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Presents a letter to the editor about adult-onset limb girdle muscular dystrophy 1B.

Presents a letter to the editor related to resonance assignments of the region 1463-1617 of the mouse p53 binding protein 1.

Presents a letter to the editor on resonance assignments of the C-terminal domain of human lamin.

Barrier-to-autointegration factor (BAF) is an essential component of the nuclear lamina. Encoded by BANF1, this DNA binding protein contributes to the regulation of gene expression, cell cycle progression, and nuclear integrity. A rare recessive BAF variant, Ala12Thr, causes the premature aging syndrome, Néstor–Guillermo progeria syndrome (NGPS). Here, we report the first dominant pathogenic BAF variant, Gly16Arg, identified in a patient presenting with progressive neuromuscular weakness. Although disease variants carry nearby amino acid substitutions, cellular and biochemical properties are distinct. In contrast to NGPS, Gly16Arg patient fibroblasts show modest changes in nuclear lamina structure and increases in repressive marks associated with heterochromatin. Structural studies reveal that the Gly16Arg substitution introduces a salt bridge between BAF monomers, reducing the conformation ensemble available to BAF. We show that this structural change increases the double-stranded DNA binding affinity of BAF Gly16Arg. Together, our findings suggest that BAF Gly16Arg has an increased chromatin occupancy that leads to epigenetic changes and impacts nuclear functions. These observations provide a new example of how a missense mutation can change a protein conformational equilibrium to cause a dominant disease and extend our understanding of mechanisms by which BAF function impacts human health. [ABSTRACT FROM AUTHOR]

Protein kinases that phosphorylate histones are ideally-placed to influence the behavior of chromosomes during cell division. Indeed, a number of conserved histone phosphorylation events occur prominently during mitosis and meiosis in most eukaryotes, including on histone H3 at threonine-3 (H3T3ph). At least two kinases, Haspin and VRK1 (NHK-1/ballchen in Drosophila), have been proposed to carry out this modification. Phosphorylation of H3 by Haspin has defined roles in mitosis, but the significance of VRK1 activity towards histones in dividing cells has been unclear. Here, using in vitro kinase assays, KiPIK screening, RNA interference, and CRISPR/Cas9 approaches, we were unable to substantiate a direct role for VRK1, or its paralogue VRK2, in the phosphorylation of threonine-3 or serine-10 of Histone H3 in mitosis, although loss of VRK1 did slow cell proliferation. We conclude that the role of VRKs, and their more recently identified association with neuromuscular disease and importance in cancers of the nervous system, are unlikely to involve mitotic histone kinase activity. In contrast, Haspin is required to generate H3T3ph during mitosis. [ABSTRACT FROM AUTHOR]

To better define the role of FOXO1 and FOXO3 transcriptional factors in breast carcinogenesis, we performed a comparative study of their expression at both the RNA and protein levels in a series of human breast tumors. We used qRT-PCR assay to quantify mRNA expression and Reverse Phase Protein Arrays (RPPA) to quantify protein expression in 218 breast tumors from patients with known clinical/pathological status and outcome. Weak correlations were observed between mRNA and protein expressions for both FOXO1 and FOXO3 genes. High expression of FOXO3 protein, but not FOXO1 protein, was a good prognostic marker, negatively correlated with KI67 and markers of activity of the PI3K/AKT/mTOR oncogenic pathway, and positively correlated with p53, a marker of apoptosis. Moreover, FOXO3 protein expression, but not FOXO1 protein expression, was also negatively correlated with various proteins involved in different DNA repair mechanisms. FOXO3 protein, but not FOXO1 protein, appears to be a tumor suppressor that inhibits breast cancer by altering DNA damage response (DDR), thereby inducing p53-dependent apoptosis. This antitumor effect appears to be suppressed by excessive activity of the PI3K/AKT/mTOR pathway. High FOXO3 protein expression could be a biomarker of deficient DDR in breast tumors. [ABSTRACT FROM AUTHOR]

