . Of note, the order of TD scores of four HLA-B alleles, B*57:01, B*27:05, B*07:02, and B*35:01, corresponds precisely, in a negative manner, to the order of their self-peptide repertoire sizes as estimated using prediction algorithms . Since there was no significant effect of TD on progression to CD4 cell counts <200/μL and AIDS-1993, definitions that precede an AIDS-defining illness , the detrimental effect of greater TD is likely to manifest later in the course of infection.The expansion or restriction of the peptide binding repertoire as a function of TD level may influence disease pathogenesis. To test this, we assigned TD scores to individuals from two completely independent HIV-1 cohorts and interrogated distinct outcomes. Cox model analysis of 954 antiretroviral therapy-naïve HIV-1 seroconverters revealed a significant association between higher global TD and more rapid progression to AIDS as defined by the CDC in 1987 (AIDS-1987), after adjusting for race and 10 increase in TD score associated with an increase of 0.21 mean log10 VL  was validated in an independent cohort of 4,306 individuals for whom longitudinal VL measurements (HIV RNA copies/mL of blood) were available prior to initiation of antiretroviral therapy. Consistent with the Cox analysis, TD correlated positively with mean VL (mVL), where every 1 logparately .HLA class I polymorphism defines multiple allele-specific features, including peptide specificity, mRNA/protein expression levels, and binding to innate immune receptors , greater promiscuity in peptide binding of chicken major histocompatibility complex (MHC) class I confers strong protection against Marek’s disease, a highly contagious disease in this species caused by an alphaherpes virus . Along tApart from presenting a broader peptide repertoire, tapasin-independent allotypes would also be relatively beneficial if tapasin function is affected by the virus, as they would continue to present viral peptides to CTL. A recent HIV-host interactome study identified tapasin, among other host proteins, to associate with HIV-1 Env , raisingn = 496 vs. 954 for survival analyses) and no validation in an independent cohort ; 2) fewer alleles for which TD values were available ; 3) inability to account for global TD levels since only HLA-B alleles were considered; 4) no functional explanation to support the genetic data. Overall, the increased power, reproducibility across cohorts, and functional data provided in the current work convincingly point to the protective effect of tapasin-independent HLA class I genotypes in HIV disease.We previously reported that tapasin-independent genotypes associated with faster progression to death after HIV infection , which cThe impact of TD on outcome after HIV infection is clearly distinct from individual allelic effects, as the most protective allotype of all, HLA-B*57:01, is highly tapasin-dependent. B*57 allotypes are known to present multiple, highly conserved protective epitopes , an attrThe range in breadth of the peptide presentation by distinct MHC allotypes described herein in humans mirrors that described in chicken, where “generalists” and “specialists” have been identified across class I allotypes . In humaCTL responses were measured in a cohort of 989 individuals from Durban, South Africa . Longitul-Glutamine Mixture (Lonza). HEK293T cells used for lentivirus production were maintained in advanced DMEM (Gibco) supplemented with 10% FBS and 2 mM l-glutamine (Gibco).The human B cell lines .220 and .221 were generated previously by γ-radiation of 721 cells and do not express HLA-A/B and may express HLA-C/E/F , 39–42. 39A*11:02, A*74:03, B*44:05, B*46:01, B*73:01, and C*18:02. Synthetically derived HLA allele fragments (LifeSct) were cloned into the modified pLVX-EF1α-IRES-Puro (Clontech) vector  vector , in whic5–106) were used for staining, and 200 μL of cells were cultured further with fresh media. For tapasin detection, cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit (Beckton Dickinson). Staining results were acquired using FACSCalibur flow cytometer (Beckton Dickinson), and analysis was performed using FlowJo software. Estimation of HLA expression levels involved several adjustments to the obtained APC MFI values: 1) background MFI of cells transduced with empty vector  were subtracted; 2) MFI values were normalized to the average MFI across samples to adjust for daily instrumental variation; 3) MFI values were normalized to the level of ZsGreen to adjust for the level of transduction; 4) the numerator in the calculation of TD for each allele was the average expression levels of the corresponding allele in .220tpn cells expressing the R240 and T240 tapasin variants.Flow cytometric analyses were performed using the following monoclonal antibodies: APC anti-DYKDDDDK Tag , HLA-ABC (W6/32) APC (eBioscience) , anti-V5HLA typing in the HIV-1 cohorts was performed using a targeted next generation sequencing method. Briefly, locus-specific primers are used to amplify polymorphic exons of HLA-A, HLA-B, HLA-C genes with Fluidigm Access Array (Fluidigm). The Fluidigm PCR amplicons were pooled and subjected to sequencing either on the Roche 454 platform (Roche) or the Illumina MiSeq platform (Illumina). HLA alleles and genotypes were called using the Omixon HLA Explore (beta version) software (Omixon). TD values determined for each HLA allotype are shown in SI Appendix, Table S1. Genotypic TD at each locus for each individual were calculated by summing the TD values of the two alleles: TD(HLA-A) = TD(A1) + TD(A2); TD(HLA-B) = TD(B1) + TD(B2); TD(HLA-C) = TD(C1) + TD(C2), where A1/A2, B1/B2, and C1/C2 represent the two alleles at HLA-A, HLA-B, and HLA-C, respectively. The global TD value for each individual was calculated by summing the TD scores at the three loci: TD(HLA-A/B/C) = TD(HLA-A) + TD(HLA-B) + TD(HLA-C). We were able to assign TD to 84%  and 86%  of individuals in the longitudinal VL and seroconverter cohorts, respectively. In the African cohort (ELISpot data), complete four-digit HLA-A/B/C genotypes were available for 853 individuals, of which TD scores were assigned to 638 (75%).www.IEDB.org). In both cases, predicted affinity values (IC50 nanomolar) were retrieved for the specific HLA allele when applicable based on the selected algorithm. HIV-1 clade C peptides consisted of overlapping 18-mers as previously described  associations between the recognition of individual peptides and the expression of particular HLA class I alleles. All HLA class I alleles expressed at a phenotypic frequency of >3% were included in the analysis.To identify HLA-peptide associations in the HIV-1 ELISpot assay A, we useHLA-A, HLA-B, and HLA-C allele, the time postenrolment, and a correction for diploid HLA allele coding were included in the models. We tested two alternative outcomes: a geometric mean HIV VL (mVL) at all measurement timepoints (mean log10VL) and the log10 transformed HIV VL at each timepoint. For survival analyses, we used the coxph function  and estimated the time to AIDS (CDC 1987 and 1993 definition), CD4+ T cell count <200 cells/μL, and death.For disease association, regression analyses were performed in R (v 3.6.0) using the following packages: lme4 and ggplot2. Analyses of VL was performed using the lmer function. Random effects due to each Correlation tests were performed using GraphPad Prism.Supplementary File