A conserved strategy for structure change and energy transduction in Hsp104 and other AAA+ protein motors.

The superfamily of massively large AAA+ protein molecular machines functions to convert the chemical energy of cytosolic ATP into physicomechanical form and use it to perform an extraordinary number of physical operations on proteins, nucleic acids, and membrane systems. Cryo-EM studies now reveal some aspects of substrate handling at high resolution, but the broader interpretation of AAA+ functional properties is still opaque. This paper integrates recent hydrogen exchange results for the typical AAA+ protein Hsp104 with prior information on several near and distantly related others. The analysis points to a widely conserved functional strategy. Hsp104 cycles through a long-lived loosely-structured energy-input "open" state that releases spent ADP and rebinds cytosolic ATP. ATP-binding energy is transduced by allosteric structure change to poise the protein at a high energy level in a more tightly structured "closed" state. The briefly occupied energy-output closed state binds substrate strongly and is catalytically active. ATP hydrolysis permits energetically downhill structural relaxation, which is coupled to drive energy-requiring substrate processing. Other AAA+ proteins appear to cycle through states that are analogous functionally if not in structural detail. These results revise the current model for AAA+ function, explain the structural basis of single-molecule optical tweezer kinetic phases, identify the separate energetic roles of ATP binding and hydrolysis, and specify a sequence of structural and energetic events that carry AAA+ proteins unidirectionally around a functional cycle to propel their diverse physical tasks.

Hsp100 Molecular Chaperone ClpB and Its Role in Virulence of Bacterial Pathogens.

This review focuses on the molecular chaperone ClpB that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and its biological function in selected bacterial pathogens, causing a variety of human infectious diseases, including zoonoses. It has been established that ClpB disaggregates and reactivates aggregated cellular proteins. It has been postulated that ClpB's protein disaggregation activity supports the survival of pathogenic bacteria under host-induced stresses (e.g., high temperature and oxidative stress), which allows them to rapidly adapt to the human host and establish infection. Interestingly, ClpB may also perform other functions in pathogenic bacteria, which are required for their virulence. Since ClpB is not found in human cells, this chaperone emerges as an attractive target for novel antimicrobial therapies in combating bacterial infections.

INO80 promotes H2A.Z occupancy to regulate cell fate transition in pluripotent stem cells.

The INO80 chromatin remodeler is involved in many chromatin-dependent cellular functions. However, its role in pluripotency and cell fate transition is not fully defined. We examined the impact of Ino80 deletion in the naïve and primed pluripotent stem cells. We found that Ino80 deletion had minimal effect on self-renewal and gene expression in the naïve state, but led to cellular differentiation and de-repression of developmental genes in the transition toward and maintenance of the primed state. In the naïve state, INO80 pre-marked gene promoters that would adopt bivalent histone modifications by H3K4me3 and H3K27me3 upon transition into the primed state. In the primed state, in contrast to its known role in H2A.Z exchange, INO80 promoted H2A.Z occupancy at these bivalent promoters and facilitated H3K27me3 installation and maintenance as well as downstream gene repression. Together, our results identified an unexpected function of INO80 in H2A.Z deposition and gene regulation. We showed that INO80-dependent H2A.Z occupancy is a critical licensing step for the bivalent domains, and thereby uncovered an epigenetic mechanism by which chromatin remodeling, histone variant deposition and histone modification coordinately control cell fate.

Fidgetin-like 2 negatively regulates axonal growth and can be targeted to promote functional nerve regeneration.

The microtubule (MT) cytoskeleton plays a critical role in axon growth and guidance. Here, we identify the MT-severing enzyme fidgetin-like 2 (FL2) as a negative regulator of axon regeneration and a therapeutic target for promoting nerve regeneration after injury. Genetic knockout of FL2 in cultured adult dorsal root ganglion neurons resulted in longer axons and attenuated growth cone retraction in response to inhibitory molecules. Given the axonal growth-promoting effects of FL2 depletion in vitro, we tested whether FL2 could be targeted to promote regeneration in a rodent model of cavernous nerve (CN) injury. The CNs are parasympathetic nerves that regulate blood flow to the penis, which are commonly damaged during radical prostatectomy (RP), resulting in erectile dysfunction (ED). Application of FL2-siRNA after CN injury significantly enhanced functional nerve recovery. Remarkably, following bilateral nerve transection, visible and functional nerve regeneration was observed in 7 out of 8 animals treated with FL2-siRNA, while no control-treated animals exhibited regeneration. These studies identify FL2 as a promising therapeutic target for enhancing regeneration after peripheral nerve injury and for mitigating neurogenic ED after RP - a condition for which, at present, only poor treatment options exist.