At the nuclear envelope, the inner nuclear membrane protein emerin contributes to the interface between the nucleoskeleton and the chromatin. Emerin is an essential actor of the nuclear response to a mechanical signal. Genetic defects in emerin cause Emery-Dreifuss muscular dystrophy. It was proposed that emerin oligomerization regulates nucleoskeleton binding, and impaired oligomerization contributes to the loss of function of emerin disease-causing mutants. We here report the first structural characterization of emerin oligomers. We identified an N-terminal emerin region from amino acid 1 to amino acid 132 that is necessary and sufficient for formation of long curvilinear filaments. In emerin monomer, this region contains a globular LEM domain and a fragment that is intrinsically disordered. Solid-state nuclear magnetic resonance analysis identifies the LEM β-fragment as part of the oligomeric structural core. However, the LEM domain alone does not self-assemble into filaments. Additional residues forming a β-structure are observed within the filaments that could correspond to the unstructured region in emerin monomer. We show that the delK37 mutation causing muscular dystrophy triggers LEM domain unfolding and increases emerin self-assembly rate. Similarly, inserting a disulfide bridge that stabilizes the LEM folded state impairs emerin N-terminal region self-assembly, whereas reducing this disulfide bridge triggers self-assembly. We conclude that the LEM domain, responsible for binding to the chromatin protein BAF, undergoes a conformational change during self-assembly of emerin N-terminal region. The consequences of these structural rearrangement and self-assembly events on emerin binding properties are discussed. [ABSTRACT FROM AUTHOR]

Many icosahedral viruses use a specialized portal vertex to control genome encapsidation and release from the viral capsid. In tailed bacteriophages, the portal system is connected to a tail structure that provides the pipeline for genome delivery to the host cell. We report the first, to our knowledge, subnanometer structures of the complete portal-phage tail interface that mimic the states before and after DNA release during phage infection. They uncover structural rearrangements associated with intimate protein-DNA interactions. The portal protein gp6 of bacteriophage SPP1 undergoes a concerted reorganization of the structural elements of its central channel during interaction with DNA. A network of protein-protein interactions primes consecutive binding of proteins gp15 and gp16 to extend and close the channel. This critical step that prevents genome leakage from the capsid is achieved by a previously unidentified allosteric mechanism: gp16 binding to two different regions of gp15 drives correct positioning and folding of an inner gp16 loop to interact with equivalent loops of the other gp16 subunits. Together, these loops build a plug that closes the channel. Gp16 then fastens the tail to yield the infectious virion. The gatekeeper system opens for viral genome exit at the beginning of infection but recloses afterward, suggesting a molecular diaphragm-like mechanism to control DNA efflux. The mechanisms described here, controlling the essential steps of phage genome movements during virus assembly and infection, are likely to be conserved among long-tailed phages, the largest group of viruses in the Biosphere. [ABSTRACT FROM AUTHOR]

The human protein tyrosine phosphatase non-receptor type 4 ( PTPN4) prevents cells death. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We demonstrate here that the PDZ domain inhibits the phosphatase activity of PTPN4. The mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We combined analytical ultracentrifugation, small angle X-ray scattering and NMR to understand how the PDZ domain controls PTPN4 activity. We show that the physiologically active PTPN4 two-domain, encompassing the PDZ and the phosphatase domains, adopts a predominant compact conformation in solution. The PDZ ligand binding restores the catalytic competence of PTPN4 disrupting the transient interdomain communication. This study strengthens the emerging notion that PDZ domains can act as regulators of enzyme activity and therefore are active players in the dynamic regulation of signaling pathways. Structured digital abstract by (), by () [ABSTRACT FROM AUTHOR]

