Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

The human AAA-ATPase VPS4A isoform and its co-factor VTA1 have a unique function in regulating mammalian cytokinesis abscission.

Mutations in the human AAA-ATPase VPS4 isoform, VPS4A, cause severe neurodevelopmental defects and congenital dyserythropoietic anemia (CDA). VPS4 is a crucial component of the endosomal sorting complex required for transport (ESCRT) system, which drives membrane remodeling in numerous cellular processes, including receptor degradation, cell division, and neural pruning. Notably, while most organisms encode for a single VPS4 gene, human cells have 2 VPS4 paralogs, namely VPS4A and VPS4B, but the functional differences between these paralogs is mostly unknown. Here, we set out to investigate the role of the human VPS4 paralogs in cytokinetic abscission using a series of knockout cell lines. We found that VPS4A and VPS4B hold both overlapping and distinct roles in abscission. VPS4A depletion resulted in a more severe abscission delay than VPS4B and was found to be involved in earlier stages of abscission. Moreover, VPS4A and a monomeric-locked VPS4A mutant bound the abscission checkpoint proteins CHMP4C and ANCHR, while VPS4B did not, indicating a regulatory role for the VPS4A isoform in abscission. Depletion of VTA1, a co-factor of VPS4, disrupted VPS4A-ANCHR interactions and accelerated abscission, suggesting that VTA1 is also involved in the abscission regulation. Our findings reveal a dual role for VPS4A in abscission, one that is canonical and can be compensated by VPS4B, and another that is regulatory and may be delivered by its monomeric form. These observations provide a potential mechanistic explanation for the neurodevelopmental defects and other related disorders reported in VPS4A-mutated patients with a fully functional VPS4B paralog.

Analysis of the Conformational Landscape of the N-Domains of the AAA ATPase p97: Disentangling the Continuous Conformational Variability in Partially Symmetrical Complexes.

Single-particle cryo-electron microscopy (cryo-EM) has been shown to be effective in defining the structure of macromolecules, including protein complexes. Complexes adopt different conformations and compositions to perform their biological functions. In cryo-EM, the protein complexes are observed in solution, enabling the recording of images of the protein in multiple conformations. Various methods exist for capturing the conformational variability through analysis of cryo-EM data. Here, we analyzed the conformational variability in the hexameric AAA + ATPase p97, a complex with a six-fold rotational symmetric core surrounded by six flexible N-domains. We compared the performance of discrete classification methods with our recently developed method, MDSPACE, which uses 3D-to-2D flexible fitting of an atomic structure to images based on molecular dynamics (MD) simulations. Our analysis detected a novel conformation adopted by approximately 2% of the particles in the dataset and determined that the N-domains of p97 sway by up to 60° around a central position. This study demonstrates the application of MDSPACE in analyzing the continuous conformational changes in partially symmetrical protein complexes, systems notoriously difficult to analyze due to the alignment errors caused by their partial symmetry.

PEX1 is essential for glycosome biogenesis and trypanosomatid parasite survival.

Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we functionally characterized a putative Trypanosoma PEX1 orthologue by bioinformatic and experimental approaches and show that it is a true PEX1 orthologue. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to the bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggest that these parasites possess a highly efficient quality control mechanism that exports the import receptors from glycosomes to the cytosol in the absence of a functional TbPEX1-TbPEX6 complex.

WRNIP1 prevents transcription-associated genomic instability.

R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.

Structural insights into the Clp protein degradation machinery.

