ABSTRACT
The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific to the leading-strand Pol (Polε). We reveal here the underlying mechanism of Ctf18-RFC specificity to Polε using cryo-electron microscopy and biochemical studies. We found that both Ctf18-RFC and Polε contain specific structural features that direct PCNA loading onto DNA. Unlike other clamp loaders, Ctf18-RFC has a disordered ATPase associated with a diverse cellular activities (AAA+) motor that requires Polε to bind and stabilize it for efficient PCNA loading. In addition, Ctf18-RFC can pry prebound Polε off of DNA, then load PCNA onto DNA and transfer the PCNA-DNA back to Polε. These elements in both Ctf18-RFC and Polε provide specificity in loading PCNA onto DNA for Polε.
Subject(s)
DNA Replication , Proliferating Cell Nuclear Antigen , Replication Protein C , Humans , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/chemistry , Cryoelectron Microscopy , DNA/chemistry , DNA/metabolism , DNA Polymerase II/metabolism , DNA Polymerase II/chemistry , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/chemistry , Nuclear Proteins , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/chemistry , Protein Binding , Replication Protein C/metabolism , Replication Protein C/chemistry , Protein DomainsABSTRACT
Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.
Subject(s)
DNA Damage , Histones , Nucleosomes , Histones/metabolism , Histones/chemistry , Nucleosomes/metabolism , Humans , Protein Binding , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Carrier Proteins/metabolism , Carrier Proteins/chemistry , Models, Molecular , ATPases Associated with Diverse Cellular Activities , DNA HelicasesABSTRACT
Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.
Subject(s)
ATPases Associated with Diverse Cellular Activities , DNA-Binding Proteins , Proliferating Cell Nuclear Antigen , Ubiquitin Thiolesterase , Ubiquitin-Specific Peptidase 7 , Ubiquitination , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/genetics , Humans , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin-Specific Peptidase 7/metabolism , Ubiquitin-Specific Peptidase 7/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Thiolester Hydrolases/metabolism , Thiolester Hydrolases/genetics , Ubiquitin/metabolism , DNA Damage , Protein Binding , Ubiquitin-Specific ProteasesABSTRACT
Genome replication is frequently impeded by highly stable DNA secondary structures, including G-quadruplex (G4) DNA, that can hinder the progression of the replication fork. Human WRNIP1 (Werner helicase Interacting Protein 1) associates with various components of the replication machinery and plays a crucial role in genome maintenance processes. However, its detailed function is still not fully understood. Here we show that human WRNIP1 interacts with G4 structures and provide evidence for its contribution to G4 processing. The absence of WRNIP1 results in elevated levels of G4 structures, DNA damage and chromosome aberrations following treatment with PhenDC3, a G4-stabilizing ligand. Additionally, we establish a functional and physical relationship between WRNIP1 and the PIF1 helicase in G4 processing. In summary, our results suggest that WRNIP1 aids genome replication and maintenance by regulating G4 processing and this activity relies on Pif1 DNA helicase.
Subject(s)
DNA Helicases , DNA Replication , G-Quadruplexes , Humans , DNA Helicases/metabolism , DNA Damage , Chromosome Aberrations , Carrier Proteins/metabolism , Carrier Proteins/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/geneticsABSTRACT
Enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles (cerebral ventriculomegaly), the cardinal feature of congenital hydrocephalus (CH), is increasingly recognized among patients with autism spectrum disorders (ASD). KATNAL2, a member of Katanin family microtubule-severing ATPases, is a known ASD risk gene, but its roles in human brain development remain unclear. Here, we show that nonsense truncation of Katnal2 (Katnal2Δ17) in mice results in classic ciliopathy phenotypes, including impaired spermatogenesis and cerebral ventriculomegaly. In both humans and mice, KATNAL2 is highly expressed in ciliated radial glia of the fetal ventricular-subventricular zone as well as in their postnatal ependymal and neuronal progeny. The ventriculomegaly observed in Katnal2Δ17 mice is associated with disrupted primary cilia and ependymal planar cell polarity that results in impaired cilia-generated CSF flow. Further, prefrontal pyramidal neurons in ventriculomegalic Katnal2Δ17 mice exhibit decreased excitatory drive and reduced high-frequency firing. Consistent with these findings in mice, we identified rare, damaging heterozygous germline variants in KATNAL2 in five unrelated patients with neurosurgically treated CH and comorbid ASD or other neurodevelopmental disorders. Mice engineered with the orthologous ASD-associated KATNAL2 F244L missense variant recapitulated the ventriculomegaly found in human patients. Together, these data suggest KATNAL2 pathogenic variants alter intraventricular CSF homeostasis and parenchymal neuronal connectivity by disrupting microtubule dynamics in fetal radial glia and their postnatal ependymal and neuronal descendants. The results identify a molecular mechanism underlying the development of ventriculomegaly in a genetic subset of patients with ASD and may explain persistence of neurodevelopmental phenotypes in some patients with CH despite neurosurgical CSF shunting.
