DNA topoisomerase II (TOP2) plays a critical role in many processes such as replication and transcription, where it resolves DNA structures and relieves torsional stress. Recent evidence demonstrated the association of TOP2 with topologically associated domains (TAD) boundaries and CCCTC-binding factor (CTCF) binding sites. At these sites, TOP2 promotes interactions between enhancers and gene promoters, and relieves torsional stress that accumulates at these physical barriers. Interestingly, in executing its enzymatic function, TOP2 contributes to DNA fragility through re-ligation failure, which results in persistent DNA breaks when unrepaired or illegitimately repaired. Here, we discuss the biological processes for which TOP2 is required and the steps at which it can introduce DNA breaks. We describe the repair processes that follow removal of TOP2 adducts and the resultant broken DNA ends, and present how these processes can contribute to disease-associated mutations. Furthermore, we examine the involvement of TOP2-induced breaks in the formation of oncogenic translocations of leukemia and papillary thyroid cancer, as well as the role of TOP2 and proteins which repair TOP2 adducts in other diseases. The participation of TOP2 in generating persistent DNA breaks and leading to diseases such as cancer, could have an impact on disease treatment and prevention. Topoisomerases relieve supercoiling in a sequence-independent manner by transiently cutting the DNA, passing the DNA strand through the break, and then re-ligating the strand(s) ,2. The tRecently, Hoa et al. demonstrated that TOP2 frequently fails to complete re-ligation, resulting in a persistent TOP2 cleavage complex (TOP2cc) that needs to be processed to prevent interference with replication and transcription machineries . Along wTOP2 was found to relieve supercoiling at origins of replication in order to preserve the underwound state of the DNA, but it does not promote replication initiation itself ,21,22,23RET (a non-transcribed gene in thyroid cells located within FRA10G) and FHIT (within FRA3B) had more aphidicolin-induced DSBs compared to non-fragile control regions. More importantly, when these cells were treated with both aphidicolin and a TOP2 catalytic inhibitor, merbarone, the replication-associated breaks were significantly reduced . T. T51]. Ter cells ,57,58,59The human genome is organized and compacted into large structures called topologically associated domains (TADs) ,61. BounTOP2 acts at TAD boundaries/CTCF-binding regions to alleviate torsional stress and/or to allow for regulatory elements to be brought into proximity with one another C. Sano eUsing genome-wide break mapping/sequencing methods such as END-seq and BLISS, TOP2-induced breaks were directly measured and indeed were concentrated at CTCF-binding sites ,19,65. TMLL and its partner genes AF9 and AF4, undergo transcription within the same transcription factories, and are, therefore, in close proximity RET upon N6-ethenoadenine lesions, which cannot base-pair and create a distortion in the DNA backbone, also promoted TOP2 cleavage [N6-ethenoadenine lesions caused a similar degree of cleavage promotion as apurinic sites [Abasic and mismatched sites in double-stranded DNA are suggested to be endogenous “poisons” to TOP2 A 13,14,,14,15,16Altogether, these studies support that helical distortions are a requirement and likely provide an energetically favorable target and, therefore, can be a mechanism for the endogenous generation of TOP2 breaks. Both secondary structure formation and these internal DNA lesions regularly occur in healthy cells ,79, presWhether TOP2ccs occur endogenously or in response to exogenous stressors, two major pathways, TDP2 (tyrosyl-DNA phosphodiesterase 2) and MRE11 in the MRE11/RAD50/NBS1 (MRN) complex, were shown to process and resolve TOP2cc adducts in human cells A.The TDP2 pathway removes TOP2ccs in a paired process in which proteasomal degradation of TOP2 leaves behind only the 5′ tyrosyl group that is covalently bound to the end of DNA . This 5′Xenopus egg extracts. Furthermore, the proper phosphorylation for binding with BRCA1 was required for resection of 5′- blocked DNA ends by MRE11 [The MRE11 pathway is able to remove the entire moiety of TOP2cc, and this can occur before proteasomal degradation . As a meby MRE11 . Additioby MRE11 ,92. Althby MRE11 . MRE11-mby MRE11 . Thus, Mby MRE11 . The ultThe different pathways that remove TOP2cc adducts result in different repair intermediates that could be captured as TOP2-induced breaks. Recently, genome-wide DSB mapping/sequencing methods that are capable of single-nucleotide resolution were developed ,95,96,97The decision for what pathway is used to repair DSBs is influenced by a combination of DNA end structure, proteins recruited to the DNA ends, and cell-cycle stages. When TOP2ccs are removed by TDP2, only the nucleotide that is bound to the tyrosine is removed, leaving ligation-compatible ends that can undergo NHEJ. The Ku complex recognizes the ends and p53 further promotes NHEJ by blocking HR-related proteins. The ligation is then carried out by the XRCC4/LigIV complex for simple, compatible ends . TOP2cc TOP2 poisons are commonly utilized in standard chemotherapy regimens. The active role of TOP2 in various biological processes enables TOP2 poisons to be incredibly effective in cancer treatment, as the resulting fork collapses/collisions and unresolved DSBs lead to apoptosis of the cancer cells. However, a common consequence of this mode of action is DNA damage to normal cells, as demonstrated by increased sensitivity of hematopoietic stem and progenitor cells (HSPCs) to DNA damaging agents when compared to more committed progenitor cells ,107. HeiKMT2A or MLL gene [MLL fused to either AF9, ENL, ELL, or AF4 [MLL [MLL is particularly sensitive to the effects of TOP2 poisons and, therefore, may explain why it is prevalent in many t-AML cases. Furthermore, we and others identified TOP2-mediated DNA breaks located at the therapy-related BCR of MLL [MLL, AF9, AF4, AF6, and ENL in human HSPCs and lymphoblastoid cells [Interestingly, the mutation most often associated with t-AML is a translocation involving chromosome 11, specifically the MLL gene ,118,119., or AF4 . Work frAF4 [MLL . ImportaAF4 [MLL ,122. ThiR of MLL ,124,125,R of MLL ,77. ReceMLL translocations, and those translocation events have breakpoints at locations that are highly similar to t-AML cases [MLL translocation in utero includes (1) identification of the translocation in neonatal blood spots of children diagnosed with AML, and (2) demonstration of monozygotic twins possessing identical AML translocations [Multiple studies implicated maternal diet and occupational exposure in incidences of pediatric/infant AML. Specifically, studies focused on the role of both synthetic and natural TOP2 inhibitors and poisons in pediatric AML. Maternal exposure to TOP2 poisons such as bioflavonoids was focused on because roughly 80% of pediatric AML cases have ML cases ,127,128.ML cases ,130. Furocations ,132,133.MLL [MLL translocations, despite conflicting opinions regarding the role of alcohol, tea, coffee, and cocoa in this process [MLL and a partner gene as classic TOP2 poisons, and they can inhibit in vitro TOP2 activity to a similar extent as etoposide and doxorubicin [MLL/AF9 translocations in hematopoietic stem cells [There are multiple chemicals and food items that are demonstrated/proposed to be TOP2 poisons. In terms of occupational exposures, benzene is of particular interest because it is a known carcinogen, with evidence supporting its role in leukemogenesis . AdditioMLL . An epidMLL . Strong MLL . There iMLL ,137,138  process ,141,142.orubicin . Recent em cells . OverallIn addition to the role of TOP2 in hematological malignancies, there is evidence to suggest TOP2 also facilitates the formation of oncogenic translocations in solid tumors. Papillary thyroid cancer (PTC) incidences increased dramatically since the 1970s according to the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) database ,145,146.RET/PTC1 rearrangement [RET/PTC3 rearrangement is more commonly the driver mutation [RET/PTC1 and RET/PTC3 rearrangements  are within known fragile sites [RET/PTC1 translocation process was first demonstrated by our laboratory when non-malignant thyroid (HTori-3) cells were treated with low doses of fragile site-inducing chemicals and only the RET/PTC1 rearrangement was observed [Sporadic rearrangement-positive PTC cases are believed to be predominantly chemical exposure-related as 50–71% of these cases possess the angement ,150,151.mutation ,152,153.le sites , indicatobserved .Due to these early indications of chemical exposure-associated PTC cases, we further examined the contribution of chemicals such as benzene and/or chemotherapeutic agents, like TOP1 and TOP2 inhibitors/poisons, to incidences of PTC. Benzene is a carcinogen with anti-TOP2 properties and is often found in cigarette smoke, gasoline, and industrial emissions ,11,156. RET BCR in intron 11 when HTori-3 cells were treated with low-dose, non-cytotoxic levels of benzene, etoposide, and doxorubicin [RET intron 11 that are predominantly distributed at or around predicted TOP1 and TOP2 cleavage sites [RET intron 11 is capable of forming stable, DNA secondary structures [RET intron 11 is due to the activity of topoisomerases at the DNA secondary structures [Recently, we observed a significant increase in the frequency of DNA breaks within the orubicin . This suge sites . RET intructures ,73. Inteructures ,69,70,71TOP2 and proteins associated with processing TOP2 adducts were also reported to be involved in B-cell immunodeficiency , autism TOP2B mutants were recently identified in patients with B-cell immunodeficiency, and, using a murine model, Broderick et al. demonstrated increased DNA damage in mutant cells [TOP2B mutations and the role of TOP2B in B-cell development.Heterozygous nt cells . Many B-Recent studies found that topoisomerases help facilitate effective transcription of the genes linked with autism spectrum disorders (ASD). Interestingly, the ASD-associated genes were found to be particularly long and AT-rich ,63, whicTOP2 is also associated with autoimmune disorders like insulin-dependent diabetes mellitus (IDDM). IDDM is classified as an autoimmune disorder because the beta cells that produce insulin are destroyed by T cells due to the presentation of autoantigens. Almost half of IDDM patients had TOP2A autoantibodies . MacrophFurthermore, TOP2-associated diseases arise from mutations in the proteins that process TOP2 adducts and TOP2-induced DNA breaks, as demonstrated by the impact of these mutant proteins in neurons. TDP2 mutations cause neurological effects, specifically, seizures, ataxia, and intellectual disabilities. TDP2 mutations are also associated with defects in transcription and ineffective repair of TOP2-induced DNA breaks, which leads to incorrect transcript levels for proteins that are required for proper neurological function and neuronal organization of the cerebellum . MutatioWe discussed how TOP2-induced DSBs are required for relieving torsional stress and resolving DNA structures during replication and transcription, as well as at TAD boundaries. Moreover, TOP2-induced breaks were shown to promote signal-induced transcription and facilitate dynamic chromatin contacts such as those between promoters and enhancers. In promoting these various processes, TOP2-associated DNA breaks could then be left unrepaired or repaired incorrectly, resulting in a variety of genomic mutations. TOP2 and the proteins associated with TOP2 end processing can directly and indirectly contribute to DNA fragility. Therefore, understanding the repercussions of impairing these proteins with environmental chemicals, dietary topoisomerase inhibitors, and/or chemotherapy regimens will allow us to mitigate exposure-related incidences of cancer in a timely manner.