The Clp protease system is important for maintaining proteostasis in bacteria. It consists of ClpP serine proteases and an AAA+ Clp-ATPase such as ClpC1. The hexameric ATPase ClpC1 utilizes the energy of ATP binding and hydrolysis to engage, unfold, and translocate substrates into the proteolytic chamber of homo- or hetero-tetradecameric ClpP for degradation. The assembly between the hetero-tetradecameric ClpP1P2 chamber and the Clp-ATPases containing tandem ATPase domains from the same species has not been studied in depth. Here, we present cryo-EM structures of the substrate-bound ClpC1:shClpP1P2 from Streptomyces hawaiiensis, and shClpP1P2 in complex with ADEP1, a natural compound produced by S. hawaiiensis and known to cause over-activation and dysregulation of the ClpP proteolytic core chamber. Our structures provide detailed information on the shClpP1-shClpP2, shClpP2-ClpC1, and ADEP1-shClpP1/P2 interactions, reveal conformational transition of ClpC1 during the substrate translocation, and capture a rotational ATP hydrolysis mechanism likely dominated by the D1 ATPase activity of chaperones.IMPORTANCEThe Clp-dependent proteolysis plays an important role in bacterial homeostasis and pathogenesis. The ClpP protease system is an effective drug target for antibacterial therapy. Streptomyces hawaiiensis can produce a class of potent acyldepsipeptide antibiotics such as ADEP1, which could affect the ClpP protease activity. Although S. hawaiiensis hosts one of the most intricate ClpP systems in nature, very little was known about its Clp protease mechanism and the impact of ADEP molecules on ClpP. The significance of our research is in dissecting the functional mechanism of the assembled Clp degradation machinery, as well as the interaction between ADEP1 and the ClpP proteolytic chamber, by solving high-resolution structures of the substrate-bound Clp system in S. hawaiiensis. The findings shed light on our understanding of the Clp-dependent proteolysis in bacteria, which will enhance the development of antimicrobial drugs targeting the Clp protease system, and help fighting against bacterial multidrug resistance.

Knockdown of hCINAP sensitizes colorectal cancer cells to ionizing radiation.

Colorectal cancer (CRC) poses a significant challenge in terms of treatment due to the prevalence of radiotherapy resistance. However, the underlying mechanisms responsible for radio-resistance in CRC have not been thoroughly explored. This study aimed to shed light on the role of human coilin interacting nuclear ATPase protein (hCINAP) in radiation-resistant HT-29 and SW480 CRC cells (HT-29-IR and SW480-IR) and investigate its potential implications. Firstly, radiation-resistant CRC cell lines were established by subjecting HT-29 and SW480 cells to sequential radiation exposure. Subsequent analysis revealed a notable increase in hCINAP expression in radiation-resistant CRC cells. To elucidate the functional role of hCINAP in radio-resistance, knockdown experiments were conducted. Remarkably, knockdown of hCINAP resulted in an elevation of reactive oxygen species (ROS) generation upon radiation treatment and subsequent activation of apoptosis mediated by mitochondria. These observations indicate that hCINAP depletion enhances the radiosensitivity of CRC cells. Conversely, when hCINAP was overexpressed, it was found to enhance the radio-resistance of CRC cells. This suggests that elevated hCINAP expression contributes to the development of radio-resistance. Further investigation revealed an interaction between hCINAP and ATPase family AAA domain containing 3A (ATAD3A). Importantly, ATAD3A was identified as an essential factor in hCINAP-mediated radio-resistance. These findings establish the involvement of hCINAP and its interaction with ATAD3A in the regulation of radio-resistance in CRC cells. Overall, the results of this study demonstrate that upregulating hCINAP expression may improve the survival of radiation-exposed CRC cells. Understanding the intricate molecular mechanisms underlying hCINAP function holds promise for potential strategies in targeted radiation therapy for CRC. These findings emphasize the importance of further research to gain a comprehensive understanding of hCINAP's precise molecular mechanisms and explore its potential as a therapeutic target in overcoming radio-resistance in CRC. By unraveling the complexities of hCINAP and its interactions, novel therapeutic approaches may be developed to enhance the efficacy of radiation therapy and improve outcomes for CRC patients.

The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity.

Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.

Methionine secreted by tumor-associated pericytes supports cancer stem cells in clear cell renal carcinoma.

Here, we identify a subset of vascular pericytes, defined by expression of platelet-derived growth factor receptor beta (PDGFR-ß) and G-protein-coupled receptor 91 (GPR91), that promote tumorigenesis and tyrosine kinase inhibitors (TKIs) resistance by functioning as the primary methionine source for cancer stem cells (CSCs) in clear cell renal cell carcinoma (ccRCC). Tumor-cell-derived succinate binds to GPR91 on pericyte to activate autophagy for methionine production. CSCs use methionine to create stabilizing N6-methyladenosine in ATPase-family-AAA-domain-containing 2 (ATAD2) mRNA, and the resulting ATAD2 protein complexes with SRY-box transcription factor 9 to assemble super enhancers and thereby dictate its target genes that feature prominently in CSCs. Targeting PDGFR-ß+GPR91+ pericytes with specific GRP91 antagonists reduce intratumoral methionine level, eliminate CSCs, and enhance TKIs sensitivity. These results unraveled the mechanisms by which PDGFR-ß+GPR91+ pericytes provide supportive niche for CSCs and could be used to develop targets for treating ccRCC.