Subject(s)
Cilia , Hydrocephalus , Microtubules , Animals , Female , Humans , Male , Mice , ATPases Associated with Diverse Cellular Activities/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/pathology , Autism Spectrum Disorder/metabolism , Cilia/metabolism , Cilia/pathology , Ependyma/metabolism , Ependyma/pathology , Hydrocephalus/genetics , Hydrocephalus/pathology , Hydrocephalus/metabolism , Katanin/metabolism , Katanin/genetics , Microtubules/metabolism , Neurons/metabolism , Pyramidal Cells/metabolism , Pyramidal Cells/pathologyABSTRACT
KEY MESSAGE: Interactor of WOX2, CDC48A, is crucial for early embryo patterning and shoot meristem stem cell initiation, but is not required for WOX2 protein turnover or subcellular localization. During Arabidopsis embryo patterning, the WUSCHEL HOMEOBOX 2 (WOX2) transcription factor is a major regulator of protoderm and shoot stem cell initiation. Loss of WOX2 function results in aberrant protodermal cell divisions and, redundantly with its paralogs WOX1, WOX3, and WOX5, compromised shoot meristem formation. To elucidate the molecular basis for WOX2 function, we searched for protein interactors by IP-MS/MS from WOX2-overexpression roots displaying reprogramming toward shoot-like cell fates. Here, we report that WOX2 directly interacts with the type II AAA ATPase molecular chaperone CELL DIVISION CYCLE 48A (CDC48A). We confirmed this interaction with bimolecular fluorescence complementation and co-immunoprecipitation and found that both proteins co-localize in the nucleus. We show that CDC48A loss of function results in protoderm and shoot meristem stem cell initiation defects similar to WOX2 loss of function. We also provide evidence that CDC48A promotes WOX2 activity independently of proteolysis or the regulation of nuclear localization, common mechanisms of CDC48A function in other processes. Our results point to a new role of CDC48A in potentiating WOX2 function during early embryo patterning.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Cell Cycle Proteins , Gene Expression Regulation, Plant , Homeodomain Proteins , Meristem , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/embryology , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Meristem/metabolism , Meristem/genetics , Meristem/embryology , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , Seeds/metabolism , Seeds/genetics , Seeds/growth & development , Plants, Genetically Modified , ATPases Associated with Diverse Cellular Activities , Transcription FactorsABSTRACT
The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across ß-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.
Subject(s)
Cytomegalovirus , Endosomal Sorting Complexes Required for Transport , Molecular Mimicry , Vacuolar Proton-Translocating ATPases , Virus Assembly , Humans , Endosomal Sorting Complexes Required for Transport/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Vacuolar Proton-Translocating ATPases/genetics , Cytomegalovirus/metabolism , Cytomegalovirus/genetics , Cytomegalovirus/physiology , Virus Assembly/physiology , Cytomegalovirus Infections/virology , Cytomegalovirus Infections/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Viral Proteins/metabolism , Viral Proteins/geneticsABSTRACT
Mitochondria are essential organelles of eukaryotic cells and thus mitochondrial proteome is under constant quality control and remodelling. Yme1 is a multi-functional protein and subunit of the homo-hexametric complex i-AAA proteinase. Yme1 plays vital roles in the regulation of mitochondrial protein homeostasis and mitochondrial plasticity, ranging from substrate degradation to the regulation of protein functions involved in mitochondrial protein biosynthesis, energy production, mitochondrial dynamics, and lipid biosynthesis and signalling. In this mini review, we focus on discussing the current understanding of the roles of Yme1 in mitochondrial protein import via TIM22 and TIM23 pathways, oxidative phosphorylation complex function, as well as mitochondrial lipid biosynthesis and signalling, as well as a brief discussion of the role of Yme1 in modulating mitochondrial dynamics.