Four-pronged negative feedback of DSB machinery in meiotic DNA-break control in mice.

In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.

Progress and prospect of single-molecular ClpX ATPase researching system-a mini-review.

ClpXP in Escherichia coli is a proteasome degrading protein substrates. It consists of one hexamer of ATPase (ClpX) and two heptamers of peptidase (ClpP). The ClpX binds ATP and translocates the substrate protein into the ClpP chamber by binding and hydrolysis of ATP. At single molecular level, ClpX harnesses cycles of power stroke (dwell and burst) to unfold the substrates, then releases the ADP and Pi. Based on the construction and function of ClpXP, especially the recent progress on how ClpX unfold protein substrates, in this mini-review, a currently proposed single ClpX molecular model system detected by optical tweezers, and its prospective for the elucidation of the mechanism of force generation of ClpX in its power stroke and the subunit interaction with each other, were discussed in detail.

Oocyte Elimination Through DNA Damage Signaling from CHK1/CHK2 to p53 and p63.

Eukaryotic organisms have evolved mechanisms to prevent the accumulation of cells bearing genetic aberrations. This is especially crucial for the germline, because fecundity and fitness of progeny would be adversely affected by an excessively high mutational incidence. The process of meiosis poses unique problems for mutation avoidance because of the requirement for SPO11-induced programmed double-strand breaks (DSBs) in recombination-driven pairing and segregation of homologous chromosomes. Mouse meiocytes bearing unrepaired meiotic DSBs or unsynapsed chromosomes are eliminated before completing meiotic prophase I. In previous work, we showed that checkpoint kinase 2 (CHK2; CHEK2), a canonical DNA damage response protein, is crucial for eliminating not only oocytes defective in meiotic DSB repair (e.g., Trip13Gt mutants), but also Spo11-/- oocytes that are defective in homologous chromosome synapsis and accumulate a threshold level of spontaneous DSBs. However, rescue of such oocytes by Chk2 deficiency was incomplete, raising the possibility that a parallel checkpoint pathway(s) exists. Here, we show that mouse oocytes lacking both p53 (TRP53) and the oocyte-exclusive isoform of p63, TAp63, protects nearly all Spo11-/- and Trip13Gt/Gt oocytes from elimination. We present evidence that checkpoint kinase I (CHK1; CHEK1), which is known to signal to TRP53, also becomes activated by persistent DSBs in oocytes, and to an increased degree when CHK2 is absent. The combined data indicate that nearly all oocytes reaching a threshold level of unrepaired DSBs are eliminated by a semiredundant pathway of CHK1/CHK2 signaling to TRP53/TAp63.

CHTF18 ensures the quantity and quality of the ovarian reserve†.

The number and quality of oocytes, as well as the decline in both of these parameters with age, determines reproductive potential in women. However, the underlying mechanisms of this diminution are incompletely understood. Previously, we identified novel roles for CHTF18 (Chromosome Transmission Fidelity Factor 18), a component of the conserved Replication Factor C-like complex, in male fertility and gametogenesis. Currently, we reveal crucial roles for CHTF18 in female meiosis and oocyte development. Chtf18-/- female mice are subfertile and have fewer offspring beginning at 6 months of age. Consistent with age-dependent subfertility, Chtf18-/- ovaries contain fewer follicles at all stages of folliculogenesis than wild type ovaries, but the decreases are more significant at 3 and 6 months of age. By 6 months of age, both primordial and growing ovarian follicle pools are markedly reduced to near depletion. Chromosomal synapsis in Chtf18-/- oocytes is complete, but meiotic recombination is impaired resulting in persistent DNA double-strand breaks, fewer crossovers, and early homolog disjunction during meiosis I. Consistent with poor oocyte quality, the majority of Chtf18-/- oocytes fail to progress to metaphase II following meiotic resumption and a significant percentage of those that do progress are aneuploid. Collectively, our findings indicate critical functions for CHTF18 in ensuring both the quantity and quality of the mammalian oocyte pool.

