The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons.

Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)1,2. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.

Structure and function of the RAD51B-RAD51C-RAD51D-XRCC2 tumour suppressor.

Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic material during meiosis. Individuals with mutations in key recombination genes, such as BRCA2 (also known as FANCD1), or the RAD51 paralogues RAD51B, RAD51C (also known as FANCO), RAD51D, XRCC2 (also known as FANCU) and XRCC3, are predisposed to breast, ovarian and prostate cancers1-10 and the cancer-prone syndrome Fanconi anaemia11-13. The BRCA2 tumour suppressor protein-the product of BRCA2-is well characterized, but the cellular functions of the RAD51 paralogues remain unclear. Genetic knockouts display growth defects, reduced RAD51 focus formation, spontaneous chromosome abnormalities, sensitivity to PARP inhibitors and replication fork defects14,15, but the precise molecular roles of RAD51 paralogues in fork stability, DNA repair and cancer avoidance remain unknown. Here we used cryo-electron microscopy, AlphaFold2 modelling and structural proteomics to determine the structure of the RAD51B-RAD51C-RAD51D-XRCC2 complex (BCDX2), revealing that RAD51C-RAD51D-XRCC2 mimics three RAD51 protomers aligned within a nucleoprotein filament, whereas RAD51B is highly dynamic. Biochemical and single-molecule analyses showed that BCDX2 stimulates the nucleation and extension of RAD51 filaments-which are essential for recombinational DNA repair-in reactions that depend on the coupled ATPase activities of RAD51B and RAD51C. Our studies demonstrate that BCDX2 orchestrates RAD51 assembly on single stranded DNA for replication fork protection and double strand break repair, in reactions that are critical for tumour avoidance.

Mechanisms of BRCA1-BARD1 nucleosome recognition and ubiquitylation.

The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination1-10. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets11-14. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks15,16. We further show that RING domains17 in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin18-22. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.

Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations.

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B-BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.

BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1.

Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.

Conformational Dynamics from Ambiguous Zinc Coordination in the RanBP2-Type Zinc Finger of RBM5.

The multi-domain RNA binding protein RBM5 is a molecular signature of metastasis. RBM5 regulates alternative splicing of apoptotic genes including the cell death receptor Fas and the initiator Caspase-2. The RBM5 RanBP2-type zinc finger (Zf1) is known to specifically recognize single-stranded RNAs with high affinity. Here, we study the structure and conformational dynamics of the Zf1 zinc finger of human RBM5 using NMR. We show that the presence of a non-canonical cysteine in Zf1 kinetically destabilizes the protein. Metal-exchange kinetics show that mutation of the cysteine establishes high-affinity coordination of the zinc. Our data indicate that selection of such a structurally destabilizing mutation during the course of evolution could present an opportunity for functional adaptation of the protein.

Structures of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR fold.

The scavenger receptor cysteine-rich (SRCR) family of proteins comprises more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ∼110 amino-acid SRCR domain, this class of proteins have widespread functions as antimicrobial molecules, scavenger receptors, and signalling receptors. Despite the high level of structural conservation of SRCR domains, no unifying mechanism for ligand interaction has been described. The SRCR protein SALSA, also known as DMBT1/gp340, is a key player in mucosal immunology. Based on detailed structural data of SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand-binding mechanism for SALSA ligands. The binding interface incorporates a dual cation-binding site, which is highly conserved across the SRCR superfamily. Along with the well-described cation dependency on most SRCR domain-ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for the SRCR class of proteins.

Cryo-EM Structure of the Human FLCN-FNIP2-Rag-Ragulator Complex.

mTORC1 controls anabolic and catabolic processes in response to nutrients through the Rag GTPase heterodimer, which is regulated by multiple upstream protein complexes. One such regulator, FLCN-FNIP2, is a GTPase activating protein (GAP) for RagC/D, but despite its important role, how it activates the Rag GTPase heterodimer remains unknown. We used cryo-EM to determine the structure of FLCN-FNIP2 in a complex with the Rag GTPases and Ragulator. FLCN-FNIP2 adopts an extended conformation with two pairs of heterodimerized domains. The Longin domains heterodimerize and contact both nucleotide binding domains of the Rag heterodimer, while the DENN domains interact at the distal end of the structure. Biochemical analyses reveal a conserved arginine on FLCN as the catalytic arginine finger and lead us to interpret our structure as an on-pathway intermediate. These data reveal features of a GAP-GTPase interaction and the structure of a critical component of the nutrient-sensing mTORC1 pathway.