In many bacterial viruses and in certain animal viruses, the double-stranded DNA genome enters and exits the capsid through a portal gatekeeper. We report a pseudoatomic structure of a complete portal system. The bacteriophage SPP1 gatekeeper is composed of dodecamers of the portal protein gp6, the adaptor gp15, and the stopper gp16. The solution structures of gp15 and gp16 were determined by NMR. They were then docked together with the X-ray structure of gp6 into the electron density of the ≈1-MDa SPP1 portal complex purified from DNA-filled capsids. The resulting structure reveals that gatekeeper assembly is accompanied by a large rearrangement of the gp15 structure and by folding of a flexible loop of gp16 to form an intersubunit parallel 13-sheet that closes the portal channel. This stopper system prevents release of packaged DNA. Disulfide cross-linking between 13-strands of the stopper blocks the key conformational changes that control genome ejection from the virus at the beginning of host infection. [ABSTRACT FROM AUTHOR]

The maintenance of genome integrity in the cell is an essential process for the accurate transmission of the genetic material. BRCA2 participates in this process at several levels, including DNA repair by homologous recombination, protection of stalled replication forks, and cell division. These activities are regulated and coordinated via cell-cycle dependent modifications. Pathogenic variants in BRCA2 cause genome instability and are associated with breast and/or ovarian cancers. BRCA2 is a very large protein of 3418 amino acids. Most well-characterized variants causing a strong predisposition to cancer are mutated in the C-terminal 700 residues DNA binding domain of BRCA2. The rest of the BRCA2 protein is predicted to be disordered. Interactions involving intrinsically disordered regions (IDRs) remain difficult to identify both using bioinformatics tools and performing experimental assays. However, the lack of well-structured binding sites provides unique functional opportunities for BRCA2 to bind to a large set of partners in a tightly regulated manner. We here summarize the predictive and experimental arguments that support the presence of disorder in BRCA2. We describe how BRCA2 IDRs mediate self-assembly and binding to partners during DNA double-strand break repair, mitosis, and meiosis. We highlight how phosphorylation by DNA repair and cell-cycle kinases regulate these interactions. We finally discuss the impact of cancer-associated variants on the function of BRCA2 IDRs and more generally on genome stability and cancer risk. [ABSTRACT FROM AUTHOR]

In vertebrates, the BRCA2 protein is essential for meiotic and somatic homologous recombination due to its interaction with the RAD51 and DMC1 recombinases through FxxA and FxPP motifs (here named A- and P-motifs, respectively). The A-motifs present in the eight BRC repeats of BRCA2 compete with the A-motif of RAD51, which is responsible for its self-oligomerization. BRCs thus disrupt RAD51 nucleoprotein filaments in vitro. The role of the P-motifs is less studied. We recently found that deletion of Brca2 exons 12-14 encoding one of them (the prototypical 'PhePP' motif), disrupts DMC1 but not RAD51 function in mouse meiosis. Here we provide a mechanistic explanation for this phenotype by solving the crystal structure of the complex between a BRCA2 fragment containing the PhePP motif and DMC1. Our structure reveals that, despite sharing a conserved phenylalanine, the A- and P-motifs bind to distinct sites on the ATPase domain of the recombinases. The P-motif interacts with a site that is accessible in DMC1 octamers and nucleoprotein filaments. Moreover, we show that this interaction also involves the adjacent protomer and thus increases the stability of the DMC1 nucleoprotein filaments. We extend our analysis to other P-motifs from RAD51AP1 and FIGNL1., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