Disrupting the association of Autographa californica multiple nucleopolyhedrovirus Ac93 with cellular ESCRT-III/Vps4 hinders nuclear egress of nucleocapsids and intranuclear microvesicles formation.

The endosomal sorting complex required for transport (ESCRT) pathway is required for efficient egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this study, we found that Ac93, a baculovirus core protein, contains a conserved MIM1-like motif. Alanine substitutions for six leucine residues in MIM1-like motif revealed that L142, L145, L146, and L149 are required for association of Ac93 with the MIT domain of Vps4. Mutations of these residues also blocked self-association and the association of Ac93 with ESCRT-III proteins or other viral core proteins Ac76 and Ac103, and resulted in a substantial reduction of infectious virus production, less efficient nuclear egress of progeny nucleocapsids, and the defect of intranuclear microvesicles formation. Combined with the localization of the association of Ac93 with ESCRT-III/Vps4 and other viral proteins at the nuclear membrane, we propose that the coordinated action of these viral proteins and ESCRT-III/Vps4 may be involved in remodeling the nuclear membrane.

A longitudinal study of retinopathy in the PEX1-Gly844Asp mouse model for mild Zellweger Spectrum Disorder.

Zellweger Spectrum Disorder (ZSD) is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly. Retinopathy leading to blindness is one of the major untreatable handicaps faced by patients with ZSD but is not well characterized, and the requirement for peroxisomes in retinal health is unknown. To address this, we examined the progression of retinopathy from 2 to 32 weeks of age in our murine model for the common human PEX1-p.Gly843Asp allele (PEX1-p.Gly844Asp) using electrophysiology, histology, immunohistochemistry, electron microscopy, biochemistry, and visual function tests. We found that retinopathy in male and female PEX1-G844D mice was marked by an attenuated cone function and abnormal cone morphology early in life, with gradually decreasing rod function. Structural defects at the inner retina occurred later in the form of bipolar cell degradation (between 13 and 32 weeks). Inner segment disorganization and enlarged mitochondria were seen at 32 weeks, while other inner retinal cells appeared preserved. Visual acuity was diminished by 11 weeks of age, while signal transmission from the retina to the brain was relatively intact from 7 to 32 weeks of age. Molecular analyses showed that PEX1-G844D is a subfunctional but stable protein, contrary to human PEX1-G843D. Finally, C26:0 lysophosphatidylcholine was elevated in the PEX1-G844D retina, while phopshoethanolamine plasmalogen lipids were present at normal levels. These characterization studies identify therapeutic endpoints for future preclinical trials, including improving or preserving the electroretinogram response, improving visual acuity, and/or preventing loss of bipolar cells.

SPG7 targets the m-AAA protease complex to process MCU for uniporter assembly, Ca2+ influx, and regulation of mitochondrial permeability transition pore opening.

The mitochondrial matrix ATPase associated with diverse cellular activities (m-AAA) protease spastic paraplegia 7 (SPG7) has been recently implicated as either a negative or positive regulatory component of the mitochondrial permeability transition pore (mPTP) by two research groups. To address this controversy, we investigated possible mechanisms that explain the discrepancies between these two studies. We found that loss of the SPG7 gene increased resistance to Ca2+-induced mPTP opening. However, this occurs independently of cyclophilin D (cyclosporine A insensitive) rather it is through decreased mitochondrial Ca2+ concentrations and subsequent adaptations mediated by impaired formation of functional mitochondrial Ca2+ uniporter complexes. We found that SPG7 directs the m-AAA complex to favor association with the mitochondrial Ca2+ uniporter (MCU) and MCU processing regulates higher order MCU-complex formation. The results suggest that SPG7 does not constitute a core component of the mPTP but can modulate mPTP through regulation of the basal mitochondrial Ca2+ concentration.

Knockdown of Fidgetin Improves Regeneration of Injured Axons by a Microtubule-Based Mechanism.

