Composition and activity of the non-canonical Gram-positive SecY2 complex

The accessory Sec system in Streptococcus gordonii DL1 is a specialized export system that transports a large serine-rich repeat protein, Hsa, to the bacterial surface. The system is composed of core proteins SecA2 and SecY2 and accessory Sec proteins Asp1–Asp5. Similar to canonical SecYEG, SecY2 forms a channel for translocation of the Hsa adhesin across the cytoplasmic membrane. Accessory Sec proteins Asp4 and Asp5 have been suggested to work alongside SecY2 to form the translocon, similar to the associated SecY, SecE, and SecG of the canonical system (SecYEG). To test this theory, S. gordonii secY2, asp4, and asp5 were co-expressed in Escherichia coli. The resultant complex was subsequently purified, and its composition was confirmed by mass spectrometry to be SecY2-Asp4-Asp5. Like SecYEG, the non-canonical complex activates the ATPase activity of the SecA motor (SecA2). This study also shows that Asp4 and Asp5 are necessary for optimal adhesion of S. gordonii to glycoproteins gp340 and fibronectin, known Hsa binding partners, as well as for early stage biofilm formation. This work opens new avenues for understanding the structure and function of the accessory Sec system

Phosphorylation-dependent BRD4 dimerization and implications for therapeutic inhibition of BET family proteins.

Funder: AstraZenecaFunder: AstraZeneca postdoc fundBromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains. Phosphorylation by casein kinase II (CK2) regulates BRD4 function, is necessary for active transcription and is involved in resistance to BRD4 drug inhibition in triple-negative breast cancer. Here, we provide the first biophysical analysis of BRD4 phospho-regulation. Using integrative structural biology, we show that phosphorylation by CK2 modulates the dimerization of human BRD4. We identify two conserved regions, a coiled-coil motif and the Basic-residue enriched Interaction Domain (BID), essential for the BRD4 structural rearrangement, which we term the phosphorylation-dependent dimerization domain (PDD). Finally, we demonstrate that bivalent inhibitors induce a conformational change within BRD4 dimers in vitro and in cancer cells. Our results enable the proposal of a model for BRD4 activation critical for the characterization of its protein-protein interaction network and for the development of more specific therapeutics

Nucleocytoplasmic mRNA redistribution accompanies RNA binding protein mislocalization in ALS motor neurons and is restored by VCP ATPase inhibition

Amyotrophic lateral sclerosis (ALS) is characterized by nucleocytoplasmic mislocalization of the RNA-binding protein (RBP) TDP-43. However, emerging evidence suggests more widespread mRNA and protein mislocalization. Here, we employed nucleocytoplasmic fractionation, RNA sequencing, and mass spectrometry to investigate the localization of mRNA and protein in induced pluripotent stem cell-derived motor neurons (iPSMNs) from ALS patients with TARDBP and VCP mutations. ALS mutant iPSMNs exhibited extensive nucleocytoplasmic mRNA redistribution, RBP mislocalization, and splicing alterations. Mislocalized proteins exhibited a greater affinity for redistributed transcripts, suggesting a link between RBP mislocalization and mRNA redistribution. Notably, treatment with ML240, a VCP ATPase inhibitor, partially restored mRNA and protein localization in ALS mutant iPSMNs. ML240 induced changes in the VCP interactome and lysosomal localization and reduced oxidative stress and DNA damage. These findings emphasize the link between RBP mislocalization and mRNA redistribution in ALS motor neurons and highlight the therapeutic potential of VCP inhibition

Multidimensional Dynamics of the Proteome in the Neurodegenerative and Aging Mammalian Brain

Neurodegenerative diseases are characterized by the abnormal accumulation of aggregated proteins in the brain. Using in vivo pulse isotope labeling, we screened the proteome for changes in protein turnover and abundance in multiple mouse models of neurodegeneration. These data suggest that the disease state of pathologically affected tissue is characterized by a proteome-wide increase in protein turnover and repair. In contrast, in healthy wild-type mice, aging in the mammalian brain is associated with a global slowdown in protein turnover.Peer reviewe

RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1–RUVBL2–RPAP3–PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, necessary for human R2TP assembly but absent from yeast. The mobile TPR domains of RPAP3 map to the opposite face of the ring, associating with PIH1D1, which mediates client protein recruitment. Thus, RPAP3 provides a flexible platform for bringing HSP90 into proximity with diverse client proteins

CryoEM of RUVBL1-RUVBL2-ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8.