AXL/WRNIP1 Mediates Replication Stress Response and Promotes Therapy Resistance and Metachronous Metastasis in HER2+ Breast Cancer.

Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that the nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2+ breast cancer cells that are resistant to HER2-targeted therapy and metastasize to the brain. In preclinical models, knocking down or pharmacologically inhibiting AXL or WRNIP1 attenuated protection of stalled replication forks. Furthermore, deficiency or inhibition of AXL and WRNIP1 also prolonged metastatic latency and delayed relapse. Together, these findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies. SIGNIFICANCE: Nuclear AXL and WRNIP1 interact and mediate replication stress response, promote therapy resistance, and support metastatic progression, indicating that targeting the AXL/WRNIP1 axis is a potentially viable therapeutic strategy for breast cancer.

Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM.

Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved 'DEAD-box' motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved.

Elevated cholesterol in ATAD3 mutants is a compensatory mechanism that leads to membrane cholesterol aggregation.

Aberrant cholesterol metabolism causes neurological disease and neurodegeneration, and mitochondria have been linked to perturbed cholesterol homeostasis via the study of pathological mutations in the ATAD3 gene cluster. However, whether the cholesterol changes were compensatory or contributory to the disorder was unclear, and the effects on cell membranes and the wider cell were also unknown. Using patient-derived cells, we show that cholesterol perturbation is a conserved feature of pathological ATAD3 variants that is accompanied by an expanded lysosome population containing membrane whorls characteristic of lysosomal storage diseases. Lysosomes are also more numerous in Drosophila neural progenitor cells expressing mutant Atad3, which exhibit abundant membrane-bound cholesterol aggregates, many of which co-localize with lysosomes. By subjecting the Drosophila Atad3 mutant to nutrient restriction and cholesterol supplementation, we show that the mutant displays heightened cholesterol dependence. Collectively, these findings suggest that elevated cholesterol enhances tolerance to pathological ATAD3 variants; however, this comes at the cost of inducing cholesterol aggregation in membranes, which lysosomal clearance only partly mitigates.

Germline specific genes increase DNA double-strand break repair and radioresistance in lung adenocarcinoma cells.

In principle, germline cells possess the capability to transmit a nearly unaltered set of genetic material to infinite future generations, whereas somatic cells are limited by strict growth constraints necessary to assure an organism's physical structure and eventual mortality. As the potential to replicate indefinitely is a key feature of cancer, we hypothesized that the activation of a "germline program" in somatic cells can contribute to oncogenesis. Our group recently described over one thousand germline specific genes that can be ectopically expressed in cancer, yet how germline specific processes contribute to the malignant properties of cancer is poorly understood. We here show that the expression of germ cell/cancer (GC) genes correlates with malignancy in lung adenocarcinoma (LUAD). We found that LUAD cells expressing more GC genes can repair DNA double strand breaks more rapidly, show higher rates of proliferation and are more resistant to ionizing radiation, compared to LUAD cells that express fewer GC genes. In particular, we identified the HORMA domain protein regulator TRIP13 to be predominantly responsible for this malignant phenotype, and that TRIP13 inhibition or expression levels affect the response to ionizing radiation and subsequent DNA repair. Our results demonstrate that GC genes are viable targets in oncology, as they induce increased radiation resistance and increased propagation in cancer cells. Because their expression is normally restricted to germline cells, we anticipate that GC gene directed therapeutic options will effectively target cancer, with limited side effects besides (temporary) infertility.

"ATAD3C regulates ATAD3A assembly and function in the mitochondrial membrane".