Fidgetin is a microtubule-severing protein that pares back the labile domains of microtubules in the axon. Experimental depletion of fidgetin results in elongation of the labile domains of microtubules and faster axonal growth. To test whether fidgetin knockdown assists axonal regeneration, we plated dissociated adult rat DRGs transduced using AAV5-shRNA-fidgetin on a laminin substrate with spots of aggrecan, a growth-inhibitory chondroitin sulfate proteoglycan. This cell culture assay mimics the glial scar formed after CNS injury. Aggrecan is more concentrated at the edge of the spot, such that axons growing from within the spot toward the edge encounter a concentration gradient that causes growth cones to become dystrophic and axons to retract or curve back on themselves. Fidgetin knockdown resulted in faster-growing axons on both laminin and aggrecan and enhanced crossing of axons from laminin onto aggrecan. Strikingly, axons from within the spot grew more avidly against the inhibitory aggrecan concentration gradient to cross onto laminin, without retracting or curving back. We also tested whether depleting fidgetin improves axonal regeneration in vivo after a dorsal root crush in adult female rats. Whereas control DRG neurons failed to extend axons across the dorsal root entry zone after injury, DRG neurons in which fidgetin was knocked down displayed enhanced regeneration of axons across the dorsal root entry zone into the spinal cord. Collectively, these results establish fidgetin as a novel therapeutic target to augment nerve regeneration and provide a workflow template by which microtubule-related targets can be compared in the future.SIGNIFICANCE STATEMENT Here we establish a workflow template from cell culture to animals in which microtubule-based treatments can be tested and compared with one another for their effectiveness in augmenting regeneration of injured axons relevant to spinal cord injury. The present work uses a viral transduction approach to knock down fidgetin from rat neurons, which coaxes nerve regeneration by elevating microtubule mass in their axons. Unlike previous strategies using microtubule-stabilizing drugs, fidgetin knockdown adds microtubule mass that is labile (rather than stable), thereby better recapitulating the growth status of a developing axon.

KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC.

During the evolution into castration or therapy resistance, prostate cancer cells reprogram the androgen responses to cope with the diminishing level of androgens, and undergo metabolic adaption to the nutritionally deprived and hypoxia conditions. AR (androgen receptor) and PKM2 (pyruvate kinase M2) have key roles in these processes. We report in this study, KDM8/JMJD5, a histone lysine demethylase/dioxygnase, exhibits a novel property as a dual coactivator of AR and PKM2 and as such, it is a potent inducer of castration and therapy resistance. Previously, we showed that KDM8 is involved in the regulation of cell cycle and tumor metabolism in breast cancer cells. Its role in prostate cancer has not been explored. Here, we show that KDM8's oncogenic properties in prostate cancer come from its direct interaction (1) with AR to affect androgen response and (2) with PKM2 to regulate tumor metabolism. The interaction with AR leads to the elevated expression of androgen response genes in androgen-deprived conditions. They include ANCCA/ATAD2 and EZH2, which are directly targeted by KDM8 and involved in sustaining the survival of the cells under hormone-deprived conditions. Notably, in enzalutamide-resistant cells, the expressions of both KDM8 and EZH2 are further elevated, so are neuroendocrine markers. Consequently, EZH2 inhibitors or KDM8 knockdown both resensitize the cells toward enzalutamide. In the cytosol, KDM8 associates with PKM2, the gatekeeper of pyruvate flux and translocates PKM2 into the nucleus, where the KDM8/PKM2 complex serves as a coactivator of HIF-1α to upregulate glycolytic genes. Using shRNA knockdown, we validate KDM8's functions as a regulator for both androgen-responsive and metabolic genes. KDM8 thus presents itself as an ideal therapeutic target for metabolic adaptation and castration-resistance of prostate cancer cells.

TRIP13 and APC15 drive mitotic exit by turnover of interphase- and unattached kinetochore-produced MCC.

The mitotic checkpoint ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), a soluble inhibitor of the anaphase-promoting complex/cyclosome (APC/C) produced by unattached kinetochores. MCC is also assembled during interphase by Mad1/Mad2 bound at nuclear pores, thereby preventing premature mitotic exit prior to kinetochore maturation and checkpoint activation. Using degron tagging to rapidly deplete the AAA+ ATPase TRIP13, we show that its catalytic activity is required to maintain a pool of open-state Mad2 for MCC assembly, thereby supporting mitotic checkpoint activation, but is also required for timely mitotic exit through catalytic disassembly of MCC. Strikingly, combining TRIP13 depletion with elimination of APC15-dependent Cdc20 ubiquitination/degradation results in a complete inability to exit mitosis, even when MCC assembly at unattached kinetochores is prevented. Thus, mitotic exit requires MCC produced either in interphase or mitosis to be disassembled by TRIP13-catalyzed removal of Mad2 or APC15-driven ubiquitination/degradation of its Cdc20 subunit.