BRCA2 tumor suppressor protein ensures genome integrity by mediating DNA repair via homologous recombination (HR). This function is executed in part by its canonical DNA binding domain located at the C-terminus (BRCA2CTD), the only folded domain of the protein. Most germline pathogenic missense variants are located in this highly conserved region which binds to single-stranded DNA (ssDNA) and to the acidic protein DSS1. These interactions are essential for the HR function of BRCA2. Here, we report that the variant R2645G, identified in breast cancer and located at the DSS1 interface, unexpectedly increases the ssDNA binding activity of BRCA2CTDin vitro. Human cells expressing this variant display a hyper-recombination phenotype, chromosomal instability in the form of chromatid gaps when exposed to DNA damage, and increased PARP inhibitor sensitivity. In mouse embryonic stem cells (mES), this variant alters viability and confers sensitivity to cisplatin and Mitomycin C. These results suggest that BRCA2 interaction with ssDNA needs to be tightly regulated to limit HR and prevent chromosomal instability and we propose that this control mechanism involves DSS1. Given that several missense variants located within this region have been identified in breast cancer patients, these findings might have clinical implications for carriers., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks (DSBs). The HSF2BP-BRME1 complex interacts with BRCA2. Its absence causes a severe reduction in recombinase loading at meiotic DSB. We previously showed that, in somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here, we report that, upon binding to BRCA2, HSF2BP forms octameric rings that are able to interlock into a large ring-shaped 24-mer. Addition of BRME1 leads to dissociation of both of these ring structures and cancels the disruptive effect of HSF2BP on cancer cell resistance to DNA damage. It also prevents BRCA2 degradation during interstrand DNA crosslink repair in Xenopus egg extracts. We propose that, during meiosis, the control of HSF2BPBRCA2 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates BRCA2 and controls its turnover, thus promoting HR.

DNA double-strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle 1 . In interphase, DSBs are mainly repaired by non-homologous end joining and homologous recombination 2 . However, these pathways are completely inhibited in mitosis 3-5 , leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta 6 (Polθ) repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair factors, Polθ function is activated in mitosis upon phosphorylation by Polo-like kinase 1 (PLK1). Phosphorylated Polθ is recruited by a direct interaction with the BRCA1 C-terminal domains of TOPBP1 to mitotic DSBs, where it mediates joining of broken DNA ends. Loss of Polθ leads to defective repair of mitotic DSBs, resulting in a loss of genome integrity. This is further exacerbated in cells that are deficient in homologous recombination, where loss of mitotic DSB repair by Polθ results in cell death. Our results identify mitotic DSB repair as the underlying cause of synthetic lethality between Polθ and homologous recombination. Together, our findings reveal the critical importance of mitotic DSB repair in the maintenance of genome integrity., (© 2023. The Author(s).)

The tumor suppressor gene WWOX is localized in an unstable chromosomal region and its expression is decreased or absent in several types of cancer. A low expression of WWOX is associated with a poor prognosis in breast cancer (BC). It has recently been shown that WWOX contributes to genome stability through its role in the DNA damage response (DDR). In breast cancer cells, WWOX inhibits homologous recombination (HR), and thus promotes the repair of DNA double-stranded breaks (DSBs) by non-homologous end joining (NHEJ). The fine-tuning modulation of HR activity is crucial. Its under or overstimulation inducing genome alterations that can induce cancer. MERIT40 is a positive regulator of the DDR. This protein is indispensable for the function of the multi-protein complex BRCA1-A, which suppresses excessive HR activity. MERIT40 also recruits Tankyrase, a positive regulator of HR, to the DSBs to stimulate DNA repair. Here, we identified MERIT40 as a new molecular partner of WWOX. We demonstrated that WWOX inhibited excessive HR activity induced by overexpression of MERIT40. We showed that WWOX impaired the MERIT40-Tankyrase interaction preventing the role of the complex on DSBs. Furthermore, we found that MERIT40 is overexpressed in BC and that this overexpression is associated to a poor prognosis. These results strongly suggest that WWOX, through its interaction with MERIT40, prevents the deleterious impact of excessive HR on BC development by inhibiting MERIT40-Tankyrase association. This inhibitory effect of WWOX would oppose MERIT40-dependent BC development., (© 2023. The Author(s).)