Mitochondrial ATAD3A is an ATPase Associated with diverse cellular Activities (AAA) domain containing enzyme, involved in the structural organization of the inner mitochondrial membrane and of increasing importance in childhood disease. In humans, two ATAD3A paralogs arose by gene duplication during evolution: ATAD3B and ATAD3C. Here we investigate the cellular activities of the ATAD3C paralog that has been considered a pseudogene. We detected unique ATAD3C peptides in HEK 293T cells, with expression similar to that in human tissues, and showed that it is an integral membrane protein that exposes its carboxy-terminus to the intermembrane space. Overexpression of ATAD3C, but not of ATAD3A, in fibroblasts caused a decrease in cell proliferation and oxygen consumption rate, and an increase of cellular ROS. This was due to the incorporation of ATAD3C monomers in ATAD3A complex in the mitochondrial membrane reducing its size. Consistent with a negative regulation of ATAD3A function in mitochondrial membrane organization, ATAD3C expression led to increased accumulation of respiratory chain dimeric CIII in the inner membrane, to the detriment to that assembled in respiratory supercomplexes. Our results demonstrate a negative dominant role of the ATAD3C paralog with implications for mitochondrial OXPHOS function and suggest that its expression regulates ATAD3A in the cell.

Abnormal Brain Protein Abundance and Cross-tissue mRNA Expression in Amyotrophic Lateral Sclerosis.

Due to the limitations of the present risk genes in understanding the etiology of amyotrophic lateral sclerosis (ALS), it is necessary to find additional causative genes utilizing novel approaches. In this study, we conducted a two-stage proteome-wide association study (PWAS) using ALS genome-wide association study (GWAS) data (N = 152,268) and two distinct human brain protein quantitative trait loci (pQTL) datasets (ROSMAP N = 376 and Banner N = 152) to identify ALS risk genes and prioritized candidate genes with Mendelian randomization (MR) and Bayesian colocalization analysis. Next, we verified the aberrant expression of risk genes in multiple tissues, including lower motor neurons, skeletal muscle, and whole blood. Six ALS risk genes (SCFD1, SARM1, TMEM175, BCS1L, WIPI2, and DHRS11) were found during the PWAS discovery phase, and SARM1 and BCS1L were confirmed during the validation phase. The following MR (p = 2.10 × 10-7) and Bayesian colocalization analysis (ROSMAP PP4 = 0.999, Banner PP4 = 0.999) confirmed the causal association between SARM1 and ALS. Further differential expression analysis revealed that SARM1 was markedly downregulated in lower motor neurons (p = 7.64 × 10-3), skeletal muscle (p = 9.34 × 10-3), and whole blood (p = 1.94 × 10-3). Our findings identified some promising protein candidates for future investigation as therapeutic targets. The dysregulation of SARM1 in multiple tissues provides a new way to explain ALS pathology.

IRX2 regulates endometrial carcinoma oncogenesis by transcriptional repressing RUVBL1.

Endometrial carcinoma (EC) is a rising concern among gynecological malignancies. Iroquois Homeobox 2 (IRX2), a member of the Iroquois homeobox gene family, demonstrates variable effects in different cancer types, emphasizing the need for extensive exploration of its involvement in EC progression. Utilizing TCGA and GEO databases, as well as performing immunohistochemistry (IHC) analysis on clinical samples, we assessed the expression levels of IRX2 and its promoter methylation in EC. To understand the functional roles of IRX2, we conducted various assays including in vitro CCK-8 assays, colony formation assays, cell invasion assays, and cell apoptosis assays. Moreover, we utilized in vivo subcutaneous xenograft mouse models. Additionally, we performed KEGG pathway and gene set enrichment analyses to gain insights into the underlying mechanisms. To validate the regulatory relationship between IRX2 and RUVBL1, we employed chromatin immunoprecipitation and luciferase reporter assays. Our results indicate significantly reduced levels of IRX2 expression in EC, correlating with higher histological grades, advanced clinical stages, and diminished overall survival. We observed that DNA methylation of the IRX2 promoter suppresses its expression in EC, with cg26333652 and cg11793269 playing critical roles as methylated sites. In contrast, ectopic overexpression of IRX2 substantially inhibits cell proliferation and invasion, and promotes cell apoptosis. Additionally, we discovered that IRX2 exerts negative regulation on the expression of RUVBL1, which is upregulated in EC and associated with a poorer prognosis. In conclusion, our findings indicate that decreased expression of IRX2 facilitates EC cell growth through the regulation of RUVBL1 expression, thereby contributing to the development of EC. Hence, targeting the IRX2-RUVBL1 axis holds promise as a potential therapeutic strategy for EC treatment.

Targeting of oncogenic AAA-ATPase TRIP13 reduces progression of pancreatic ductal adenocarcinoma.