RUVBL1, a novel C-RAF-binding protein, activates the RAF/MEK/ERK pathway to promote lung cancer tumorigenesis.

Lung cancer remains the leading cause of cancer-related deaths in the world. The RAF/MEK/ERK pathway controls many fundamental cellular functions and plays key roles in lung carcinogenesis. However, the proteins that regulate this pathway remain largely unknown. Here, we identified a novel C-RAF-binding protein, RUVBL1, which activates the RAF/MEK/ERK pathway by inhibiting phosphorylation of the C-RAF protein at serine 259. RUVBL1 expression was elevated in lung adenocarcinoma tissues. In addition, knocking out RUVBL1 effectively inhibited the proliferation and invasion of A549 cells. In vivo experiments, RUVBL1 deficiency significantly decreased the tumorigensis of lung cancer. In conclusion, we have shown that RUVBL1 could activate the RAF/MEK/ERK pathway by inhibiting phosphorylation of the C-RAF protein at serine 259, to promote lung cancer progression. Therefore, RUVBL1 could represent a novel therapeutic target for lung cancer treatment.

Liver Reptin/RUVBL2 controls glucose and lipid metabolism with opposite actions on mTORC1 and mTORC2 signalling.

OBJECTIVE: The AAA+ ATPase Reptin is overexpressed in hepatocellular carcinoma and preclinical studies indicate that it could be a relevant therapeutic target. However, its physiological and pathophysiological roles in vivo remain unknown. This study aimed to determine the role of Reptin in mammalian adult liver. DESIGN AND RESULTS: We generated an inducible liver-specific Reptin knockout (RepinLKO ) mouse model. Following Reptin invalidation, mice displayed decreased body and fat mass, hypoglycaemia and hypolipidaemia. This was associated with decreased hepatic mTOR protein abundance. Further experiments in primary hepatocytes demonstrated that Reptin maintains mTOR protein level through its ATPase activity. Unexpectedly, loss or inhibition of Reptin induced an opposite effect on mTORC1 and mTORC2 signalling, with: (1) strong inhibition of hepatic mTORC1 activity, likely responsible for the reduction of hepatocytes cell size, for decreased de novo lipogenesis and cholesterol transcriptional programmes and (2) enhancement of mTORC2 activity associated with inhibition of the gluconeogenesis transcriptional programme and hepatic glucose production. Consequently, the role of hepatic Reptin in the pathogenesis of insulin resistance (IR) and non-alcoholic fatty liver disease consecutive to a high-fat diet was investigated. We found that Reptin deletion completely rescued pathological phenotypes associated with IR, including glucose intolerance, hyperglycaemia, hyperlipidaemia and hepatic steatosis. CONCLUSION: We show here that the AAA +ATPase Reptin is a regulator of mTOR signalling in the liver and global glucido-lipidic homeostasis. Inhibition of hepatic Reptin expression or activity represents a new therapeutic perspective for metabolic syndrome.

Mitochondrial ATAD3A regulates milk biosynthesis and proliferation of mammary epithelial cells from dairy cow via the mTOR pathway.

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein, which is essential for cell growth and metabolism. The mechanism by which ATAD3A acts is still not fully understood. In this study, we explored the regulatory role of ATAD3A on milk biosynthesis and proliferation of bovine mammary epithelial cell. We showed that ATAD3A is localized in mitochondria and the expression of ATAD3A was up-regulated in response to extracellular stimuli such as amino acids and hormones. We observed that ATAD3A positively regulated milk protein, fat, and lactose biosynthesis, and cell proliferation. We further revealed that ATAD3A promoted the expressions of mTOR, SREBP-1c, and Cyclin D1, and triggers mTOR phosphorylation. In summary, our data reveal that ATAD3A regulates the mTOR, SREBP-1c, and Cyclin D1 signaling pathways for milk biosynthesis and cell proliferation.

Vacuolar Protein Sorting 4B (VPS4B) Regulates Apoptosis of Chondrocytes via p38 Mitogen-Activated Protein Kinases (MAPK) in Osteoarthritis.