EGFR, PIK3CA, and HRAS, or loss-of-function in p53 and PTEN are often associated with mTOR hyperactivation, whereas mutations identified from The Cancer Genome Atlas (TCGA) dataset that potentially lead to aberrant mTOR signalling are found in the EIF4G1, PLD1, RAC1, and SZT2 genes. In this review, we discuss how these mutant genes could affect mTOR signalling and highlight their impact on metabolic processes, as well as suggest potential targets for therapeutic intervention, primarily in head and neck cancer.The mammalian target of rapamycin (mTOR) signalling pathway is a central regulator of metabolism in all cells. It senses intracellular and extracellular signals and nutrient levels, and coordinates the metabolic requirements for cell growth, survival, and proliferation. Genetic alterations that deregulate mTOR signalling lead to metabolic reprogramming, resulting in the development of several cancers including those of the head and neck. Gain-of-function mutations in  Head and neck squamous cell carcinoma (HNSCC) is currently the sixth most frequently diagnosed malignancy worldwide . It is tTP53, CDKN2A, FAT1, NOTCH1, EGFR, HRAS, and PI3KCA [+ subtype patients, high-risk HPV infection has been associated with the abnormal expression of proteins associated with cell cycle regulation, including p53 and p16 (CDKN2A) [The recent application of next generation sequencing to study patient cancer genomes has revolutionized medical oncology. In silico analyses provide great insights into the diverse genomic alterations within each cancer sample, allowing for a functional understanding of the drivers behind deregulated oncogenic pathways and biological mechanisms involved in cancer progression. Importantly, these approaches are being exploited to potentially personalise suitable treatment regimens, tailored towards targeting key oncogenic drivers based on the individual’s mutational profile. On this basis, The Cancer Genome Atlas (TCGA) has been extensively interrogated for a comprehensive genomic characterization of HNSCC, whereby several reports have identified hundreds of mutations in each cancer subtype ,20,21,22d PI3KCA ,25,26,27(CDKN2A) ,29. Func(CDKN2A) .EGFR and HRAS often lead to aberrant signalling and deregulation of important proto-oncogenic networks, such as the PI3K–mTOR pathway. For instance, PIK3CA mutations that directly activate the PI3K–mTOR signalling pathway have been reported in HNSCC at rates ranging from 2.6% to 19% [Furthermore, FAT1 is involved in the migration and invasion of HNSCC cells through the activation of the β-catenin pathway . In addi% to 19% . OverallThe mammalian target of rapamycin (mTOR) is a serine-threonine kinase that senses growth factor cues, nutrient and oxygen status, and directs appropriate changes to maintain cellular and tissue homeostasis . The mTOConstitutive mTOR activation is known to promote metabolic changes, including dysregulation in glucose, fatty acid, amino acid, and lipid metabolism. For instance, cancer cells largely rely on glucose as the major source of cellular energy to sustain proliferation and survival. In the context of aberrant mTOR signalling, glucose metabolism is dysregulated as a result of increased synthesis of glucose transporter proteins and glycolytic enzyme activation, followed by lactic acid fermentation even when oxygen is available—a phenomenon known as the “Warburg effect” ,42. The The mTOR pathway is known to be hyperactivated in several cancers, including HNSCC, and both mTOR complexes play essential roles in HNSCC tumorigenesis. Interestingly, mTOR deregulation in HNSCC is the most commonly seen genomic alteration (~80–90% HNSCC) involved in aberrant mitogenic signalling, compared to other known pathways such as the JAK/STAT and MAPK, which harbour mutations in less than 10% of lesions ,54. PIK3CA mutations [In vivo analyses of mTOR signalling in HNSCC are commonly studied, and chemically-induced HNSCC mouse models have long been established. These include the widely used carcinogens DMBA-TPA and 4-nitroquinoline-1-oxide (4NQO), which have both been reported to result in persistent mTOR activation, leading to tumour development, and regression is observed after the administration of the mTOR inhibitor rapamycin ,56,57. Futations . The comutations . Furtherutations . mTORC1 interacts with the Rag GTPases, which promote its translocation and activation at the lysosomal surface in response to amino acids ,65. InhiHPV infection is known to activate mTOR signalling in HNSCC and is further sustained through deregulation of metabolic pathways. For instance, HPV-positive cells utilise mitochondrial respiration, as evidenced by increased oxygen consumption in comparison to HPV-negative HNSCC cells, which exhibit increased glucose metabolism, as evidenced by the over production of lactate . HPV-negmTOR inhibitors have shown promising anti-cancer effects in HPV-positive HNSCC mouse models. The mTOR inhibitors Rapamycin and RAD001 reduced tumour burden in HPV-positive HNSCC xenografts through the inhibition of mTOR activity . MoreovePIK3CA mutations or amplifications in over half of HPV-positive HNSCC, leading to drug resistance. Brand et al. showed that PI3K inhibition resulted in increased expression of the HER3 receptor and, in turn, elevated the abundance of E6 and E7 oncoproteins to promote resistance to PI3K inhibition [Furthermore, the E6 and E7 HPV oncoproteins are known to correlate with hibition . This sthibition . PIK3CA, and HRAS, as well as others found in potential genes such as EIF4G1, RAC1, SZT2, and PLD1, can result in aberrant mTOR signalling , including afatinib, appear clinically promising ,98,99. TMutations that activate the catalytic unit of phosphoinositide-3-kinase (PI3K) have been implicated in several cancers, including HNSCC. Gain of function mutations of PIK3CA, the most common activator of the PI3K pathway, is detected in approximately 6–20% of HNSCC cases ,22. Lui AKT1 (downstream of PI3K) or EIF4E (downstream of mTORC1), suggesting a direct link between SOX2 regulation and PI3K. Additionally, SOX2 knocks down suppressed ALDH transcripts and protein levels. Moreover, Suda et al. revealed that copy-number amplification of PIK3CA, within 3q (found in up to 30% of HNSCC) is associated with a poor prognosis of HNSCC patients [Aberrant PI3K-mTOR signalling was shown to also regulate the properties of key cancer stem cell (CSC) factors, including the sex determining region Y box 2 (SOX2) ,103. SOXpatients  and partpatients , and a spatients . The elePten [PTEN gene are rare in human skin SCC and common in HNSCC, which could be a prognostic marker for patients with tongue cancer [Conversely, inactivation of phosphatase and tensin homologue (PTEN), a potent tumour suppressor and negative regulator of PI3K, also leads to hyperactivation of PI3K-driven mTOR signalling . AlthougPten ,116,117.Pten . InhibitPten . Interese cancer ,119,120.e cancer . PTEN loss also promotes cancer progression by enhancing glucose metabolism and reducing DNA repair and checkpoint pathways. Martin et al. observed PTEN loss in prostate cancer cell lines and increased pAKT expression and enhanced glucose metabolism, resulting in the survival of tumour cells . Mathur HRAS belongs to the Ras oncogene family, and HRAS mutants are known to aberrantly activate mTOR signalling in HNSCC tumours [HRAS mutations in 5% of samples [HRAS mutations [HRAS mutations in 86 OSCC patients (8.6%) of Greek descent, and defined a role for HRAS in driving PI3K-mTOR signalling in OSCC [HRAS in 10 out of 56 Vietnamese OSCC patients (18%) and associated these events with an advanced tumour stage [HRAS mutations have been shown to play a role in altering metabolic processes. Zheng et al. suggest that the HRAS transformed breast cancer cell line MCF10a, derived from an early stage cancer, exhibits a profound alteration in glucose metabolism and is strongly regulated by the oncogenic proteins HIF-1α and c-Myc [ tumours . The Can tumours . Nakagak samples . Su et autations . Further in OSCC . Adding ur stage . In addind c-Myc . HRAS mutant HNSCC cell lines did not respond to PI3K inhibition (BYL719). This inhibitor induced constitutive MAPK signalling suggests feedbacks between MAPK and PI3K, resulting in persistent mTOR activity [HRAS mutations and resistance to the EGFR tyrosine kinase inhibitor erlotinib in a panel of HNSCC cell lines [HRAS mutations in 7 out of 55 samples (12.7%) that were associated with a poorer response to Cetuximab treatment. Additional investigations have implicated the HRAS protein in driving resistance to therapies in HNSCC. In a recent study conducted by Ruicci et al., activity . Hah et ll lines . Likewisll lines . FurtherEukaryotic translation initiation factor (EIF) 4 gamma 1 (EIF4G1) plays a crucial role downstream of mTOR signalling. EIF4G1 functions as a modular scaffold in the translational initiation complex, interacting with EIF3, EIF4A, EIF4E, poly (A)-binding proteins, and MNK1 ,135. WheAberrant over-expression of EIF4G1 is tightly linked with the prognosis of several cancers, such as lung squamous cell carcinoma, inflammatory breast cancer, cervical cancers, and nasopharyngeal carcinoma ,140,141.TIAM-1) expression, which all contribute to tumour growth [The Rac family of small GTPase 1 (RAC1) functions downstream of the mTOR signalling to regulate the reorganization of F-actin, lamellipodia formation, and cell motility . mTORC1-r growth ,151. Incr growth ,153. ColRAC1 expression is positively associated with cancer progression [Overexpression of RAC1 is frequently observed in oral, breast, gastric, testicular, and prostate cancers and increased gression ,156,157.gression ,159. Altgression . Moreovegression ,160,161.gression  and EHT gression . Alongsigression . AlthougSZT2 gene shows a relatively high somatic mutation frequency (3.6%) in HNSCC in the TCGA database. In addition, low expression of SZT2 is correlated with a low five-year survival rate of HNSCC patients [Deficiency of seizure threshold 2 protein homolog (SZT2) is commonly detected in patients with intellectual disability, epilepsy, and autism ,167,168.patients . Future PLD1 expression is frequently detected in various cancers, including glioma, pancreatic ductal adenocarcinoma, colorectal cancer, hepatocellular carcinoma, breast cancer, and melanoma [Phospholipase D1 (PLD1) is an established upstream regulator of mTOR signalling . Once acmelanoma ,177,178.melanoma .Multiple inhibitors targeting PLD1 have been developed, such as VU-0155069 and VU-0359595, which directly bind to the N-terminus, allosterically suppressing the catalytic activity of PLD1 . In addiBecause multiple mutant genes are directly associated with the oncogenic activation of the mTOR pathway, it is not surprising that multiple clinical trials are currently targeting aberrant mTOR signalling in cancer . For insPIK3CA mutations received the PI3K inhibitor taselisib, patients harbouring EGFR mutations received the EGFR inhibitor afatinib, while patients with loss or mutated PTEN received the PI3K-beta inhibitor GSK2636771. In addition to monotherapies, combination treatments are scheduled with the PI3K/mTOR inhibitor gedatolisib and the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor palbociclib, and HNSCC patients are currently being recruited for this Phase I trial (NCT03065062). Several clinical trials are currently recruiting HNSCC patients for the assessment of other mTOR pathway inhibitors. Following promising results obtained in vitro and in vivo with BYL719, a potent PI3Kα inhibitor, this drug has progressed to Phase II trials for the treatment of recurrent or metastatic HNSCC patients who have previously failed to respond to platinum-based therapy (NCT02145312) . MoreoveDespite mTOR signalling driving aberrant metabolic processes, there is currently no clinical trial investigating combinational treatment against both mTOR and dysregulated metabolism in HNSCC patients. Nonetheless, there are several studies targeting key transporters involved in metabolism and HNSCC progression. In a recent study conducted by Mehibel et al., the authors investigated the use of simvastatin  and AZD3965 (which inhibits monocarboxylate transporter 1 and results in enhanced glycolysis) in HNSCC cell lines . They foPTEN loss, PTEN mutations, NOTCH1 mutation, and EGFR expression in other cancers, but could be further established in HNSCC. Furthermore, the functional characterisation of these mutant genes and the molecular dissection of their associated oncogenic networks could provide targets for combinatorial therapies to alleviate resistance to mTOR inhibition.The mTOR pathway integrates multiple intrinsic genetic alterations and extrinsic cues, leading to aberrant signalling and metabolic alterations. Since the validated and potential mutant genes, as identified from the TCGA dataset, directly affect mTOR activation status in cancer, they could be used as biomarkers for response and mTOR targeted inhibition in a tissue-specific manner. In fact, multiple biomarkers for predicting drug sensitivity have been proposed, including those related to


Death associated protein kinase (DAPK) is a calcium/calmodulin-regulated serine/threonine kinase; its main function is to regulate cell death. DAPK family proteins consist of DAPK1, DAPK2, DAPK3, DAPK-related apoptosis-inducing protein kinases (DRAK)-1 and DRAK-2. In this review, we discuss the roles and regulatory mechanisms of DAPK family members and their relevance to diseases. Furthermore, a special focus is given to several reports describing cross-talks between DAPKs and mitogen-activated protein kinases (MAPK) family members in various pathologies. We also discuss small molecule inhibitors of DAPKs and their potential as therapeutic targets against human diseases. DAPK2 contains an N-terminal kinase domain with high homology to DAPK1 catalytic domain [Death-associated protein kinase (DAPK) family proteins are closely related, Caunctions ,2. DAPK unctions ,4,5,6,7 c domain . The kinc domain . All of c domain ,6, excepc domain . Mitogen-activated protein kinases (MAPKs) are an important sub-family of non-receptor serine-threonine kinases. MAPKs mediate signal transduction pathways that are involved in cellular responses to a diverse range of stimuli, such as mitogens, hormones, osmotic stress, heat shock, proinflammatory cytokines, and significant developmental changes in organisms. They mediate cellular functions including proliferation, differentiation, mitosis, gene expression, and apoptosis . ExtraceThe mutual regulation between MAPK and DAPK family proteins plays a role in apoptosis regulation and several diseases. In this review, we introduce the reports showing the regulatory mechanisms and various functions of DAPK family proteins. Furthermore, we refer to reports indicating the relationship between DAPK and MAPK family proteins in multiple diseases.Increased activity of DAPK family proteins results in pronounced death-associated cellular changes, which include cell rounding, membrane blebbing, detachment from extracellular matrix, and formation of autophagic vesicles ,17,18,19Among DAPK family proteins, DAPK1 controls cell cycle, apoptosis, autophagy, tumor metastasis, and oxidative stress. Several reports demonstrate that complex regulation of DAPK1 activity by various signaling pathways modulates the balance between pro-apoptotic and pro-survival pathways . FurtherDAPK2 is known to be involved in pro-inflammatory responses mediated by granulocytes, which might be linked to the mechanism of myosin light chain (MLC) phosphorylation by DAPK2 . BesidesThe biological role of DAPK3 has been gradually investigated . DAPK3 iDAPK1, DAPK2, and DAPK3 are all ubiquitously expressed in various tissues, such as heart, lung, spleen, and brain. In particular, DAPK1 is highly expressed in the hippocampus ,5. DAPK1 acts as a positive mediator of apoptosis induced by several death stimuli, such as interferon (IFN)-γ, Fas, transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, ceramide, oncogene expression, and DNA damaging agents ,28,29,30DAPK2 is regarded as a tumor suppressor in non-solid tumors and is implicated in apoptotic cell death . After s2+/CaM regulatory domain, and its activity is regulated independently of intracellular Ca2+ levels. There are reports showing that DAPK3 is activated via interacting with DAPK1, forming a death-associated multi-protein complex [On the contrary, DAPK3 lacks a Ca complex . DAPK3 w complex , and to  complex . DAPK3 i complex ,37. The These studies suggest that the activity of DAPK proteins is regulated not only by the upstream signals but also the interactions by themselves. Increased activity of DAPK family proteins results in pronounced death-associated cellular changes, which include cell rounding, membrane blebbing, detachment from the extracellular matrix, and formation of autophagic vesicles ,17,18,19Among DAPK family proteins, DAPK1 controls cell cycle, apoptosis, autophagy, tumor metastasis, and oxidative stress. Several reports demonstrate that complex regulation of DAPK1 activity by various signaling pathways modulates the balance between pro-apoptotic and pro-survival pathways . FurtherDAPK2 is known to be involved in pro-inflammatory responses mediated by granulocytes, which might be linked to the mechanism of myosin light chain (MLC) phosphorylation by DAPK2 . BesidesThe biological role of DAPK3 has been gradually investigated . DAPK3 iAmong DAPK proteins, the relationship between DAPK1 and diseases has been often reported A. In theTwo main characteristics of AD are amyloid-beta (Aβ) senile plaques and tau neurofibrillary tangles. The metabolism of amyloid precursor protein (APP) results in extracellular deposition of Aβ protein that leads to the formation of Aβ senile plaques. In AD, tau is commonly hyperphosphorylated prior to tangle formation and neurodegeneration. ,51. HoweSeveral studies showed that DAPK1 contributes to the pathogenesis of AD through excessive processing of APP and  triggeriIn addition to regulating APP and tau proteins, DAPK1 mediates the neuronal cell death in AD model animals. DAPK1 binds N-myc downstream regulated gene 2 (NDRG2), which triggers its phosphorylation at Ser350 and induces neuronal cell death in AD model mice. In contrast, DAPK1 inhibition prevented NDRG2-mediated neuronal cell death . These dIn a wide variety of tumors, DAPK1 expression is frequently suppressed and the tumor-suppressive function of DAPK1 is linked to its role in cell death via apoptosis and autophagy . It was DAPK1 also has a role in cellular antiviral immune response. Once the viral infections occur, the viruses inhibit INF-mediated signals including INF-α/β for their proliferation ,61. AfteBesides, DAPK1 is involved in the regulation of myofibril degeneration and myocyte apoptosis induced by chronic stimulation with β1-adrenergic receptors. The result implies that DAPK1 activation might contribute to the pathogenesis of β-adrenergic receptor-related signaling during the development of heart failure . DAPK1 seems to have both pro- and anti-inflammatory functions. It positively contributes to production and secretion of interleukin (IL)-1β in macrophages , while iAnother disease that DAPK1 may be involved in is atherosclerosis. Although DAPK1 expression was increased in atherosclerotic plaques, the detailed mechanisms are not known . In addiIn contrast to DAPK1 and DAPK3, DAPK2 has not been identified as a tumor suppressor in solid tumors. Interestingly, DAPK2 was predominantly found in the hematopoietic compartment  and emerDAPK3 is proposed to be a tumor suppressor, suggesting that mutations in DAPK3 could result in the loss of function. It was reported that the DAPK3 gene is frequently methylated or mutated in various types of cancer , resultiOn the other hand, a recent study demonstrated that DAPK3 promotes cancer cell proliferation rather than the promotion of apoptosis in many types of cancer cells. In prostate cancer cells, DAPK3 promoted proliferation . Further2+-independent Myosin light chain kinase (MLCK) activity [In our previous study, it was found that the expression level of DAPK3 protein was increased in the mesenteric artery from spontaneously hypertensive rats (SHR) . Moreoveactivity . In our activity . Furtheractivity . These dNevertheless, the data of DAPK proteins in diseases are preliminary. Further studies are needed to clarify the expression and functional correlation of DAPK proteins with these diseases.ERKs are a group of MAPKs (ERK1–8) and ERK1/2 are the first discovered members of the MAPK family. Once activated (phosphorylated), ERK1/2 moves from the cytoplasm to the nucleus, which is critical for many cellular functions, such as gene transcription, cell proliferation, and differentiation . Since EERK1/2 was identified as a DAPK1-interacting protein  Figure . DAPK1 iMechanistically, it was shown that DAPK1 prevented activation of ribosomal S6 kinase through inhibition of ERK1/2 nuclear translocation, indicating that DAPK1 may play an inhibitory role in the survival function of ERK1/2 signaling . It was ERK1/2 regulates activation of the p53 pathway , and DAPDAPK1-ERK1/2 signals also regulate neuronal apoptosis following ischemia-reperfusion . In the p38 plays roles in inflammation, cell proliferation, differentiation, survival, and cell death ,90,91,92Several studies provided direct evidence for the interaction of DAPK1 with p38 signaling in inflammation-associated colorectal cancer cells . That stIn our previous study, it was clarified that DAPK3 mediates platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of SMC through activation of p38/heat shock protein (HSP)27 signals, which leads to vascular structural remodeling including neointimal hyperplasia  Figure B. Since JNK pathways are activated by various stress stimuli, such as heat shock, osmotic shock, ultraviolet irradiation, and cytokines ,91,92, wJNK is a key node of the cell death network activated under oxidative stress ,96,97, aIn contrast, DAPK2 inhibits JNK activation through the prevention of oxidative stress in osteosarcoma and lung cancer cells  Figure B. In theIn our previous study, it was demonstrated that DAPK3 mediated TNF-α-induced activation of JNK, p38, and Akt through a generation of ROS  Figure B,5C. In Discovery of DAPK inhibitors has been performed using previously described experimental approaches ,101. SevAlkylated 3-amino-6-phenylpyridazine inhibited DAPK1 catalytic activity in cultured neuron cells and mouse models . FurtherZ)-4-(3-pyridylmethylene)-2-styryl-oxazol-5-one, was known to inhibit DAPK1 activity effectively and selectively (IC50 = 69 nM) [Another compound, (4= 69 nM) ,53,102. 