Architecture of the human GATOR1 and GATOR1-Rag GTPases complexes.

Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.

Neural migration. Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism.

Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted in colorectal cancer (DCC) is a well-established netrin-1 receptor mediating attractive responses. We provide evidence that its close relative neogenin is also a functional netrin-1 receptor that acts with DCC to mediate guidance in vivo. We determined the structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC. Netrin-1 has a rigid elongated structure containing two receptor-binding sites at opposite ends through which it brings together receptor molecules. The ligand/receptor complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous ligand/receptor assembly. The differences result from different lengths of the linker connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes.

Interaction of mutant p53 with p73: a Surface Plasmon Resonance and Atomic Force Spectroscopy study.

BACKGROUND: TP53 tumor suppressor gene is mutated in more than 50% of human tumors. Mutated p53 proteins could sequestrate and inactivate p73 reducing the apoptotic and anti-proliferative effects of the transcription factor, and yielding cancer cells more aggressive and chemoresistant. The possibility of using drugs to prevent the mutant p53/p73 complex formation preserving the p73 function, calls for a deeper insight into the molecular and biochemical mechanisms of mutant p53/p73 protein interaction. METHODS: The kinetics of the mutant p53R175H/p73 complex was investigated with innovative and complementary techniques, operating in real time, in near physiological conditions and without any labeling. Specifically, Atomic Force Spectroscopy and Surface Plasmon Resonance working at single-molecule level and in bulk condition, respectively, were used. RESULTS: The two techniques revealed that a stable complex is formed between mutant p53R175H and p73 proteins; the complex being characterized by a high interaction force and a dissociation equilibrium constant in the order of 10(-7)M, as expected for specific interactions. No binding was instead observed between p73 and wild type p53. CONCLUSIONS: Mutant p53R175H protein, unlike wild type p53, can form a stable complex with p73. The mutant p53R175H/p73 protein complex could be a target for innovative pharmaceutical drugs that, by dissociating it or preventing biomolecule interaction thus preserving the p73 function, could enhance the response of cancerous cells carrying mutant p53R175H protein to common chemotherapeutic agents. GENERAL SIGNIFICANCE: The kinetic information obtained in vitro may help to design specific pharmaceutical drugs directed against cancerous cells carrying mutant p53 proteins.

3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography.

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

Client-loading conformation of the Hsp90 molecular chaperone revealed in the cryo-EM structure of the human Hsp90:Hop complex.

Hsp90 is an essential molecular chaperone required for the folding and activation of many hundreds of cellular "client" proteins. The ATP-dependent chaperone cycle involves significant conformational rearrangements of the Hsp90 dimer and interaction with a network of cochaperone proteins. Little is known about the mechanism of client protein binding or how cochaperone interactions modulate Hsp90 conformational states. We have determined the cryo-EM structure of the human Hsp90:Hop complex that receives client proteins from the Hsp70 chaperone. Hop stabilizes an alternate Hsp90 open state, where hydrophobic client-binding surfaces have converged and the N-terminal domains have rotated and match the closed, ATP conformation. Hsp90 is thus simultaneously poised for client loading by Hsp70 and subsequent N-terminal dimerization and ATP hydrolysis. Upon binding of a single Hsp70, the Hsp90:Hop conformation remains essentially unchanged. These results identify distinct functions for the Hop cochaperone, revealing an asymmetric mechanism for Hsp90 regulation and client loading.

Functional proteomic analysis of promyelocytic leukaemia nuclear bodies in irradiation-induced MCF-7 cells.