Barrier-to-Autointegration Factor (BAF) is a highly conserved DNA binding protein important for genome integrity. Its localization and function are regulated through phosphorylation. Previously reported structures of BAF suggested that it is fully ordered, but our recent NMR analysis revealed that its N-terminal region is flexible in solution and that S4/T3 di-phosphorylation by VRK1 reduces this flexibility. Here, molecular dynamics (MD) simulation was used to unveil the conformational ensembles accessible to the N-terminal region of BAF either unphosphorylated, mono-phosphorylated on S4 or di-phosphorylated on S4/T3 (pBAF) and to reveal the interactions that contribute to define these ensembles. We show that the intrinsic flexibility observed in the N-terminal region of BAF is reduced by S4 phosphorylation and to a larger extent by S4/T3 di-phosphorylation. Thanks to the atomic description offered by MD supported by the NMR study of several BAF mutants, we identified the dynamic network of salt bridge interactions responsible for the conformational restriction involving pS4 and pT3 with residues located in helix α1 and α6. Using MD, we showed that the flexibility in the N-terminal region of BAF depends on the ionic strength and on the pH. We show that the presence of two negative charges of the phosphoryl groups is required for a substantial decrease in flexibility in pBAF. Using MD supported by NMR, we also showed that H7 deprotonation reduces the flexibility in the N-terminal region of BAF. Thus, the conformation of the intrinsically disordered N-terminal region of BAF is highly tunable, likely related to its diverse functions., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Nestor-Guillermo progeria syndrome (NGPS) is caused by a homozygous alanine-to-threonine mutation at position 12 (A12T) in barrier-to-autointegration factor (BAF). It is characterized by accelerated aging with severe skeletal abnormalities. BAF is an essential protein binding to DNA and nuclear envelope (NE) proteins, involved in NE rupture repair. Here, we assessed the impact of BAF A12T on NE integrity using NGPS-derived patient fibroblasts. We observed a strong defect in lamin A/C accumulation to NE ruptures in NGPS cells, restored upon homozygous reversion of the pathogenic BAF A12T mutation with CRISPR/Cas9. By combining in vitro and cellular assays, we demonstrated that while the A12T mutation does not affect BAF 3D structure and phosphorylation by VRK1, it specifically decreases the interaction between BAF and lamin A/C. Finally, we revealed that the disrupted interaction does not prevent repair of NE ruptures but instead generates weak points in the NE that lead to a higher frequency of NE re-rupturing in NGPS cells. We propose that this NE fragility could directly contribute to the premature aging phenotype in patients., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity - the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR., (© 2021. The Author(s).)

Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection.

Bacteriophage SPP1 is a double-stranded DNA virus of the Siphoviridae family that infects the bacterium Bacillus subtilis. This family of phages features a long, flexible, non-contractile tail that has been difficult to characterize structurally. Here, we present the atomic structure of the tail tube of phage SPP1. Our hybrid structure is based on the integration of structural restraints from solid-state nuclear magnetic resonance (NMR) and a density map from cryo-EM. We show that the tail tube protein gp17.1 organizes into hexameric rings that are stacked by flexible linker domains and, thus, form a hollow flexible tube with a negatively charged lumen suitable for the transport of DNA. Additionally, we assess the dynamics of the system by combining relaxation measurements with variances in density maps.

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors.

The Breast Cancer susceptibility protein 2 (BRCA2) is involved in mechanisms that maintain genome stability, including DNA repair, replication and cell division. These functions are ensured by the folded C-terminal DNA binding domain of BRCA2 but also by its large regions predicted to be disordered. Several studies have shown that disordered regions of BRCA2 are subjected to phosphorylation, thus regulating BRCA2 interactions through the cell cycle. The N-terminal region of BRCA2 contains two highly conserved clusters of phosphorylation sites between amino acids 75 and 210. Upon phosphorylation by CDK, the cluster 1 is known to become a docking site for the kinase PLK1. The cluster 2 is phosphorylated by PLK1 at least at two positions. Both of these phosphorylation clusters are important for mitosis progression, in particular for chromosome segregation and cytokinesis. In order to identify the phosphorylated residues and to characterize the phosphorylation sites preferences and their functional consequences within BRCA2 N-terminus, we have produced and analyzed the BRCA2 fragment from amino acid 48 to amino acid 284 (BRCA2 48-284 ). Here, we report the assignment of 1 H, 15 N, 13 CO, 13 Cα and 13 Cβ NMR chemical shifts of this region. Analysis of these chemical shifts confirmed that BRCA2 48-284 shows no stable fold: it is intrinsically disordered, with only short, transient α-helices.