50 = 69 and 225 nM, respectively), which makes it the most potent DAPK1 inhibitor discovered to date [The role of DAPK activity in the histone deacetylase inhibitor (LBH589)-induced apoptosis in HCT116 wildtype colon cancer cells was clarified by using a selective DAPK1 inhibitor, 2-phenyl-4-(pyridin-3-ylmethylidene)-4,5-dihydro-1,3-oxazol-5-one, known as TC-DAPK6, for inhibition of DAPK1 catalytic activity ,103. It  to date .H-purine (6d). Eighteen analogs exhibiting anti-proliferative and pro-apoptotic properties on an acute T cell leukemia cell line were obtained . These aSeveral other selective inhibitors of DAPK have also been investigated . The nonLaurus nobilis, a DAPK1 non-selective inhibitor of plant origin, significantly improved the ischemic neuronal death through keeping DAPK1 in an inactivate form after oxygen and glucose deprivation in human SH-SY5Y neuroblastoma cell lines and organotypic hippocampal slice tissue. Moreover, it also reduced the infarct size and neurological deficit of middle cerebral artery occlusion in vivo [Besides, natural DAPK inhibitors have been developed. The chloroform fraction of  in vivo .Recently, a dual Pim/DAPK3 inhibitor (HS56) was synthesized using crystal structure-guided medicinal chemistry techniques . HS56 re50 value to that of DAPK1 and DAPK3 [While specific inhibitors for DAPK2 remain to be developed, DAPK1 and DAPK3 inhibitors have been tested for DAPK2 activity. Since the catalytic domain of the three kinases shares high-sequence homology, the most potent derivative showed comparable ICnd DAPK3 .The potency of most DAPK inhibitors in in vitro kinase assays toward DAPK proteins is not very high. Improvement of the structures of these compounds is needed to gain more potent inhibitors and to perform clinical trials. Although there are only a few detailed studies focusing on DAPK proteins as a drug target, it would be helpful to use selective inhibitors against unmet medical needs.Several recent studies have unraveled pathologically relevant mechanisms involving DAPK-MAPK cross-talks in neuronal diseases, cancer, and cardiovascular diseases. With the availability of more specific and effective DAPK inhibitors, the interface between DAPK and MAPKs might become promising targets in the treatment of various diseases.


EGFR and KRAS mutations were detected in 18 (24%) and 20 (26%) patients, respectively. No ALK alterations were found, but in two patients ROS1 translocation was identified. Of 22 cases tested, 17 were positive for PD-L1 staining. Octogenarian patients who received tyrosine kinase inhibitors (TKIs) based on molecular analysis showed clinical benefits, with long progression-free survival as expected in TKI-treated younger cohorts. Conclusions: This study highlights the utility of molecular profiling in all advanced-stage NSCLCs, regardless of the age at diagnosis, to drive personalized treatment. The prevalence of druggable alterations and the clinical benefits obtained by biologically-driven therapies in octogenarians were comparable to those of the younger NSCLC population.Background: There is a pressing need to expand the evidence base in geriatric lung oncology. Most non-small cell lung cancers (NSCLCs) are diagnosed in the elderly, with approximately 15% of cases affecting octogenarians. Treatment-related decisions are challenging in this population, and the role of biologically driven therapies is still underrated. Methods: A single-institution cohort of 76 NSCLCs from octogenarian patients was submitted to molecular analysis using a next-generation sequencing (NGS) multigene panel, fluorescence in situ hybridization (FISH) analyses, and immunohistochemistry for PD-L1 assessment. Treatment and clinical outcome data were available for 33 patients. Results: Most cases ( EGFR mutations or ALK and ROS1 rearrangements) has changed the treatment paradigm and natural history of non-small cell lung cancer (NSCLC) harboring these aberrations [Lung cancer is the leading cause of cancer-specific mortality worldwide , and is rrations ,5. To darrations ,7. NeverEGFR, KRAS, and BRAF genes, in addition to fluorescence in situ hybridization (FISH) analyses for ALK, ROS1, MET, and immunohistochemical (IHC) evaluation of PD-L1 expression. We sought to (i) evaluate the biological characteristics of NSCLC occurring in octogenarian patients, which could lead to personalized treatments; and (ii) investigate the potential clinical benefit of molecularly driven therapies in a subgroup of patients treated with targeted treatment.In the present study, we report the molecular characterization of advanced NSCLC from 76 consecutive octogenarian patients who were referred to our institution over 19 months for molecular diagnosis, following clinical requests. The molecular testing was performed using a next-generation sequencing (NGS) panel including EGFR, KRAS, BRAF, ALK, ROS1, MET, and PD-L1 according to the international guidelines for molecular testing in lung cancer [Of the 890 patients with advanced-stage (IIIb and IV) NSCLCs referred to the Molecular Diagnostics Unit of the European Institute of Oncology from August 2016 to February 2018, 76 patients over 80 years were included in this study. Molecular tests and IHC evaluation were required by thoracic oncologists, and included the analysis of g cancer . Patientg cancer .All patients gave written informed consent regarding the storage of any biological specimens collected in the course of diagnosis and the use of these samples for research purposes.n = 63) and cytoblocks (n = 8) or smears (n = 5) were used for the analyses. The DNA was extracted automatically with the Promega Maxwell instrument  using the Promega Maxwell RSC DNA FFPE kit, and was quantified with the Quantus fluorometer . The NGS mutational analysis was performed with the CE-IVD  Oncomine Solid Tumour DNA kit . This panel allowed for the simultaneous evaluation of the mutational status  of 22 genes, namely EGFR, ALK, ERBB2, ERBB4, FGFR1, FGFR2, FGFR3, MET, DDR2, KRAS, PIK3CA, BRAF, AKT1, PTEN, NRAS, MAP2K1, STK11, NOTCH1, CTNNB1, SMAD4, FBXW7, and TP53. The targeted NGS analysis was performed following the manufacturer’s instructions. Briefly, 10 ng of genomic DNA was used for the library preparation. Sequencing was performed on a Personal Genome Machine (PGM) sequencer or Ion S5 System , and data were analyzed using the Ion Reporter Analysis software v.5.2-5.10 . Quality metric filters were used as previously described [http://cancer.sanger.ac.uk/cosmic; ClinVar: https://www.ncbi.nlm.nih.gov/clinvar; TCGA Cancer Genome: http://cancergenome.nih.gov) were reported. Benign variants or polymorphisms were not reported. Mutations were classified as level I variants, level II variants, and level III variants according to their clinical relevance and/or availability of targeted therapy and the type of cancer under investigation [Five-micrometer-thick sections from representative formalin-fixed paraffin-embedded (FFPE) tissue blocks . In each case, at least 100 tumor nuclei were evaluated. Cells were considered positive if a break-apart pattern of orange and green signals, at least one single orange signal, or a combination of both patterns were seen. Tumors with at least 15% of cells with ALK or ROS1 rearrangements were defined as positive. In ambiguous or equivocal cases, ALK or ROS1 immunohistochemical stains  were performed. The presence of MET gene amplification was evaluated using the MET IQFISH Probe with CEP7 . Amplification was reported in cases with a MET Probe/CEP7 Ratio ≥2 and/or gene copy number ≥5.PD-L1 expression was evaluated on tumor cells using the immunohistochemistry assay CE-IVD PD-L1 IHC 22C3 pharmDx with the Agilent-Dako 22C3 clone developed on the Dako Autostainer Link 48  as previously described . The TumTP53 (37%), KRAS (26%), and EGFR (24%)  cases harbored at least one gene alteration, including clinically relevant and cancer-related variants. The most frequently mutated genes were FR (24%) .EGFR, ROS1, KRAS, BRAF (1 case V600E), MET, NRAS, ERBB2, PIK3CA, and AKT1 genes  or clinical trials (level II), accounted for 62/106 (58.5%) of genomic aberrations identified and involved T1 genes .KRAS and EGFR mutations were reported in 20 and 18 NSCLC specimens, respectively. All KRAS mutations identified affected exon 2.In detail, EGFR included both TKI-sensitive mutations (n = 16), such as exon 19 deletions (n = 8) and exon 21 L858R point mutation (n = 8); and TKI-resistant mutations, such as exon 20 insertions (n = 2). In four cases, a secondary T790M resistant mutation was detected during TKI treatment. Concurrent EGFR, T790M, and L858R mutations were present at diagnosis in one patient with no history of TKI therapy.Mutations in ALK fusions were detected in our study cohort. ROS1 gene rearrangements were identified in two cases, with a concurrent NRAS mutation or KRAS mutation.No MET alterations were found in 12% of cases and included missense variants (n = 4), exon 14 skipping mutations (n = 1), and gene amplifications (n = 4).Moreover, TP53 mutations with either EGFR (n = 7) or KRAS (n = 4) mutations. Moreover, EGFR mutations were found in association with alterations of MET (n = 2) and STK11 (n = 1) genes. Concurrent KRAS and STK11 mutations were identified in two cases.Overall concurrent genetic aberrations were found in 33 cases , includin = 22, 29%), including five negative cases (25%), seven cases (25%) with intermediate TPS between 1% and 49%, and ten cases (50%) with diffuse PD-L1 expression (TPS > 50%)  . Among tEGFR mutations. The other actionable alterations  were found in NSCLC outpatients or inpatients with a poor PS, not eligible for enrollment in a clinical trial or active treatment.Treatment and follow-up data were available for thirty-three inpatients. Twenty-three of these patients received an active treatment . In ten EGFR mutations, the majority were never-smokers or long-term ex-smokers . Smoking habits were not assessed in the remaining cases. A different distribution between males and females was not seen . Treatment and follow-up data were available for ten patients who received tyrosine kinase inhibitors (TKIs), including erlotinib, gefitinib, or osimertinib, when a concurrent EGFR T790M mutation was detected. One patient