Biogenesis of the U5 small nuclear ribonucleoprotein (snRNP) is an essential and highly regulated process. In particular, PRPF8, one of U5 snRNP main components, requires HSP90 working in concert with R2TP, a cochaperone complex containing RUVBL1 and RUVBL2 AAA-ATPases, and additional factors that are still poorly characterized. Here, we use biochemistry, interaction mapping, mass spectrometry and cryoEM to study the role of ZNHIT2 in the regulation of the R2TP chaperone during the biogenesis of PRPF8. ZNHIT2 forms a complex with R2TP which depends exclusively on the direct interaction of ZNHIT2 with the RUVBL1-RUVBL2 ATPases. The cryoEM analysis of this complex reveals that ZNHIT2 alters the conformation and nucleotide state of RUVBL1-RUVBL2, affecting its ATPase activity. We characterized the interactions between R2TP, PRPF8, ZNHIT2, ECD and AAR2 proteins. Interestingly, PRPF8 makes a direct interaction with R2TP and this complex can incorporate ZNHIT2 and other proteins involved in the biogenesis of PRPF8 such as ECD and AAR2. Together, these results show that ZNHIT2 participates in the assembly of the U5 snRNP as part of a network of contacts between assembly factors required for PRPF8 biogenesis and the R2TP-HSP90 chaperone, while concomitantly regulating the structure and nucleotide state of R2TP.Agencia Estatal de Investigación (AEI/10.13039/501100011033), Ministerio de Ciencia e Innovación and co-funded by the European Regional Development Fund (ERDF-UE) [SAF2017-82632-P and PID2020-114429RB-I00 to O.L.]; Autonomous Region of Madrid and co-funded by the European Social Fund and the European Regional Development Fund [Y2018/BIO4747 and P2018/NMT4443 to O.L., and which support the contracts of S.C. and A.G-C.]; Funding for open access charge: Agencia Estatal de Investigación (AEI/10.13039/501100011033), Ministerio de Ciencia e Innovación, co-funded by the European Regional Development Fund (ERDF-UE) [SAF2017-82632-P to O.L.]; S.C. contract is funded by the CNIO Friends Program philanthropic initiative since June 2021.S

Trivalent RING assembly on retroviral capsids activates TRIM5 ubiquitination and innate immune signaling

Summary: TRIM5 is a RING domain E3 ubiquitin ligase with potent antiretroviral function. TRIM5 assembles into a hexagonal lattice on retroviral capsids, causing envelopment of the infectious core. Concomitantly, TRIM5 initiates innate immune signaling and orchestrates disassembly of the viral particle, yet how these antiviral responses are regulated by capsid recognition is unclear. We show that hexagonal assembly triggers N-terminal polyubiquitination of TRIM5 that collectively drives antiviral responses. In uninfected cells, N-terminal monoubiquitination triggers non-productive TRIM5 turnover. Upon TRIM5 assembly on virus, a trivalent RING arrangement allows elongation of N-terminally anchored K63-linked ubiquitin chains (N-K63-Ub). N-K63-Ub drives TRIM5 innate immune stimulation and proteasomal degradation. Inducing ubiquitination before TRIM5 assembly triggers premature degradation and ablates antiviral restriction. Conversely, driving N-K63 ubiquitination after TRIM5 assembly enhances innate immune signaling. Thus, the hexagonal geometry of TRIM5’s antiviral lattice converts a capsid-binding protein into a multifunctional antiviral platform

The structure of the R2TP complex defines a platform for recruiting diverse client proteins to the HSP90 molecular chaperone system

The R2TP complex, comprising the Rvb1p-Rvb2p AAA-ATPases, Tah1p, and Pih1p in yeast, is a special- ized Hsp90 co-chaperone required for the assembly and maturation of multi-subunit complexes. These include the small nucleolar ribonucleoproteins, RNA polymerase II, and complexes containing phosphati- dylinositol-3-kinase-like kinases. The structure and stoichiometry of yeast R2TP and how it couples to Hsp90 are currently unknown. Here, we determine the 3D organization of yeast R2TP using sedimenta- tion velocity analysis and cryo-electron microscopy. The 359-kDa complex comprises one Rvb1p/Rvb2p hetero-hexamer with domains II (DIIs) forming an open basket that accommodates a single copy of Tah1p-Pih1p. Tah1p-Pih1p binding to multiple DII do- mains regulates Rvb1p/Rvb2p ATPase activity. Using domain dissection and cross-linking mass spectro- metry, we identified a unique region of Pih1p that is essential for interaction with Rvb1p/Rvb2p. These data provide a structural basis for understanding how R2TP couples an Hsp90 dimer to a diverse set of client proteins and complexes

A bipartite structural organization defines the SERINC family of HIV-1 restriction factors

The human integral membrane protein SERINC5 potently restricts HIV-1 infectivity and sensitizes the virus to antibody-mediated neutralization. Here, using cryo-EM, we determine the structures of human SERINC5 and its orthologue from Drosophila melanogaster at subnanometer and near-atomic resolution, respectively. The structures reveal a novel fold comprised of ten transmembrane helices organized into two subdomains and bisected by a long diagonal helix. A lipid binding groove and clusters of conserved residues highlight potential functional sites. A structure-based mutagenesis scan identified surface-exposed regions and the interface between the subdomains of SERINC5 as critical for HIV-1-restriction activity. The same regions are also important for viral sensitization to neutralizing antibodies, directly linking the antiviral activity of SERINC5 with remodeling of the HIV-1 envelope glycoprotein

Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication