The mechanistic target of rapamycin (mTOR) is a master regulator of cell growth and metabolism, integrating environmental signals to regulate anabolic and catabolic processes, regulating lipid synthesis, growth factor-induced cell proliferation, cell survival, and migration. These activities are performed as part of two distinct complexes, mTORC1 and mTORC2, each with specific roles. mTORC1 and mTORC2 are elaborated dimeric structures formed by the interaction of mTOR with specific partners. mTOR functions only as part of these large complexes, but their assembly and activation require a dedicated and sophisticated chaperone system. mTOR folding and assembly are temporarily separated with the TELO2-TTI1-TTI2 (TTT) complex assisting the cotranslational folding of mTOR into a native conformation. Matured mTOR is then transferred to the R2TP complex for assembly of active mTORC1 and mTORC2 complexes. R2TP works in concert with the HSP90 chaperone to promote the incorporation of additional subunits to mTOR and dimerization. This review summarizes our current knowledge on how the HSP90-R2TP-TTT chaperone system facilitates the maturation and assembly of active mTORC1 and mTORC2 complexes, discussing interactions, structures, and mechanisms.

Human adenoviruses (HAdVs) are small DNA viruses that generally cause mild disease. Certain strains, particularly those belonging to species B HAdVs, can cause severe pneumonia and have a relatively high mortality rate. Little is known about the molecular aspects of how these highly pathogenic species affect the infected cell and how they suppress innate immunity. The present study provides molecular insights into how species B adenoviruses suppress the interferon signaling pathway. Our study shows that these viruses, unlike HAdV-C2, are resistant to type I interferon. This resistance likely arises due to the highly efficient suppression of interferon-stimulated gene expression. Unlike in HAdV-C2, HAdV-B7 and B14 sequester STAT2 and RNA polymerase II from interferon-stimulated gene promoters in infected cells. This results in suppressed interferon- stimulated gene activation. In addition, we show that RuvBL1 and RuvBL2, cofactors important for RNA polymerase II recruitment to promoters and interferon-stimulated gene activation, are redirected to the cytoplasm forming high molecular weight complexes that, likely, are unable to associate with chromatin. Proteomic analysis also identified key differences in the way these viruses affect the host cell, providing insights into species B-associated high pathogenicity. Curiously, we observed that at the level of protein expression changes to the infected cell, HAdV-C2 and B7 were more similar than those of the same species, B7 and B14. Collectively, our study represents the first such study of innate immune suppression by the highly pathogenic HAdV-B7 and B14, laying an important foundation for future investigations.IMPORTANCEHuman adenoviruses form a large family of double-stranded DNA viruses known for a variety of usually mild diseases. Certain strains of human adenovirus cause severe pneumonia leading to much higher mortality and morbidity than most other strains. The reasons for this enhanced pathogenicity are unknown. Our study provides a molecular investigation of how these highly pathogenic strains might inactivate the interferon signaling pathway, highlighting the lack of sensitivity of these viruses to type I interferon in general while providing a global picture of how viral changes in cellular proteins drive worse disease outcomes.

Macromolecular complexes play essential roles in various cellular processes. The assembly of macromolecular assemblies within the cell must overcome barriers imposed by a crowded cellular environment which is characterized by an estimated concentration of biological macromolecules amounting to 100-450 g/L that take up approximately 5-40% of the cytoplasmic volume. The formation of the macromolecular assemblies is facilitated by molecular chaperones in cooperation with their co-chaperones. The R2TP protein complex has emerged as a co-chaperone of Hsp90 that plays an important role in macromolecular assembly. The R2TP complex is composed of a heterodimer of RPAP3:P1H1DI that is in turn complexed to members of the ATPase associated with diverse cellular activities (AAA +), RUVBL1 and RUVBL2 (R1 and R2) families. What makes the R2TP co-chaperone complex particularly important is that it is involved in a wide variety of cellular processes including gene expression, translation, co-translational complex assembly, and posttranslational protein complex formation. The functional versatility of the R2TP co-chaperone complex makes it central to cellular development; hence, it is implicated in various human diseases. In addition, their roles in the development of infectious disease agents has become of interest. In the current review, we discuss the roles of these proteins as co-chaperones regulating Hsp90 and its partnership with Hsp70. Furthermore, we highlight the structure-function features of the individual proteins within the R2TP complex and describe their roles in various cellular processes.

DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigation involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA + family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries. In the present study, we identified DPCD as a new R1R2 partner in vivo. We show that DPCD interacts directly with R1 and R2 in vitro and in cells. We characterized the physico-chemical properties of DPCD in solution and built a 3D model of DPCD. In addition, we used a variety of orthogonal biophysical techniques including small-angle X-ray scattering, structural mass spectrometry and electron microscopy to assess the molecular determinants of DPCD interaction with R1R2. Interestingly, DPCD disrupts the dodecameric state of R1R2 complex upon binding and this interaction occurs mainly via the DII domains of R1R2.

NFAT5 is the only known mammalian tonicity-responsive transcription factor with an essential role in cellular adaptation to hypertonic stress. It is also implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity, but the underlying mechanisms remain elusive. Here, we demonstrate that NFAT5 enters the nucleus via the nuclear pore complex. We found that NFAT5 utilizes a unique nuclear localization signal (NFAT5-NLS) for nuclear import. siRNA screening revealed that only karyopherin β1 (KPNB1), but not karyopherin α, is responsible for the nuclear import of NFAT5 via direct interaction with the NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is driven by exportin-T (XPOT), where the process requires RuvB-like AAA-type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified an unconventional tonicity-dependent nucleocytoplasmic trafficking pathway for NFAT5 that represents a critical step in orchestrating rapid cellular adaptation to change in extracellular tonicity. These findings offer an opportunity for the development of novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with heat shock protein 90 (HSP90), functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, small nuclear ribonucleoprotein particles (snRNPs), and phosphatidylinositol 3-kinase (PI3K)-like kinases (PIKKs) such as TOR and SMG1. PIKK stabilization depends on an additional complex of TELO2, TTI1, and TTI2 (TTT), whose structure and function are poorly understood. The cryoelectron microscopy (cryo-EM) structure of the human R2TP-TTT complex, together with biochemical experiments, reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Taken together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.