In line with their high accessibility, disordered proteins are exquisite targets of kinases. Eukaryotic organisms use the so-called intrinsically disordered proteins (IDPs) or intrinsically disordered regions of proteins (IDRs) as molecular switches carrying intracellular information tuned by reversible phosphorylation schemes. Solvent-exposed serines and threonines are abundant in IDPs, and, consistently, kinases often modify disordered regions of proteins at multiple sites. In this context, nuclear magnetic resonance (NMR) spectroscopy provides quantitative, residue-specific information that permits mapping of phosphosites and monitoring of their individual kinetics. Hence, NMR monitoring emerges as an in vitro approach, complementary to mass-spectrometry or immuno-blotting, to characterize IDP phosphorylation comprehensively. Here, we describe in detail generic protocols for carrying out NMR monitoring of IDP phosphorylation, and we provide a number of practical insights that improve handiness and reproducibility of this method.

BRCA1 mutations have been identified that increase the risk of developing hereditary breast and ovarian cancers. Genetic screening is now offered to patients with a family history of cancer, to adapt their treatment and the management of their relatives. However, a large number of BRCA1 variants of uncertain significance (VUS) are detected. To better understand the significance of these variants, a high-throughput structural and functional analysis was performed on a large set of BRCA1 VUS. Information on both cellular localization and homology-directed DNA repair (HR) capacity was obtained for 78 BRCT missense variants in the UMD-BRCA1 database and measurement of the structural stability and phosphopeptide-binding capacities was performed for 42 mutated BRCT domains. This extensive and systematic analysis revealed that most characterized causal variants affect BRCT-domain solubility in bacteria and all impair BRCA1 HR activity in cells. Furthermore, binding to a set of 5 different phosphopeptides was tested: all causal variants showed phosphopeptide-binding defects and no neutral variant showed such defects. A classification is presented on the basis of mutated BRCT domain solubility, phosphopeptide-binding properties, and VUS HR capacity. These data suggest that HR-defective variants, which present, in addition, BRCT domains either insoluble in bacteria or defective for phosphopeptide binding, lead to an increased cancer risk. Furthermore, the data suggest that variants with a WT HR activity and whose BRCT domains bind with a WT affinity to the 5 phosphopeptides are neutral. The case of variants with WT HR activity and defective phosphopeptide binding should be further characterized, as this last functional defect might be sufficient per se to lead to tumorigenesis. IMPLICATIONS: The analysis of the current study on BRCA1 structural and functional defects on cancer risk and classification presented may improve clinical interpretation and therapeutic selection., (©2018 American Association for Cancer Research.)

Proton detection and fast magic-angle spinning have advanced biological solid-state NMR, allowing for the backbone assignment of complex protein assemblies with high sensitivity and resolution. However, so far no method has been proposed to detect intermolecular interfaces in these assemblies by proton detection. Herein, we introduce a concept based on methyl labeling that allows for the assignment of these moieties and for the study of protein-protein interfaces at atomic resolution., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson-Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR 1 H, 15 N, 13 CO, 13 Cα and 13 Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR 1 H, 15 N, 13 CO, 13 Cα and 13 Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.

Human emerin is an inner nuclear membrane protein involved in the response of the nucleus to mechanical stress. It contributes to the physical connection between the cytoskeleton and the nucleoskeleton. It is also involved in chromatin organization. Its N-terminal region is nucleoplasmic and comprises a globular LEM domain from residue 1 to residue 43. The three-dimensional structure of this LEM domain in complex with the chromatin BAF protein was solved from NMR data. Apart from the LEM domain, the nucleoplasmic region of emerin, from residue 44 to residue 221, is predicted to be intrinsically disordered. Mutations in this region impair binding to several emerin partners as lamin A, actin or HDAC3. However the molecular details of these recognition defects are unknown. Here we report (1)H, (15)N, (13)CO, (13)Cα and (13)Cβ NMR chemical shift assignments of the emerin fragment from residue 67 to residue 170, which is sufficient for nuclear localization and involved in lamin A binding. Chemical shift analysis confirms that this fragment is intrinsically disordered in 0 and 8 M urea.