Subject(s)
Mitochondria , Mitochondrial Dynamics , Mitochondrial Proteins , Oxidative Phosphorylation , Protein Transport , Proteostasis , Humans , Mitochondrial Proteins/metabolism , Mitochondria/metabolism , Animals , ATPases Associated with Diverse Cellular Activities/metabolism , Lipids/biosynthesis , Lipids/chemistry , Lipid Metabolism , Homeostasis , Signal Transduction , ATP-Dependent Proteases/metabolismABSTRACT
Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.
Subject(s)
Cockayne Syndrome , DNA Helicases , DNA Repair Enzymes , DNA Repair , Poly-ADP-Ribose Binding Proteins , RNA Polymerase II , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/genetics , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Humans , Animals , Mice , DNA Helicases/metabolism , DNA Helicases/genetics , Cockayne Syndrome/genetics , Cockayne Syndrome/metabolism , Transcription, Genetic , Phosphorylation , Casein Kinase II/metabolism , Casein Kinase II/genetics , Mice, Knockout , DNA Damage , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Chromatin/metabolism , Ubiquitination , Excision RepairABSTRACT
OBJECTIVE: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC). DESIGN: To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumours in mice. RESULTS: Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immunoevasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer. CONCLUSION: One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.
Subject(s)
ATPases Associated with Diverse Cellular Activities , Carcinoma, Pancreatic Ductal , DNA Helicases , Pancreatic Neoplasms , Proto-Oncogene Proteins c-myc , Animals , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Mice , Humans , DNA Helicases/genetics , DNA Helicases/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , Cell Line, Tumor , Carrier Proteins/metabolism , Carrier Proteins/geneticsABSTRACT
Exploring novel diagnostic and therapeutic biomarkers is extremely important for osteosarcoma. YME1 Like 1 ATPase (YME1L), locating in the mitochondrial inner membrane, is key in regulating mitochondrial plasticity and metabolic activity. Its expression and potential functions in osteosarcoma are studied in the present study. We show that YME1L mRNA and protein expression is significantly elevated in osteosarcoma tissues derived from different human patients. Moreover, its expression is upregulated in various primary and immortalized osteosarcoma cells. The Cancer Genome Atlas database results revealed that YME1L overexpression was correlated with poor overall survival and poor disease-specific survival in sarcoma patients. In primary and immortalized osteosarcoma cells, silencing of YME1L through lentiviral shRNA robustly inhibited cell viability, proliferation, and migration. Moreover, cell cycle arrest and apoptosis were detected in YME1L-silenced osteosarcoma cells. YME1L silencing impaired mitochondrial functions in osteosarcoma cells, causing mitochondrial depolarization, oxidative injury, lipid peroxidation and DNA damage as well as mitochondrial respiratory chain complex I activity inhibition and ATP depletion. Contrarily, forced YME1L overexpression exerted pro-cancerous activity and strengthened primary osteosarcoma cell proliferation and migration. YME1L is important for Akt-S6K activation in osteosarcoma cells. Phosphorylation of Akt and S6K was inhibited after YME1L silencing in primary osteosarcoma cells, but was strengthened with YME1L overexpression. Restoring Akt-mTOR activation by S473D constitutively active Akt1 mitigated YME1L shRNA-induced anti-osteosarcoma cell activity. Lastly, intratumoral injection of YME1L shRNA adeno-associated virus inhibited subcutaneous osteosarcoma xenograft growth in nude mice. YME1L depletion, mitochondrial dysfunction, oxidative injury, Akt-S6K inactivation, and apoptosis were detected in YME1L shRNA-treated osteosarcoma xenografts. Together, overexpressed YME1L promotes osteosarcoma cell growth, possibly by maintaining mitochondrial function and Akt-mTOR activation.
Subject(s)
Bone Neoplasms , Cell Proliferation , Mice, Nude , Osteosarcoma , Animals , Female , Humans , Male , Mice , Apoptosis/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Bone Neoplasms/genetics , Cell Line, Tumor , Cell Movement/genetics , Gene Expression Regulation, Neoplastic , Mice, Inbred BALB C , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Osteosarcoma/pathology , Osteosarcoma/genetics , Osteosarcoma/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.