To aim of this study is to investigate the expression of VPS4B (vacuolar protein sorting 4B) in articular cartilage with osteoarthritis (OA) and to analyze the relationship between VPS4B and chondrocyte apoptosis. We established an OA rat model by the MLI (meniscal/ligamentous injury) modeling method, and we observed the expression of VPS4B in articular cartilage through immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Human SW1353 chondrosarcoma cells were treated with IL-1ß to mimic the OA-like chondrocyte injury in vitro, and Western blot was employed to examine the IL-1ß-induced expression of VPS4B, phosphorylated p38, and apoptotic markers, namely active caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The co-localization of VPS4B and active caspase 3 was confirmed through immunofluorescence. We knocked down VPS4B expression through RNA interference. Western blot was carried out to detect the knockdown efficiency of VPS4B and evaluate its effects on IL-1ß-stimulated expression of apoptotic markers and phosphorylated p38 in SW1353 cells. Annexin V/propidium iodide (PI) staining was used to detect chondrocyte apoptosis. VPS4B expression was significantly upregulated in articular cartilage of OA rat model. IL-1ß stimulation increased the expression of VPS4B, apoptotic markers, and phosphorylated p38 in SW1353 cells. VPS4B co-localized with active caspase 3 in IL-1ß-treated SW1353 cells. VPS4B inhibition significantly reduced IL-1ß-stimulated expression of apoptotic markers and phosphorylated p38 in SW1353 cells. Moreover, flow cytometry assay demonstrated that VPS4B knockdown alleviated IL-1ß-induced apoptosis. Our results suggested that VPS4B might facilitate chondrocyte apoptosis in OA via p38 MAPK signaling pathway. This study may provide a novel insight into the pathophysiology of OA and a potential therapeutic target for its treatment.

Role of CCT chaperonin in the disassembly of mitotic checkpoint complexes.

The mitotic checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. When this checkpoint is active, a mitotic checkpoint complex (MCC), composed of the checkpoint proteins Mad2, BubR1, Bub3, and Cdc20, is assembled. MCC inhibits the ubiquitin ligase anaphase promoting complex/cyclosome (APC/C), whose action is necessary for anaphase initiation. When the checkpoint signal is turned off, MCC is disassembled, a process required for exit from checkpoint-arrested state. Different moieties of MCC are disassembled by different ATP-requiring processes. Previous work showed that Mad2 is released from MCC by the joint action of the TRIP13 AAA-ATPase and the Mad2-binding protein p31comet Now we have isolated from extracts of HeLa cells an ATP-dependent factor that releases Cdc20 from MCC and identified it as chaperonin containing TCP1 or TCP1-Ring complex (CCT/TRiC chaperonin), a complex known to function in protein folding. Bacterially expressed CCT5 chaperonin subunits, which form biologically active homooligomers [Sergeeva, et al. (2013) J Biol Chem 288(24):17734-17744], also promote the disassembly of MCC. CCT chaperonin further binds and disassembles subcomplexes of MCC that lack Mad2. Thus, the combined action of CCT chaperonin with that of TRIP13 ATPase promotes the complete disassembly of MCC, necessary for the inactivation of the mitotic checkpoint.

ATAD2 is an epigenetic reader of newly synthesized histone marks during DNA replication.

ATAD2 (ATPase family AAA domain-containing protein 2) is a chromatin regulator harboring an AAA+ ATPase domain and a bromodomain, previously proposed to function as an oncogenic transcription co-factor. Here we suggest that ATAD2 is also required for DNA replication. ATAD2 is co-expressed with genes involved in DNA replication in various cancer types and predominantly expressed in S phase cells where it localized on nascent chromatin (replication sites). Our extensive biochemical and cellular analyses revealed that ATAD2 is recruited to replication sites through a direct interaction with di-acetylated histone H4 at K5 and K12, indicative of newly synthesized histones during replication-coupled chromatin reassembly. Similar to ATAD2-depletion, ectopic expression of ATAD2 mutants that are deficient in binding to these di-acetylation marks resulted in reduced DNA replication and impaired loading of PCNA onto chromatin, suggesting relevance of ATAD2 in DNA replication. Taken together, our data show a novel function of ATAD2 in cancer and for the first time identify a reader of newly synthesized histone di-acetylation-marks during replication.