LAP2-emerin-MAN1 (LEM)-domain proteins are modular proteins characterized by the presence of a conserved motif of about 50 residues. Most LEM-domain proteins localize at the inner nuclear membrane, but some are also found in the endoplasmic reticulum or nuclear interior. Their architecture has been analyzed by predicting the limits of their globular domains, determining the 3D structure of these domains and in a few cases calculating the 3D structure of specific domains bound to biological targets. The LEM domain adopts an α-helical fold also found in SAP and HeH domains of prokaryotes and unicellular eukaryotes. The LEM domain binds to BAF (barrier-to-autointegration factor; BANF1), which interacts with DNA and tethers chromatin to the nuclear envelope. LAP2 isoforms also share an N-terminal LEM-like domain, which binds DNA. The structure and function of other globular domains that distinguish LEM-domain proteins from each other have been characterized, including the C-terminal dimerization domain of LAP2α and C-terminal WH and UHM domains of MAN1. LEM-domain proteins also have large intrinsically disordered regions that are involved in intra- and intermolecular interactions and are highly regulated by posttranslational modifications in vivo., (© 2016 Elsevier Inc. All rights reserved.)

Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.

Tailed bacteriophages use a portal system for genome entry and exit from viral capsids. Here, we review the mechanisms how these movements are controlled by the genome gatekeeper that assembles at the portal structure. Phage DNA is packaged at high pressure inside the viral capsid by a powerful motor. The viral genome is translocated through the central channel of the portal protein found at a single vertex of the capsid. Packaging is normally terminated by endonucleolytic cleavage of the substrate DNA followed by disassembly of the packaging motor and closure of the portal system, preventing leakage of the viral genome. This can be achieved either by conformational changes in the portal protein or by sequential addition of proteins that extend the portal channel (adaptors) and physically close it preventing DNA exit (stoppers). The resulting connector structure provides the interface for assembly of short tails (podoviruses) or for attachment of preformed long tails (siphoviruses and myoviruses). The connector maintains the viral DNA correctly positioned for ejection that is triggered by interaction of the phage particle with bacterial receptors. Recent exciting advances are providing new molecular insights on the mechanisms that ensure precise coordination of these critical steps required both for stable viral genome packaging and for its efficient release to initiate infection.

Context: Mutations in PPARG are associated with insulin resistance and familial partial lipodystrophy, a disease characterized by altered distribution of sc fat and symptoms of the metabolic syndrome. The encoded protein, peroxisome proliferator-activated receptor (PPAR)-gamma, plays a pivotal role in regulating lipid and glucose metabolism, the differentiation of adipocytes, and other cellular regulatory processes., Objectives: The objective of the study was to detect a novel PPARG mutation in a kindred with partial lipodystrophy and analyze the functional characteristics of the mutant protein., Patients and Methods: In three subjects with partial lipodystrophy, one unaffected family member, and 124 unaffected subjects, PPARG was screened for mutations by direct sequencing. Body composition, laboratory abnormalities, and hepatic steatosis were assessed in each affected subject. Transcriptional activity was determined, and EMSA was performed to investigate DNA binding capacity of the mutant protein., Results: We identified a PPARG mutation, C190S, causing partial lipodystrophy with metabolic alterations in three affected family members. The mutation was absent in the unaffected family member and unaffected controls. The mutation is located within zinc-finger 2 of the DNA binding domain. C190S PPARgamma has a significantly lower ability to activate a reporter gene than wild-type PPARgamma in absence and presence of rosiglitazone. A dominant-negative effect was not observed. Compared with wild-type PPARgamma, C190S PPARgamma shows a reduced capacity to bind DNA., Conclusion: Mutation of a zinc-binding amino acid of PPARgamma leads to an altered protein-DNA binding pattern, resulting in a partial loss of function, which in turn is associated with partial lipodystrophy.