Subject(s)
ATPases Associated with Diverse Cellular Activities , DNA-Binding Proteins , Histones , Lysine , Histones/metabolism , Histones/chemistry , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/chemistry , Humans , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/chemistry , Lysine/metabolism , Lysine/chemistry , Acetylation , Protein Processing, Post-Translational , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/chemistry , Protein Binding , Protein Domains , Models, Molecular , Binding SitesABSTRACT
The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how the folded substrate is bound to and released from Bcs1L has been unclear, and there has been ongoing debate as to whether subunits of Bcs1L act in sequence or in unison hydrolyzing ATP when moving the protein cargo. Here, we captured Bcs1L conformations by cryo-EM during active ATP hydrolysis in the presence or absence of ISP substrate. In contrast to the threading mechanism widely employed by AAA proteins in substrate translocation, subunits of Bcs1L alternate uniformly between ATP and ADP conformations without detectable intermediates that have different, co-existing nucleotide states, indicating that the subunits act in concert. We further show that the ISP can be trapped by Bcs1 when its subunits are all in the ADP-bound state, which we propose to be released in the apo form.
Subject(s)
ATPases Associated with Diverse Cellular Activities , Electron Transport Complex III , Humans , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/chemistry , Cryoelectron Microscopy , Electron Transport Complex III/metabolism , Electron Transport Complex III/chemistry , Hydrolysis , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/chemistry , Models, Molecular , Protein Conformation , Protein Folding , Protein TransportABSTRACT
The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique ß-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.
Subject(s)
ATPases Associated with Diverse Cellular Activities , Cryoelectron Microscopy , Nuclear Proteins , Proliferating Cell Nuclear Antigen , Replication Protein C , Humans , Cryoelectron Microscopy/methods , DNA/metabolism , DNA/chemistry , DNA Replication , Models, Molecular , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/chemistry , Protein Binding , Replication Protein C/metabolism , Replication Protein C/chemistryABSTRACT
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.
Subject(s)
ATPases Associated with Diverse Cellular Activities , Cytokinesis , Endosomal Sorting Complexes Required for Transport , Vacuolar Proton-Translocating ATPases , Humans , Endosomal Sorting Complexes Required for Transport/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Vacuolar Proton-Translocating ATPases/metabolism , Vacuolar Proton-Translocating ATPases/genetics , HeLa Cells , Protein Isoforms/metabolism , Protein Isoforms/geneticsABSTRACT
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.
Subject(s)
Hydrazines , Kidney Neoplasms , Triazoles , Wilms Tumor , Humans , Exportin 1 Protein , Active Transport, Cell Nucleus , Karyopherins/genetics , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Cell Line, Tumor , Apoptosis , Neoplasm Recurrence, Local , Doxorubicin/pharmacology , Wilms Tumor/drug therapy , Wilms Tumor/genetics , Kidney Neoplasms/drug therapy , Kidney Neoplasms/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , Cell Cycle Proteins/metabolismABSTRACT
Radiotherapy effectiveness in breast cancer is limited by radioresistance. Nevertheless, the mechanisms behind radioresistance are not yet fully understood. RUVBL1 and RUVBL2, referred to as RUVBL1/2, are crucial AAA+ ATPases that act as co-chaperones and are connected to cancer. Our research revealed that RUVBL1, also known as pontin/TIP49, is excessively expressed in MMTV-PyMT mouse models undergoing radiotherapy, which is considered a murine spontaneous breast-tumor model. Our findings suggest that RUVBL1 enhances DNA damage repair and radioresistance in breast cancer cells both in vitro and in vivo. Mechanistically, we discovered that DTL, also known as CDT2 or DCAF2, which is a substrate adapter protein of CRL4, promotes the ubiquitination of RUVBL1 and facilitates its binding to RUVBL2 and transcription cofactor ß-catenin. This interaction, in turn, attenuates its binding to acetyltransferase Tat-interacting protein 60 (TIP60), a comodulator of nuclear receptors. Subsequently, ubiquitinated RUVBL1 promotes the transcriptional regulation of RUVBL1/2-ß-catenin on genes associated with the non-homologous end-joining (NHEJ) repair pathway. This process also attenuates TIP60-mediated H4K16 acetylation and the homologous recombination (HR) repair process. Expanding upon the prior study's discoveries, we exhibited that the ubiquitination of RUVBL1 by DTL advances the interosculation of RUVBL1/2-ß-catenin. And, it then regulates the transcription of NHEJ repair pathway protein. Resulting in an elevated resistance of breast cancer cells to radiation therapy. From the aforementioned, it is evident that targeting DTL-RUVBL1/2-ß-catenin provides a potential radiosensitization approach when treating breast cancer.