was first-line treated with osimertinib because of the co-occurrence of L858R and T790M mutations at diagnosis  or platinum-based therapy (n = 3). The median PFS of these patients was 2 months  and KRAS (26%) mutations. In our previous series, level I and II variants were detected in 63.6% of cases, with KRAS and EGFR mutations present in 31% and 22% of the cases, respectively [2 test p > 0.05) and showed that the presence of specific tumor genetic aberrations was not related to the age of the patients. Conversely, ALK fusions were not detected in our cohort, consistent with the reported association between ALK translocation and the patients’ younger age [ROS1 translocations in two patients. This rarest druggable alteration has been reported in 1%–2% of NSCLCs [ROS1 translocation, and both had concurrent clinically relevant mutations (ROS1+/KRAS+ and ROS1+/NRAS+). Similar anecdotal cases of concurrent ROS1 rearrangements and KRAS mutations were recently reported [ROS1 translocations and NRAS variants have been described so far. ROS1 gene rearrangements could be targeted by TKIs, but KRAS/NRAS gene point mutations could lead to ineffective inhibition through inactivation of the RAF/MEK/ERK signaling pathway. Given that these patients were both outpatients, no data about therapeutic regimens or clinical outcomes were available.In the present study, we focused on a single-institution cohort of 76 consecutive octogenarian patients affected by advanced NSCLC. All tumors were analyzed using an NGS multi-gene panel that expands the tumor genetic portrait, including genes such as ndations ,9. Most ndations . Indeed,ectively . The difnger age ,18. Surpf NSCLCs ,20, usuaf NSCLCs . In our reported ,22, wherMET, BRAF, and ERBB2 genes, which may be targeted by specific therapies in a clinical setting or clinical trials [TP53 mutations have recently been associated with prognosis or therapy response in patients with concomitant EGFR or KRAS mutations, or ALK rearrangement [EGFR and TP53 mutations showed a worse response rate to TKIs and worse progression-free survival [+-TP53+KRAS tumors displayed a higher level of inflammatory markers and improved relapse-free survival [In a subgroup of octogenarian patients, we also identified alterations in l trials ,24. Moreangement . Patientsurvival . KRAS+-Tsurvival .EGFR mutated status was associated with non-smoking, as in the general population of NSCLC patients. A TKI treatment was administered to ten EGFR mutation-positive patients, who showed a PFS consistent with data from larger series of NSCLC patients [EGFR-mutated NSCLC [In the octogenarians included in this study, patients ,33,34,35patients  or erlotpatients . Howeverpatients  to 75 yepatients . Recentled NSCLC . The autDuring the past few years, the spectrum of therapeutic options for NSCLC patients with no identified genomic aberrations has been enlarged. Besides standard chemotherapy regimens, treatments with immune checkpoint inhibitors alone or in combination with chemotherapy have been shown to be effective in selected patients with advanced NSCLC enrolled in clinical trials. However, in the largest series, elderly patients were underrepresented, especially octogenarians . The ELDA major limitation of the present study is the relatively small cohort of patients analyzed. However, we collected consecutive octogenarian patients affected by NSCLC who underwent tumor molecular analyses for diagnostic purposes in a single institution. Another limitation is that treatment and follow-up data were not available for all patients and were collected retrospectively from patients’ medical records. We reported data for thirty-three inpatients of the Division of Thoracic Oncology of the European Institute of Oncology, including twenty-three patients who received molecularly driven treatments. Although the small cohort prevented identification of any clinical or statistical correlations and the retrospective analysis was not selected for the probability of survival, we reported interesting insights into the clinical course of octogenarian patients actively treated based on tumor molecular characteristics.This study highlights the utility of molecular analyses for all patients with advanced-stage NSCLC, regardless of age at diagnosis, to guide clinical decisions. Except for ALK translocation, the distribution of molecular alterations in the octogenarian population under investigation was very similar to that in a younger population. Molecular analyses should be performed with multi-gene panels, following the recent recommendations for molecular testing in NSCLC ,9, to wi


Homopolymeric amino acid repeats (AARs) like polyalanine (polyA) and polyglutamine (polyQ) in some developmental proteins (DPs) regulate certain aspects of organismal morphology and behavior, suggesting an evolutionary role for AARs as developmental “tuning knobs.” It is still unclear, however, whether these are occasional protein-specific phenomena or hints at the existence of a whole AAR-based regulatory system in DPs. Using novel approaches to trace their functional and evolutionary history, we find quantitative evidence supporting a generalized, combinatorial role of AARs in developmental processes with evolutionary implications. We observe nonrandom AAR distributions and combinations in HOX and other DPs, as well as in their interactomes, defining elements of a proteome-wide combinatorial functional code whereby different AARs and their combinations appear preferentially in proteins involved in the development of specific organs/systems. Such functional associations can be either static or display detectable evolutionary dynamics. These findings suggest that progressive changes in AAR occurrence/combination, by altering embryonic development, may have contributed to taxonomic divergence, leaving detectable traces in the evolutionary history of proteomes. Consistent with this hypothesis, we find that the evolutionary trajectories of the 20 AARs in eukaryotic proteomes are highly interrelated and their individual or compound dynamics can sharply mark taxonomic boundaries, or display clock-like trends, carrying overall a strong phylogenetic signal. These findings provide quantitative evidence and an interpretive framework outlining a combinatorial system of AARs whose compound dynamics mark at the same time DP functions and evolutionary transitions. If such a system exists, its contours are still essentially obscure and have to be traced at both the quantitative and qualitative levels.In fact, despite the frequent occurrence of repeats of different amino acids in DPs , the speFurthermore, while homopolymeric repeats of multiple amino acids, which can be structurally or functionally related, such as polyQ, polyA, and polyS, frequently co-occur in one same protein , the oveFinally, the quantitative evolutionary dynamics of AARs and their combinations are not clearly understood, and their broad fluctuations across species/taxa  are largTo address these issues, we use here novel approaches to trace the functional and evolutionary trajectories of the repeats of the 20 amino acids throughout phylogenesis and find quantitative and qualitative evidence supporting the existence of a generalized combinatorial system of AARs in developmental processes with evolutionary implications.www.uniprot.org; canonical isoforms; gene symbols: DLX1, DLX2, DLX3, DLX4, DLX5, DLX6, FOXA1, FOXA2, FOXA3, FOXB1, FOXB2, FOXC1, FOXC2, FOXD1, FOXD2, FOXD3, FOXD4, FOXE1, FOXE3, FOXF1, FOXF2, FOXG1, FOXH1, FOXI1, FOXI2, FOXI3, FOXJ1, FOXJ2, FOXJ3, FOXK1, FOXK2, FOXL1, FOXL2, FOXM1, FOXN1, FOXN2, FOXN3, FOXN4, FOXO1, FOXO3, FOXO4, FOXO6, FOXP1, FOXP2, FOXP3, FOXP4, FOXQ1, FOXR1, FOXR2, FOXS1, FOXD4L1, FOXD4L3, FOXD4L4, FOXD4L5, FOXD4L6, HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXB13, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, HOXC13, HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13, IRX1, IRX2, IRX3, IRX4, IRX5, IRX6, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, NKX1-1, NKX1-2, NKX2-1, NKX2-2, NKX2-3, NKX2-4, NKX2-5, NKX2-6, NKX2-8, NKX3-1, NKX3-2, NKX6-1, NKX6-2, NKX6-3, PAX1, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PAX9, POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F2, POU3F3, POU3F4, POU4F1, POU4F2, POU4F3, POU5F1, POU5F1B, POU5F2, POU6F1, POU6F2, SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21, SOX30, SRY). Reference proteomes were retrieved from Uniprot without isoforms for the following 55 species: H. sapiens (Hom sap), Pan troglodytes (Pan tro), Pongo abelii (Pon abe), Callithrix jacchus , Otolemur garnetti (Oto gar), Mus musculus (Mus mus), Rattus norvegicus (Rat nor), Heterocephalus glaber (Het gla), Ailuropoda melanoleuca (Ail mel), Felis catus (Fel cat), Bos taurus (Bos tau), Ovis aries (Ovi ari), Sus scrofa (Sus scr), Monodelphis domestica (Mon dom), Sarcophilus harrisii (Sar har), Ficedula albicollis , Taeniopygia guttata (Tae gut), Gallus gallus , Meleagris gallopavo , Anas platyrhynchos (Ana pla), Anolis carolinensis (Ano car), Ophiophagus hannah (Oph han), Astyanax mexicanus (Ast mex), Danio rerio (Dan rer), Oryzias latipes (Ory lat), Xiphophorus maculatus (Xip mac), Oreochromis niloticus (Ore nil), Gasterosteus aculeatus (Gas acu), Takifugu rubripes (Tak rub), Tetraodon nigroviridis (Tet nig), Lepisosteus oculatus (Lep ocu), Apis mellifera (Api mel), Camponotus floridanus (Cam flo), Acromyrmex echinatior (Acr ech), Atta cephalotes (Att cep), Solenopsis invicta (Sol inv), Anopheles gambiae (Ano gam), Anopheles darlingi (Ano dar), Aedes aegypti (Aed aeg), Drosophila pseudoobscura (Dro pse), Drosophila persimilis (Dro per), Drosophila mojavensis (Dro moj), Drosophila virilis (Dro vir), Drosophila grimshawi (Dro gri), Drosophila melanogaster (Dro mel), Drosophila sechellia (Dro sec), Drosophila simulans (Dro sim), Caenorhabditis remanei (Cae rem), Caenorhabditis brenneri (Cae bre), Caenorhabditis briggsae (Cae bri), Caenorhabditis elegans (Cae ele), Caenorhabditis japonica (Cae jap), Komagataella pastoris (Kom pas), Saccharomyces cerevisiae (Sac cer), and Schizosaccharomyces pombe (Sch pom). Protein sequences were then analyzed using ad hoc Perl scripts (www.perl.org). Standard eukaryotic phylogenies and phylogenetic distances were derived from www.timetree.org  and pairwise AAR combinations . Percent values [%X4 and %(X4+Z4)] were calculated by normalizing to the number of proteins in the proteome.For each proteome, we calculated the number of proteins containing a repeat of at least 4 units of each amino acid (X2 test with a Benjamini-Hochberg correction (FDR = 0.05). We also calculated all the possible pairwise repeats ratios (RR parameters) between %X4s  as an index of the relative occurrence of two AARs in a proteome (4+Z4)/%X4] and then normalizing this value to the size (%X4) of the group containing the given repeat [i.e. OV(X4+Z4) = %(X4+Z4)/(%X4)2; The statistical significance of the pairwise co-occurrence of AARs in each proteome was calculated by using the χproteome . Moreove4/X4) parameters, which all equal 1 and do not carry any signal for cluster analyses, and by the 20 OV(X4+X4) parameters whose value corresponds to 1/X4. These latter cases were also included in the cluster analyses as they carry some phylogenetic signal related to X4s. Cluster analyses were thus performed using all the 800 parameters . RRs and OVs in which a value at the denominator of a division operation was equal to 0 were assigned a null value of 0. The same approach was used to calculate the same set of parameters for sets of 20 random tetrapeptides (RND4), generated either by randomly reshuffling the 20 homopetides or by replacing the second, third, and fourth residue of each homopeptide with randomly selected amino acids  of 167 DPs and 167 control MPs were derived from BioGrid . MPs wer4, RR(X4/Z4), and OV(X4+Z4) parameters, or their mean values in taxa of interest, were used to define clusters of covarying phylogenetic profiles of AAR occurrence/cooccurrence across taxa, and clusters of taxa/species based on AAR occurrence/cooccurrence profiles, using Cluster 3.0 cluster association, as described in the Results section.We investigated the preferential associations of AARs with specific DP functions by determining whether protein sets associated with a specific developmental GO term, or with a cluster of semantically related GO terms, contained a significant overrepresentation of AAR-containing proteins, as assessed by means of χ2 tests on 2 × 2 contingency tables.To analyze dynamic phylogenetic changes of the preferential AARs/DP function associations, we focused on 5 representative GO terms  and two control terms . The Uniprot identifiers of proteins associated with these GO terms in species of interest were downloaded from Panther . The folP < 0.05 was considered as statistically significant in all instances. The Benjamini-Hochberg procedure , Prism (GraphPad), and SPSS 21 (IBM) software. Appropriate statistical tests were performed as indicated in the Results section and rocedure , where aGorilla gorilla by T. Michael Keesey (after Colin M. L. Burnett), Mus musculus by Daniel Jaron, Bos primigenius taurus by Steven Traver, Sauropsida by Nobu Tamura , and Eupercaria by Lily Hughes.Graphs and figures were generated using Excel (Microsoft), Igor Pro 6.1 (WaveMetrics Inc.), Cytoscape, Java TreeView , MyDomai4, where X is for any amino acid). We performed this count for all of the 20 amino acids , other contain pairwise or higher order combinations . Notably, a considerable number of pairwise AAR combinations are also highly overrepresented, from 2.9 to 20.8 times, with respect to the whole proteome . For the other four AARs, the differences were not statistically significant, likely due to the relatively low number of proteins in each group, although quite large absolute variations were evident in the percentage of these AARs across DP families.Among the DP families, the HOX, FOX, SOX, and POU proteins display higher degrees of overall AAR occurrence , combinations of two or more small, polar, and cyclic AARs are significantly more frequent in the antero-posterior group , charged , small , and cyclic (P) residues are differentially overrepresented in human DP families and subfamilies, in a combinatorial manner, with a high prevalence of pairwise and higher order AAR combinations.AARs can mediate homotypic interactions with other AARs of the same kind in other proteins, and heterotypic interactions with conventional protein–protein interaction (PPI) domains e.g. . Thus, iD) and their direct interactors (DI), and those between a control set of 167 metabolic proteins , randomly chosen among those involved in carbohydrate/lipid metabolism , and their interactors (MI). The average number of interactions formed by each protein in the two groups was comparable . We found that, unlike MPs, DPs have a considerably higher proportion of interactors containing A, E, G, H, P, Q, R, S repeats  of at least four residues in human protein sets associated with the development of anatomical (sub)systems and their parts  online.We obtained from the AmiGO database  sets of 2 tests for the protein sets within each (sub)cluster in order to detect enrichments of AAR-containing proteins in comparison with the whole proteome.We then performed χ2 value that is, to the degree of overrepresentation of proteins bearing a given AAR in them. These dots were grouped in rings representing each GO term cluster. This analysis highlighted how certain AARs are enriched in numerous protein sets involved in the development of some anatomical systems but not of others (2 tests with a Benjamini-Hochberg correction (FDR = 0.05). This analysis revealed complex, combinatorial patterns of AAR enrichment in protein sets associated with the development of specific anatomical systems and their parts/organs cluster association systems, we generated networks in which nodes represent AARs and GO term (sub)clusters connected by edges whose thickness is proportional to the χociation  online.The analysis of these data revealed four main features of the AAR functional associations.First, protein sets regulating the development of certain systems  are considerably more enriched with AARs and their combinations than protein sets associated with other systems .Second, some AARs are more broadly overrepresented in protein sets related to the development of multiple anatomical (sub)systems , while others appear to be involved in more limited functional associations with specific protein sets .Third, many associations of AARs with specific anatomical systems are combination-dependent. Thus, combinations of one same AAR with other AARs can be either positive or negative predictors of the association with a certain anatomical system. For example, polyA or polyP repeats are much more significantly associated with the nervous system when they are combined with polyG, while the opposite is true when they are combined with polyE.Fourth, a combination of two AARs  can be significantly overrepresented in protein sets associated with the development of a certain (sub)system , even when the two AARs individually are not significantly overrepresented in the same proteins.Taken together, these findings indicate that AARs in the human proteome have preferential, combinatorial associations with protein sets involved in the development of specific anatomical systems and/or their parts/organs.These findings raised the questions whether the combinatorial associations of AARs with subsets of DPs controlling the development of specific systems/organs that we observed in the human proteome, are phylogenetically conserved, and whether they can vary quantitatively throughout phylogenesis.Homo sapiens and in seven other species representative of major vertebrate taxa that diverged from Primates at progressively more distant times  and of two nondevelopmental GO terms  in P < 0.001 in all instances, χ2 test) in protein sets related to “pattern specification process” . These enrichments are both AAR- and GO term-specific. Thus, polyE and polyK proteins are underrepresented in proteins sets associated with the same two GO terms , or vice versa (G-A)  is increasingly more associated to “pattern specification process” from sa (G-A) B. SimilaB and C, right panels, Takifugu to Homo, while the opposite is true when polyA is combined with polyH  than when they do (A + E groups) , nematodes (nem), insects (ins), fishes (fis), birds and reptiles , metatherian  and eutherian mammals belonging to Laurasiatheria (lau), Rodentia (rod), and Primates . We indicate with %X4 each one of these percentages, where X is one of the 20 amino acids, and with m%X4 the mean %X4 in multiple species of one taxon. This analysis revealed distinctive phylogenetic trends in the occurrence of the 20 AAR types , and many have intermediate features between these clock-like and taxon-specific trends . Notably, changes in the occurrence of certain repeats sharply mark evolutionary transitions. Thus, %E4 has a neat biphasic profile marking the vertebrate/invertebrate divide  to yeast (y), whereas the second describes the tendency of AAR trends to display taxon-specific peaks. Strikingly, the data points of the 20 AARs have a quasi-circular distribution, indicating that AAR occurrences mostly vary according to graded combinations of the two main principal components.A PCA confirmed these results and revealed two major components to %Xvariance C. The fi4s further confirmed these findings. This analysis revealed two major clusters of covarying %X4s  were excluded to limit variability, the dendrogram reproduced an unrooted phylogenetic tree recapitulating the correct relationships between taxa , thus retaining the same overall amino acid composition of the homopolymeric tetrapeptides, or by adding three random amino acids after each one of the 20 amino acids (f–j), as a further control with a completely randomized amino acid composition.A cluster analysis of the m%Xing %X4s A, mar we 4; B–E,  online tThese findings revealed that the variations in the occurrence of the 20 AARs throughout phylogenesis are highly interrelated and carry a phylogenetic signal strong enough to detect the lineage relationships between major vertebrate and invertebrate taxa.Next, we extended the evolutionary analysis to AAR combinations. Toward this aim, we preliminarily analyzed the combinatorial landscape of AARs in species of representative eukaryotic taxa  online.P < 0.01 in all instances, χ2 test, FDR 0.05; Homo are also significant in five metazoan species and a core set of them also in yeast  significantly exceeds what expected by chance given the number of proteins bearing either one of the two repeats in the proteome. Of the 190 possible pairwise combinations of the 20 AARs, 40 are significantly overrepresented (in yeast B. Howeve4/%A4) in the whole proteome that is, the “repeat ratio” . The other expresses the co-occurrence of the two AARs in the same proteins , as the normalized overlap between the Q4 and the A4 protein groups  discriminate broadly vertebrates (>4) from invertebrates (<4), while thresholds of other RR/OV parameters identify more specific phylogenetic boundaries, even between closely related species . Interestingly, several RRs and OVs display a multiphasic behavior, and multiple threshold values of these parameters identify multiple taxa  or correlating with divergence times , correctly grouped them according to their actual phylogetic relationships .This phylogenetic signal further improved using a limited subset of RR and OV parameters (128 over 800) resulting from polyA/E/G/H/P/Q/R/S that is, those AARs most represented in DPs. Using this approach, the phylogenetic trees encompassing all taxa from yeast to humans, as well as the primate and Drosophila trees were all correctly solved  and in the combinatorial patterns of cooccurrence (OVs) of AARs, especially of those enriched in DPs, mark taxonomic differences throughout phylogenesis even at the level of closely related species.The results of our analyses indicate that AARs in DPs, DP interactomes, and proteomes display nonrandom combination patterns, functional associations, and interrelated phylogenetic dynamics. The observed combinatorial distribution and evolution of AARs in proteomes configure a system of markers of DP functions and evolutionary transitions, consistent with a generalized role of AARs as a whole system of regulatory sequences in developmental processes with evolutionary implications. These findings are novel and establish a quantitative and qualitative framework tracing the functional and evolutionary history of AARs in proteomes as a whole system of interrelated sequences, rather than sporadic functional regulators in some DPs. From this perspective, AARs appear to define a combinatorial regulatory system of specific developmental processes. Our findings also define a novel set of quantitative parameters (RR and OV) that mark evolutionary transitions.Our findings indicate that DPs pervasively contain AARs in variable combinations, with differential distributions across functionally distinct DP families and subfamilies, such as the anterior, central and posterior HOX genes. Also at the proteome level, we found evidence of preferential occurrences of AARs, and their combinations, in protein sets regulating specific developmental processes, ranging from embryo patterning to the morphogenesis of specific systems/organs. Overall, the differential occurrence of distinct AARs in functionally specialized DP families and subfamilies  and the androgen receptor  skeletal and/or neurodevelopmental syndromes, with endocrine dysfunction in some cases , and ourWe found extensive evidence of taxon- and species-specific changes in the relative occurrence (RR parameters) and combination (OV parameters) of AARs, which may signal overall rearrangements of DP interaction networks that contributed to the divergence of species/taxa. In fact, our analyses indicate that RR and OV parameters vary throughout phylogenesis marking a variety of taxonomic boundaries. Although some vary with monotonic trends, as clock-like parameters, many of them show distinctive taxonomic fluctuations marking specific taxa or major taxonomic divides. These compound AAR dynamics carry overall a considerable phylogenetic signal which is significantly stronger than that carried by random tetrapeptides. Notably, this signal is even stronger when considering only a subset of RR/OV parameters derived from those AARs enriched in DPs which may have a more direct influence on development.In principle, the observed phylogenetic dynamics of AARs may be attributed purely to the occurrence of neutral AAR-encoding indel mutations and genetic drift. While these processes will certainly have contributed to at least some of the observed patterns, existing evidence indicates that the origin of AAR evolutionary dynamics conceivably lies in a complex interplay of mutational and selective forces. AARs originate and expand/contract as a result of replication slippage or unequal crossing-over , leadingHuntley and Golding 2006Besides their contribution to the understanding of AAR functions, our findings may potentially be useful also in phylogenetic studies by providing sets of novel, alignment-free quantitative parameters that may help to resolve ambiguities in conventional phylogenetic analyses based on sequence alignments e.g. .The interrelated changes in the occurrence and combination of AARs that we observed throughout phylogenesis may represent evolutionary traces of AAR-mediated regulatory changes in developmental processes contributing to morphological and behavioral evolution.In yeast, recent evidence indicates that AAR variability increases evolvability also through the rewiring of protein interactomes . Our finhunchback in Drosophila, have been directly related to morphological and behavioral evolution , rather than their length variation  which allows additional levels of regulation .cis-regulatory gene regions (e.g., Thus, the structural/functional properties of AARs may ultimately underlie the modulation of the activity/interactions of DPs, consistent with an evolutionary paradigm that views mutations in the coding part of genes as important players in evolutionary processes  togetherns e.g., . In thisThe mutation rate of AAR-encoding repeats is considerably greater than for point mutations , and thuIn conclusion, our analyses define novel quantitative evidence and a proteome-wide interpretive framework supporting the notion of a combinatorial role of AARs as a system of regulatory sequences that mark functionally related DPs, and whose interrelated evolutionary dynamics signal evolutionary distances and transitions. These findings may provide critical guidance for the informed experimental dissection of the functional roles of specific AARs, and their combinations, in evolution and development.Genome Biology and Evolution online.evz216_Supplementary_DataClick here for additional data file.


There is considerable interest in tankyrase because of its potential use in cancer therapy. Tankyrase catalyzes the ADP-ribosylation of a variety of target proteins and regulates various cellular processes. The anti-cancer effects of tankyrase inhibitors are mainly due to their suppression of Wnt signaling and inhibition of telomerase activity, which are mediated by AXIN and TRF1 stabilization, respectively. In this review, we describe the underappreciated effects of another substrate, SH3 domain-binding protein 2 (SH3BP2). Specifically, SH3BP2 is an adaptor protein that regulates intracellular signaling pathways. Additionally, in the human genetic disorder cherubism, the gain-of-function mutations in SH3BP2 enhance osteoclastogenesis. The pharmacological inhibition of tankyrase in mice induces bone loss through the accumulation of SH3BP2 and the subsequent increase in osteoclast formation. These findings reveal the novel functions of tankyrase influencing bone homeostasis, and imply that tankyrase inhibitor treatments in a clinical setting may be associated with adverse effects on bone mass. The poly(ADP-ribose) polymerase (PARP) superfamily catalyzes the ADP-ribosylation of target proteins and regulates various cellular processes, including the maintenance of genome stability, mitosis, and signal transduction ,2. TankySeveral tankyrase substrates have been identified, including TRF1 ,9,10, AXOne of the thoroughly studied substrates is AXIN, which is a negative regulator of the canonical Wnt pathway . A previTankyrase has attracted attention as a novel and promising target for cancer treatments . Indeed,In addition, the mechanism of synthetic lethality should be noted. Cancers with mutation of BRCA1/2 are treatable by PARP inhibitors ,30,31. PBecause canonical Wnt signaling is an essential mediator of fibroblast activation and tissue fibrosis ,41, tank50 values of 95, 5, 74, and 27 nM, respectively, because of the highly conserved nicotinamide subsite among PARP family members [Tankyrase inhibitors have been classified into two types. Specifically, one type targets the nicotinamide subsite of the tankyrase protein, which is conserved in various PARPs, and the other type targets a unique adenosine subsite that is more potent and specific to tankyrase . Tankyra members ,47. ThusSH3BP2 gene is responsible for the human genetic disease cherubism (OMIM#118400) [SH3BP2 is an adaptor protein that is predominantly expressed in immune cells, including macrophages/osteoclasts and osteoblasts ,52,53,54#118400) . Cherubi#118400) ,62. Cher#118400) ) prevent#118400) ,64  ,56,57,58Sh3bp2 locus [The mechanisms underlying cherubism were elucidated by analyzing SH3BP2 cherubism mutant mice, in which the P416R mutation  was introduced in the murine p2 locus . Analysip2 locus . The mutp2 locus ,65,66,67p2 locus ,57. Addip2 locus ,68,69,70p2 locus .Poly(ADP-ribose) is expressed in bone tissue, especially in the nucleus and cytoplasm of bone cells , suggestBecause we previously reported the importance of SH3BP2 for bone homeostasis ,57, we eTankyrase inhibitors also affect osteoblast differentiation and maturation. Tankyrase inhibitor treatments can suppress Wnt signaling, as indicated by increased AXIN contents and the subsequent suppression of the nuclear translocation of β-catenin . We reveBone mass is maintained by balancing bone formation and resorption, which is mediated by osteoblasts and osteoclasts, respectively. To evaluate the in vivo effect of tankyrase inhibition, seven-week-old wild-type mice were orally administered G007-LK for four weeks . This trIn our in vivo experiment , the G00Apart from the roles of tankyrase in bone, it should be noted that there is some evidence that another member of the PARP family, PARP1, also affects bone homeostasis. In terms of osteoclastogenesis, PARP1 is reported to be a negative regulator. PARP1 suppresses Tcirg1 gene expression, which encodes the a3 isoform of the V-ATPase subunit, in murine pre-osteoclastic RAW264.7 cells . AdditioIn terms of osteoblasts, PARP1 has a promoting effect on osteoblast differentiation . During SH3BP2 promoter and regulates SH3BP2 gene expression [Sh3bp2 promoter activity and SH3BP2 protein expression are significantly suppressed [Regarding the effect of PARP1 on bone, SH3BP2 might act as a modifier of PARP1-mediated bone homeostasis, not just tankyrase-mediated bone homeostasis. PARP1 is reported to bind the human pression . In PARPppressed . Though Other than the effect of tankyrase inhibitors on bone, we might need to also pay attention to their effect on immune cells. As described in the previous section, SH3BP2 is ubiquitously expressed in various immune cells, such as macrophages and lymphocytes. Previous studies revealed that excessive amounts of SH3BP2 in macrophages enhance the production of inflammatory cytokines ,65,68 anTankyrase inhibitors may be toxic to the intestinal tract. When a tankyrase inhibitor (G-631) was administered in a murine xenograft colorectal cancer model, the inhibitor exhibited an anti-cancer effect, but it simultaneously induced severe intestinal toxicity, characterized by multifocal-regionally extensive necrotizing and ulcerative enteritis . Wnt sigTankyrase inhibitors are widely recognized as Wnt-specific/selective inhibitors because of their regulatory effects on AXIN . HoweverIn conclusion, the characterization of tankyrase-mediated cellular processes, especially the AXIN-regulated pathway, has resulted in advances in preclinical research on the utility of tankyrase inhibitors for treating cancers and fibrotic diseases. The next step will involve a more in-depth examination of the relevant cellular processes regulated by SH3BP2 and other substrates to enable researchers to predict and prevent avoidable adverse events associated with tankyrase inhibitors as well as to discover other potential therapeutic applications via modified tankyrase activity.