It is well established that promyelocytic leukaemia nuclear bodies (PML NBs) play important roles in DNA damage responses (DDR). After irradiation, PML NBs dynamically recruit or release important proteins involved in cell-cycle regulation, DNA repair and apoptosis. As PML protein is the key molecule of PML NBs' dynamic assembling, we aimed to characterize the PML-interacting proteins in (60)Co-irradiated MCF-7 cells. A proteomic approach using CoIP, mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 124 proteins that may associate with PML after irradiation. Bioinformatic analysis of the identified proteins showed that most of them were related to characterized PML functions, such as transcriptional regulation, cell-cycle regulation, cell-death regulation and response to stress. Four proteins, B23, MVP, G3BP1 and DHX9, were verified to co-localize with PML differentially before and after ionizing radiation (IR) treatment. The proteins identified in this study will significantly improve our understanding of the dynamic organization and multiple functions of PML NBs in DDR.

Three-dimensional organization of promyelocytic leukemia nuclear bodies.

Promyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50- to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PML-NBs serves to concentrate different biological activities while allowing for an efficient exchange of components.

The number of PML nuclear bodies increases in early S phase by a fission mechanism.

Promyelocytic leukemia (PML) nuclear bodies have been implicated in a variety of cellular processes including apoptosis, tumour suppression, anti-viral response, DNA repair and transcriptional regulation. PML nuclear bodies are both positionally and structurally stable over extended periods of interphase. As demonstrated in this study, the structural stability is lost as cells enter S phase, evidenced both by distortions in shape and by fission and fusion events. At the end of this period of structural instability, the number of PML nuclear bodies has increased by a factor of twofold. Association of the fission products with chromatin implies that the PML nuclear bodies respond to changes in chromatin organisation or topology, and thus could play a role in monitoring genome integrity during DNA synthesis or in the continued maintenance of functional chromosomal domains prior to mitosis.

Novel spatiotemporal patterns of epithelial tumor invasion in Drosophila discs large egg chambers.

Loss of Discslarge (Dlg) in early Drosophila egg chambers causes invasion of tumor follicle cells from the anterior epithelium, a pattern that resembles developmental border cell migration during mid-oogenesis. Here, we have analyzed novel spatial and temporal patterns of dlg invasion. Even though Dlg is ubiquitously expressed in all follicle cells, invasions are biased at the anterior and posterior termini. The patterns of invasion correlate with both a higher rate of follicle cell proliferation and with a greater frequency of loss of epithelial polarity at the termini compared with central regions of the egg chamber. Nonetheless, the average number of cells that invade per invasion event from terminal vs. central regions is approximately equal. Of interest, patterns of dlg invasion appear to coincide with boundaries established by proto-oncogene signals responsible for anterior-posterior patterning. The Drosophila egg chamber may thus be a useful model for exploring how epithelial tumor invasion might be a neomorphogenetic process organized by signals essential for developmental pattern formation.

Disruption of oxygen-regulated responses underlies pathological changes in the placentas of women who smoke or who are passively exposed to smoke during pregnancy.

Previously, we showed that maternal smoking harms human placental development by changing the balance between cytotrophoblast (CTB) proliferation and differentiation. To understand the mechanisms involved, we studied the effects of maternal smoking and in vitro exposure of CTBs to nicotine and on CTB expression of molecules that govern cellular responses to oxygen tension: the von Hippel-Lindau tumor suppressor protein (pVHL), the hypoxia-inducible transcription factors (HIFs), and the vascular endothelial growth factors (VEGFs). We previously reported that hypoxia upregulates CTB pVHL expression (1). Here we show that in vitro exposure of CTBs to nicotine has the same effect. Maternal smoking also dysregulated CTB expression of all three molecules. Remarkably, we found that passive exposure to cigarette smoke had many of the same effects as active smoking, a graphic demonstration of the ill effects of cigarette smoke, even secondhand, on placental development. Together, these findings explain, in part, how smoking damages the placenta by altering expression of key mediators of placental development.