Subject(s)
Mammary Neoplasms, Animal , beta Catenin , Animals , Mice , ATPases Associated with Diverse Cellular Activities/genetics , beta Catenin/genetics , DNA Helicases/genetics , Gene Expression Regulation , Ubiquitin , Ubiquitination , Nuclear ProteinsABSTRACT
INTRODUCTION: Heimler syndrome (HS) is a rare disorder that includes sensorineural hearing loss (SNHL), nail abnormalities, and enamel hypoplasia. Patients with this syndrome can also exhibit ocular manifestations. At present, only a few cases of HS have been reported, existing knowledge of this syndrome is limited, and many cases have been misdiagnosed or even missed. This is the first report of Heimler syndrome with blurred vision as the first complaint, which was diagnosed by genetic analysis in the ophthalmology department. CASE DESCRIPTION: An 8-year-old girl complained of bilateral visual blur and night blindness from birth. Ophthalmic examinations revealed bilateral retinitis pigmentosa with cystoid macular edema, visual impairment with hyperopia and astigmatism. Hearing test revealed bilateral severe sensorineural hearing loss. Dental examinations revealed enamel hypoplasia. In addition, whole-exome sequencing (WES) identified two pathogenic variants in PEX1: the previously reported missense variant c.2966T > C (p.I989â T), and the novel frameshift variant c.1671_1672del (p.G558Sfs*33). CONCLUSION: Heimler syndrome is caused by compound heterozygous PEX1 pathogenic variants, c.2966T > C (p.I989â T) and c.1671_1672del (p.G558Sfs*33), which contributed to the diversity of clinical and genetic profiles in this patient. The main clinical manifestations include bilateral retinitis pigmentosa with cystoid macular edema, sensorineural hearing loss, and enamel hypoplasia. Systemic examinations are suggested for patients suspected of having pigmentary retinal dystrophy, especially combined with hearing-related impairments. Genetic testing can help us to make a definitive diagnosis.
Subject(s)
Amelogenesis Imperfecta , Hearing Loss, Sensorineural , Nails, Malformed , Humans , Female , Child , Hearing Loss, Sensorineural/genetics , Hearing Loss, Sensorineural/diagnosis , Nails, Malformed/genetics , Nails, Malformed/diagnosis , Amelogenesis Imperfecta/genetics , Amelogenesis Imperfecta/diagnosis , Visual Acuity , Heterozygote , DNA Mutational Analysis , Exome Sequencing , Tomography, Optical Coherence , Vision Disorders/genetics , Vision Disorders/diagnosis , Membrane Proteins/genetics , DNA/genetics , ATPases Associated with Diverse Cellular ActivitiesABSTRACT
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.
Subject(s)
ATPases Associated with Diverse Cellular Activities , Apoptosis , Colorectal Neoplasms , Gene Knockdown Techniques , Radiation Tolerance , Reactive Oxygen Species , Humans , Colorectal Neoplasms/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/radiotherapy , Radiation Tolerance/genetics , Apoptosis/radiation effects , Apoptosis/genetics , Reactive Oxygen Species/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Cell Line, Tumor , Radiation, Ionizing , Mitochondria/metabolism , Mitochondria/radiation effects , HT29 CellsABSTRACT
Proliferating cell nuclear antigen (PCNA) is a eukaryotic replicative DNA clamp. Furthermore, DNA-loaded PCNA functions as a molecular hub during DNA replication and repair. PCNA forms a closed homotrimeric ring that encircles the DNA, and association and dissociation of PCNA from DNA are mediated by clamp-loader complexes. PCNA must be actively released from DNA after completion of its function. If it is not released, abnormal accumulation of PCNA on chromatin will interfere with DNA metabolism. ATAD5 containing replication factor C-like complex (RLC) is a PCNA-unloading clamp-loader complex. ATAD5 deficiency causes various DNA replication and repair problems, leading to genome instability. Here, we review recent progress regarding the understanding of the action mechanisms of PCNA unloading complex in DNA replication/repair pathways.