Plasmodium lifecycle two infectious stages of parasites, merozoites, and sporozoites, efficiently infect mammalian host cells, erythrocytes, and hepatocytes, respectively. The apical structure of merozoites and sporozoites contains rhoptry and microneme secretory organelles, which are conserved with other infective forms of apicomplexan parasites. During merozoite invasion of erythrocytes, some rhoptry proteins are secreted to form a tight junction between the parasite and target cell, while others are discharged to maintain subsequent infection inside the parasitophorous vacuole. It has been questioned whether the invasion mechanisms mediated by rhoptry proteins are also involved in sporozoite invasion of two distinct target cells, mosquito salivary glands and mammalian hepatocytes. Recently we demonstrated that rhoptry neck protein 2 (RON2), which is crucial for tight junction formation in merozoites, is also important for sporozoite invasion of both target cells. With the aim of comprehensively describing the mechanisms of sporozoite invasion, the expression and localization profiles of rhoptry proteins were investigated in Plasmodium berghei sporozoites. Of 12 genes representing merozoite rhoptry molecules, nine are transcribed in oocyst-derived sporozoites at a similar or higher level compared to those in blood-stage schizonts. Immuno-electron microscopy demonstrates that eight proteins, namely RON2, RON4, RON5, ASP/RON1, RALP1, RON3, RAP1, and RAMA, localize to rhoptries in sporozoites. It is noteworthy that most rhoptry neck proteins in merozoites are localized throughout rhoptries in sporozoites. This study demonstrates that most rhoptry proteins, except components of the high-molecular mass rhoptry protein complex, are commonly expressed in merozoites and sporozoites in Plasmodium spp., which suggests that components of the invasion mechanisms are basically conserved between infective forms independently of their target cells. Combined with sporozoite-stage specific gene silencing strategies, the contribution of rhoptry proteins in invasion mechanisms can be described.In the  During the malaria lifecycle, three invasive forms, ookinetes, sporozoites, and merozoites, invade different types of cells in mosquito vectors and mammalian hosts. Among them, sporozoites, which transmit malaria disease from mosquitoes to mammalian hosts, firstly invade mosquito salivary glands prior to be injected into the mammalian skin during a blood meal. Sporozoites actively migrate through the skin to enter blood vessels and finally infect hepatocytes, where they develop into several thousand merozoites within a parasitophorous vacuole membrane (PVM). Merozoites invade erythrocytes by similarly forming a PVM, while ookinetes, the invasive form which develops in the midgut lumen after fertilization, simply traverse midgut epithelial cells without PVM formation.Plasmodium merozoites and sporozoites which proliferate inside the PVM, and are absent in ookinetes, raising the possibility that rhoptry secretory proteins are involved in cell infection  promoter without any drug resistance gene  to obtain asexual stage parasites. To harvest mature schizonts, infected mouse erythrocytes with 0.5–1% parasitemia were cultured for 16 h and purified using Nycoprep 1.077 solution  mosquitoes were maintained on a 5% sucrose solution during adult stages at 25°C. After feeding on P. berghei infected ICR mice, fully engorged mosquitoes were selected and kept at 20°C until dissection under a 12 h-light/12 h-dark cycle. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Ehime University and the experiments were conducted according to the Ethical Guidelines for Animal Experiments of Ehime University.A transgenic ef1α (PBANKA_1133300) mRNA levels and were compared using the delta, delta-Ct method  were collected in RNAlater  and stored at 4°C until RNA isolation. Total RNA was extracted using an RNeasy kit  and treated with DNaseI (Qiagen). Reverse transcription was conducted using a PrimeScript RT reagent Kit  with gDNA Eraser. Real-time RT-PCR reactions were performed using SYBR Premix Ex Taq (Takara Bio). The primer sequences used are listed in  Pfaffl, .To generate transgenic parasites expressing a rhoptry protein tagged with c-Myc at its C-terminus, the native locus of the targeted rhoptry molecule in the WT-GFP genome was replaced by single crossover homologous recombination with an expression cassette of the C-terminus of the rhoptry protein fused with a c-Myc tag, similar to the generation of RON2-c-Myc expressing parasites -N1  at EcoRV and BamHI sites to produce recombinant ASP/RON1 proteins and at XhoI and BamHI sites to produce recombinant RON3 with GST tag at their N-terminus. The GST-tagged recombinant proteins were produced using the wheat germ cell-free protein expression system (CellFree Sciences) and purified using a glutathione-Sepharose 4B column  containing 5% 2-mercaptoethanol. Sporozoites collected from midguts of infected mosquitoes at days 24–26 post-feeding were purified by density gradient centrifugation using 17% Accudenz solution , 1:100 for anti-c-Myc rabbit antibodies (A-14), and 1:200 for anti RON2, ASP/RON1, and RAMA rabbit polyclonal antibodies) in blocking solution at 37°C for 2 h, followed by Alexa Fluor 488–conjugated goat anti-rabbit IgG and Alexa Fluor 568-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) at 37°C for 30 min. Nuclei were stained with 1 mg/ml of 4′,6-diamidino-2-phenylindole (DAPI). The samples were mounted in ProLong Gold antifade reagent (Thermo Fisher Scientific) and observed with an inverted fluorescence microscope .Cultivated schizonts of WT-GFP or transgenic parasites expressing c-Myc tagged rhoptry proteins were purified by density gradient centrifugation. Infected midguts (day 17 or 21 post-feeding) or salivary glands (day 24 or 26 post-feeding) were dissected. The samples were fixed in 1% paraformaldehyde, 0.2% glutaraldehyde and embedded in LR-White resin . Ultrathin sections were blocked in PBS containing 5% non-fat dry milk and 0.01% Tween 20 (PBS-MT), then incubated at 4°C overnight with anti-c-Myc antibody (Santa Cruz biotechnology) or specific antibodies against ASP/RON1 (1:25), RAMA (1:100), or RON3 (1:200). The sections were washed with PBS containing 0.4% Block Ace  and 0.01% Tween 20 (PBS-BT), and the grids were incubated for 1 h at 37°C with goat anti-rabbit IgG conjugated with 15 nm gold particles  diluted 1:20 in PBS-MT, rinsed with PBS-BT, and fixed in 2% glutaraldehyde for 10 min at 4°C to stabilize the gold particles. The sections were then stained with 2% uranyl acetate in 50% methanol and lead citrate. Samples were examined using a transmission electron microscope .ron2, PBANKA_1315700; ron4, PBANKA_0932000; ron5, PBANKA_0713100; ron6, PBANKA_0311700; ralp1, PBANKA_0619700; asp/ron1, PBANKA_1003600; rap1, PBANKA_1032100; ron3, PBANKA_1464900; rama, PBANKA_0804500; rhoph1a, PBANKA_1400600; rhoph2, PBANKA_0830200; and rhoph3, PBANKA_0416000.The sequence information of genes in this article can be found in the PlasmoDB database (PlasmoDB.org) under the following gene ID numbers: P. berghei ANKA strain expressing GFP under the control of ef1α promoter , and apical sushi protein (asp)/ron1) are also transcribed in sporozoites (rhoptry-associated protein 1 (rap1), ron3, and rhoptry associated membrane antigen (rama) are also transcribed in sporozoites . Ten transgenic parasite lines were successfully isolated. Specific antibodies against recombinant protein were prepared for ASP/RON1 and RAMA, since they are predicted to have C-terminal glycosylphosphatidylinositol (GPI) anchored domains  and rhoptry bulb proteins   using anti-c-Myc or specific antibodies  was performed using schizont stage merozoites and oocyst sporozoites. In k region . In addirozoites . RAMA isIn sporozoites formed inside oocysts, it was confirmed that three components for the RhopH complex do not accumulate in rhoptries, as expected from the observation of far less amounts of transcripts and proteins in sporozoites compared to merozoites . Other tP. berghei. Our results on mRNA expression in schizonts and sporozoites of rhoptry molecules are mostly confirmed by single cell RNA-seq analyses in P. berghei (Reid et al., P. yoelii, another rodent malaria parasite, are transcribed in oocyst-derived sporozoites (Tarun et al., Plasmodium falciparum were detected in salivary gland residing sporozoites (Lindner et al., Plasmodium species.In this study it is demonstrated that most rhoptry proteins reported in merozoites are expressed in sporozoites, with the exception of components of the RhopH complex. Together with three reported proteins, RAP2/3, RON11, and RON12 (Tufet-Bayona et al., Plasmodium, Toxoplasma, Babesia, and Cryptosporidium; however, proteins localized to the rhoptry bulb tend to be species specific. Since it has been proposed that rhoptry neck proteins, such as RON2, RON4, and RON5, are discharged to form the tight junction between parasites and host cells, this invasion mechanism could be conserved across the Apicomplexa phylum. Recently it was reported that the Toxoplasma genome contains paralogous genes for RON2 and AMA1, selectively expressed in sporozoites, suggesting that Toxoplasma has developed stage-specific invasion mechanisms (Poukchanski et al., Plasmodium genome contains only one set of genes encoding RON2 and AMA1, it would be interesting to examine whether the rhoptry neck protein complex is formed during sporozoite invasion of mosquito salivary glands and mammalian hepatocytes. In contrast, it has been revealed that many of the Toxoplasma rhoptry bulb proteins are involved in interaction with host signaling pathways (Lim et al., Plasmodium and Toxoplasma, the sporozoite-specific gene knockdown system by promoter swapping is a powerful tool to reveal functions of rhoptry proteins in sporozoites (Ishino et al., It is noteworthy that all examined commonly expressed rhoptry proteins localize to the neck region in merozoites, that is RON2, RON4, RON5, RALP1, ASP1/RON1, RON11, and RON12; while some rhoptry bulb proteins, RhopH1A, RhopH2, and RhopH3, are expressed predominantly in merozoites. The orthologs of proteins localized to the rhoptry neck are highly conserved across the infective stages of apicomplexan parasites, such as P. berghei (Bushell et al., The mRNA amounts of ASP/RON1 and RON3 are clearly greater in sporozoites than in schizonts. This suggests that ASP/RON1 and RON3 might mainly have roles or additional roles in sporozoites. Indeed, unlike other rhoptry proteins, ASP/RON1 is dispensable for parasite proliferation during the intraerythrocytic stage in Plasmodium proteins. The c-Myc tag, a peptide of 10 amino acids and ~1,200 Da, is relatively small and unlikely to interfere with protein folding and function following addition to the C-terminus of target proteins that do not possess a C-terminal GPI-anchor domain. All the transgenic parasite lines, in which native rhoptry proteins were replaced by c-Myc tagged rhoptry proteins, proliferate normally during the intra-erythrocytic stage. Considering that most rhoptry proteins are refractory to gene deletion due to their necessity for parasite proliferation in the blood stage (Tufet-Bayona et al., This is the first profiling data showing the expression and localization of rhoptry proteins in both merozoites and sporozoites by immuno-electron microscopy. By generation of transgenic parasite lines expressing c-Myc tagged rhoptry proteins, comprehensive localization analyses by immuno-electron microscopy could be performed using anti-c-Myc antibodies, thus overcoming the difficulty to obtain specific and high titer antibodies against P. berghei ANKA, as well as in P. falciparum, RON2, RON4, RON5, RALP1, and ASP /RON1 are localized to the rhoptry neck, while RAP1, RON3, RAMA, RhopH1A, RhopH2, and RhopH3 are localized to the rhoptry bulb. It has been proposed that sub-localization of rhoptry proteins reflects the order of discharge during merozoite invasion; that is, rhoptry neck proteins are secreted prior to invasion and followed by release of rhoptry bulb proteins (Zuccala et al., In merozoites of All datasets generated for this study are included in the manuscript/All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Ehime University and the experiments were conducted according to the Ethical Guidelines for Animal Experiments of Ehime University.NT, MTo, and TI conceived and designed the experiments. NT, MN, MTa, MB, KM, MTo, and TI performed the experiments and analyzed the data. MN, MTo, and TI wrote manuscript. NT, MTa, MB, KM, and TT critically reviewed it.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.