Thyroid hormone receptor-interacting protein 13 (TRIP13) is involved in cancer progression, but its role in pancreatic ductal adenocarcinoma (PDAC) is unknown. Thus, we assessed the expression, functional role, and mechanism of action of TRIP13 in PDAC. We further examined the efficacy of TRIP13 inhibitor, DCZ0415, alone or in combination with gemcitabine on malignant phenotypes, tumor progression, and immune response. We found that TRIP13 was overexpressed in human PDACs relative to corresponding normal pancreatic tissues. TRIP13 knockdown or treatment of PDAC cells with DCZ0415 reduced proliferation and colony formation, and induced G2/M cell cycle arrest and apoptosis. Additionally, TRIP13 knockdown or targeting with DCZ0415 reduced the migration and invasion of PDAC cells by increasing E-cadherin and decreasing N-cadherin and vimentin. Pharmacologic targeting or silencing of TRIP13 also resulted in reduce expression of FGFR4 and STAT3 phosphorylation, and downregulation of the Wnt/ß-catenin pathway. In immunocompromised mouse models of PDAC, knockdown of TRIP13 or treatment with DCZ0415 reduced tumor growth and metastasis. In an immunocompetent syngeneic PDAC model, DCZ0415 treatment enhanced the immune response by lowering expression of PD1/PDL1, increasing granzyme B/perforin expression, and facilitating infiltration of CD3/CD4 T-cells. Further, DCZ0415 potentiated the anti-metastatic and anti-tumorigenic activities of gemcitabine by reducing proliferation and angiogenesis and by inducing apoptosis and the immune response. These preclinical findings show that TRIP13 is involved in PDAC progression and targeting of TRIP13 augments the anticancer effect of gemcitabine.

Identification of ATAD3A as a key regulator in non-small cell lung cancer by promoting STAT3-induced cell proliferation and tumor angiogenesis.

Malignant proliferation and abundant angiogenesis are major causes of lung adenocarcinoma (LUAD) with high morbidity and mortality. Therefore, the exploration of the key regulatory mechanisms of malignant proliferation and angiogenesis in LUAD provides an opportunity for the development of targeted precision therapy. In this study, we found that the high expression of ATPase family AAA domain-containing protein 3A (ATAD3A) in LUAD was positively associated with the poor survival of patients, while its high expression was positively associated with the angiogenesis of LUAD. Further knockdown of ATAD3A in LUAD significantly inhibited cell proliferation and suppressed expression of vascular endothelial growth factor A, FGF-2, ANG-1, and TGF-ß. The opposite effect was observed with ATAD3A overexpression. Furthermore, ATAD3A knockdown significantly inhibited tumor growth and angiogenesis in an in vivo subcutaneous xenograft tumor model. Mechanistic studies suggest that ATAD3A may promote signal transducer and activator of transcription 3 activation, a key signal regulating lung cancer cell proliferation and transcriptional secretion of proangiogenic factors. Therefore, targeted inhibition of ATAD3A may be an effective strategy for LUAD therapy, and ATAD3A may be a potential biomarker for predicting malignant progression.

Diverse nature of ClpX degradation motifs in Streptococcus mutans.

IMPORTANCE: Cytoplasmic Clp-related proteases play a major role in maintaining cellular proteome in bacteria. ClpX/P is one such proteolytic complex that is important for conserving protein homeostasis. In this study, we investigated the role of ClpX/P in Streptococcus mutans, an important oral pathogen. We identified several putative substrates whose cellular levels are regulated by ClpX/P in S. mutans and subsequently discovered several recognition motifs that are critical for degradation. Our study is the first comprehensive analysis of determining ClpX/P motifs in streptococci. We believe that identifying the substrates that are regulated by ClpX/P will enhance our understanding about virulence regulation in this important group of pathogens.

The N1 domain of the peroxisomal AAA-ATPase Pex6 is required for Pex15 binding and proper assembly with Pex1.

The heterohexameric ATPases associated with diverse cellular activities (AAA)-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motor's N-terminal domains or N terminally bound cofactors. Here, we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast, Saccharomyces cerevisiae. We found that although Pex1/ΔN1-Pex6 is an active ATPase in vitro, it does not support Pex1/Pex6 function at the peroxisome in vivo. An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N-terminal domains of PEX1, CDC48, and NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of cofactors and substrates with Pex1/Pex6 ATPase activity.