The NS1 protein of influenza A virus (IAV) plays important roles in viral pathogenesis and host immune response. Through a proteomic approach, we have identified RuvB-like proteins 1 and 2 (RuvBL1 and RuvBL2) as interacting partners of the NS1 protein of IAVs. Infection of human lung A549 cells with A/PR/8/34 (PR8) virus resulted in reductions in the protein levels of RuvBL2 but not RuvBL1. Further studies with RuvBL2 demonstrated that the NS1-RuvBL2 interaction is RNA-independent, and RuvBL2 binds the RNA-binding domain of the NS1. Infection of interferon (IFN)-deficient Vero cells with wild-type or delNS1 PR8 virus reduced RuvBL2 protein levels and induced apoptosis; delNS1 virus caused more reductions in RuvBL2 protein levels and induced more apoptosis than did wild-type virus. Knockdown of RuvBL2 by siRNAs induced apoptosis and overexpression of RuvBL2 resulted in increased resistance to infection-induced apoptosis in Vero cells. These results suggest that a non-NS1 viral element or elements induce apoptosis by suppressing RuvBL2 protein levels, and the NS1 inhibits the non-NS1 viral element-induced apoptosis by maintaining RuvBL2 abundance in infected cells in the absence of IFN influence. In contrast to Vero cells, infection of IFN-competent A549 cells with PR8 virus caused reductions in RuvBL2 protein levels but did not induce apoptosis. Concomitantly, pretreatment of Vero cells with a recombinant IFN resulted in resistance to infection-induced apoptosis. These results demonstrate that the infection-induced, RuvBL2-regulated apoptosis in infected cells is counterbalanced by IFN survival signals. Our results reveal a novel mechanism underlying the infection-induced apoptosis that can be modulated by the NS1 and type I IFN signaling in IAV-infected cells.

Previous studies have shown that Reptin is overexpressed in hepatocellular carcinoma and that it is necessary for in vitro proliferation and cell survival. However, its pathophysiological role in vivo remains unknown. We aimed to study the role of Reptin in hepatocyte proliferation after regeneration using a liver Reptin knock-out model (ReptinLKO ). Interestingly, hepatocyte proliferation is strongly impaired in ReptinLKO mice 36 h after partial hepatectomy, associated with a decrease of cyclin-A expression and mTORC1 and MAPK signalling, leading to an impaired liver regeneration. Moreover, in the ReptinLKO model, we have observed a progressive loss of Reptin invalidation associated with an atypical liver regeneration. Hypertrophic and proliferative hepatocytes gradually replace ReptinKO hypotrophic hepatocytes. To conclude, our results show that Reptin is required for hepatocyte proliferation in vivo and liver regeneration and that it plays a crucial role in hepatocyte survival and liver homeostasis.

Adipocyte differentiation is an essential part of adipose tissue development, and is closely related to obesity and obesity-related diseases. In this study, we found that the expression of PPARγ, RUVBL2 and Adiponectin were concurrently obviously increased in the 5th-7th day of 3T3-L1 cell differentiation. PPARγ overexpression or the PPARγ activator facilitated Adiponectin trafficking and secretion and upregulated RUVBL2 expression as well as AS160 phosphorylation during adipogenic differentiation of 3T3-L1 cells. Consistently RUVBL2 overexpression also enhanced the polymerization and secretion of Adiponectin, in contrast, RUVBL2 knockdown reduced Adiponectin secretion. Further, PPARγ significantly enhanced RUVBL2 promoter activity and transcription. The progressive deletions and mutations of RUVBL2 promoter for PPARγ binding sites suggested that the PPARγ binding motif situated at -804/-781 bp is an essential component required for RUVBL2 promoter activity. Chromatin immunoprecipitation (ChIP) assays determined that PPARγ can directly interact with the RUVBL2 promoter DNA. Taken together, these data suggest that PPARγ promotes the expression, polymerization and secretion of Adiponectin by activating RUVBL2 transcriptionally, which accelerates 3T3-L1 cell differentiation.

Mutations in histone modifying enzymes and histone variants were identified in multiple cancers in The Cancer Genome Atlas (TCGA) studies. However, very little progress and understanding has been made in identifying the contribution of epigenetic factors in head and neck squamous cell carcinoma (HNSCC). Here, we report the identification of RUVBL1 (TIP49a), a component of the TIP60 histone modifying complex as being amplified and overexpressed in HNSCC. RUVBL1 plays a key role in incorporating histone variant H2AZ in chromatin thereby regulating transcription of key genes involved in differentiation, cancer cell proliferation and invasion. H2AZ is also overexpressed in HNSCC tumors thereby regulating RUVBL1/H2AZ dependent transcriptional programs. Patient data analysis of multiple cohorts including TCGA and single cell HNSCC data indicated RUVBL1 overexpression as a poor prognostic marker and predicts poor survival. In vitro experiments indicate a pro-proliferative role for RUVBL1/H2AZ in HNSCC cells. RUVBL1 inversely correlates with differentiation program and positively correlates with oncogenic programs, making it a key contributor to tumorigenesis and a vulnerable therapeutic target in HNSCC patients.