Biology subjects

Jamshad, M.

Publications and source records attributed to Jamshad, M..

10 recordsLinked to original sources

bioRxiv2022

Pin1-promoted SUMOylation of RNF168 restrains its chromatin accumulation.

The E3 ubiquitin ligase RNF168 is a rate-limiting component of DNA double-strand break signalling that acts to amplify histone ubiquitylation. The confining of RNF168 chromatin spreading around DNA break sites has been related to constraining its expression levels and to the removal of ubiquitin-conjugates to which it binds. Here we identify a new mechanism that suppresses RNF168 amplification at chromatin. We show that depletion of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) or mutation of the Pin1-binding site of RNF168 increases DNA-damage site accumulation of RNF168 to supraphysiological levels without impacting RNF168 expression levels. Pin1 promotes SUMOylation of RNF168 by SUMO2/3, and a SUMO conjugation site on RNF168 is required to restrict RNF168 accumulations. We find Pin1-SUMO mediated-regulation of RNF168 is associated with cellular radioresistance. These data demonstrate that the regulation of chromatin ubiquitylation is actively suppressed by modification of RNF168 to counteract excessive RNF168-ubiquitin spreading.

molecular biology↗

bioRxiv2022

Modification of the SUMO activating enzyme subunit SAE2 directs SUMO isoform bias required for mitotic fidelity.

Mammalian cells possess three conjugatable SUMO variants: SUMO1 and the largely indistinguishable SUMO2 and SUMO3 (designated SUMO2/3). Some SUMOylated substrates are modified by both SUMO1 and SUMO2/3, while others show biased modifications towards SUMO1 or SUMO2/3. How preferential SUMO protein conjugation is coordinated is poorly understood. Here, we examine a modification of the catalytic component of the human SUMO Activation Enzyme, SAE2. We observe that lysine 164 of SAE2 is deacetylated during mitosis in an HDAC6-dependent manner. We find that an acetyl-analogue mutant, SAE2-K164Q, biases the activation and conjugation of SUMO2>SUMO1 and discriminates SUMO1 and SUMO2/3 through their C-terminal tails. Complementation of SAE2-depleted or inhibited cells with SAE2-K164Q restricts mitotic SUMO1-conjugates and increases multipolar spindle formation. We confirm the SUMO E1-dependent modification of the nuclear mitotic apparatus, NuMA, and find that the mitotic defects of both SAE2-K164Q complemented cells and HDAC6-inhibitor-treated cells are corrected by either over-expression of SUMO1 or by expression of a GFP-SUMO1-NuMA-K1766R fusion protein. Our observations suggest a model in which the SAE1:SAE2 enzyme is deacetylated on early mitosis to encourage the conjugation of SUMO1 to support mitotic fidelity. These surprising data reveal that the SUMO-activating enzyme can bias SUMO variant conjugation.

molecular biology↗

bioRxiv2020

AscA (YecA) is a molecular chaperone involved in Sec-dependent protein translocation in Escherichia coli

Proteins that are translocated across the cytoplasmic membrane by Sec machinery must be in an unfolded conformation in order to pass through the protein-conducting channel during translocation. Molecular chaperones assist Sec-dependent protein translocation by holding substrate proteins in an unfolded conformation in the cytoplasm until they can be delivered to the membrane-embedded Sec machinery. For example, in Escherichia coli, SecB binds to a subset of unfolded Sec substrates and delivers them to the Sec machinery by interacting with the metal-binding domain (MBD) of SecA, an ATPase required for translocation in bacteria. Here, we describe a novel molecular chaperone involved Sec-dependent protein translocation, which we have named AscA (for accessory Sec component). AscA contains a metal-binding domain (MBD) that is nearly identical to the MBD of SecA. In vitro binding studies indicated that AscA binds to SecB and ribosomes in an MBD-dependent fashion. Saturated transposon mutagenesis and genetics studies suggested that AscA is involved in cell-envelope biogenesis and that its function overlaps with that of SecB. In support of this idea, AscA copurified with a range of proteins and prevented the aggregation of citrate synthase in vitro. Our results suggest that AscA is molecular chaperone and that it enhances Sec-dependent protein translocation by delivering its substrate proteins to SecB. IMPORTANCEThis research describes the discovery of a novel molecular chaperone, AscA (YecA). The function of AscA was previously unknown. However, it contains a small domain, known as the MBD, suggesting it could interact with the bacterial Sec machinery, which is responsible for transporting proteins across the cytoplasmic membrane. The work described this study indicates that the MBD allows AscA to bind to both the protein synthesis machinery and the Sec machinery. The previously function of the previously uncharacterised N-terminal domain is that of a molecular chaperone, which binds to unfolded substrate proteins. We propose that AscA binds to protein substrates as they are still be synthesised by ribosomes in order to channel them into the Sec pathway.

molecular biology↗

bioRxiv2019

Iron Is a Physiological Ligand of SecA-like Metal Binding Domains

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modelling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

biochemistry↗

bioRxiv2018

The deSUMOylase SENP2 coordinates homologous recombination and non-homologous end joining by independent mechanisms.

SUMOylation in the DNA double-strand break (DSB) response regulates recruitment, activity and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and non-homologous enjoining (NHEJ) through the investigation of the deSUMOylase SENP2. We find regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast we show HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 foci retention and increases NHEJ and radioresistance. Collectively our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

cell biology↗

bioRxiv2018

PIN1 isomerisation of BRCA1 promotes replication fork protection.

BRCA1, BRCA2 and a subset of Fanconis Anaemia proteins act to promote RAD51-mediated protection of newly synthesised DNA at stalled replication forks from degradation by nucleases. How BRCA1 contributes, how it is regulated and whether replication fork protection relates to, or differs from, the roles BRCA1 has in homologous recombination is not clear. Here we show that the canonical BRCA1-PALB2 interaction is not required for fork protection and instead we demonstrate that the ability of BRCA1 to protect nascent DNA is regulated in an unexpected fashion through conformational change mediated by the phosphorylation-directed prolyl isomerase, PIN1. BRCA1 isomerisation enhances BRCA1-BARD1 interaction with RAD51 and consequently RAD51 presence at stalled replication structures. Our data suggest BRCA1-BARD1 promotes fork protection in part by enhancing the RAD51 synapse. Patient missense variants in the regulated BRCA1-BARD1 regions similarly show poor nascent strand protection but proficient homologous recombination, defining novel domains required for fork protection in BRCA1-BARD1 associated with cancer predisposition. Together these findings reveal a previously unrecognised pathway that governs BRCA1-mediated replication fork protection.

cell biology↗

bioRxiv2018

The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

In bacteria, the translocation of a subset of proteins across the cytoplasmic membrane by the Sec machinery requires SecA. Although SecA can recognise nascent polypeptides, the mechanism of cotranslational substrate protein recognition is not known. Here, we investigated the role of the C-terminal tail (CTT) of SecA, which consists of a flexible linker (FLD) and a small metal-binding domain (MBD), in its interaction with nascent polypeptides. Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the entire CTT or the MBD alone had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function. Autophotocrosslinking, mass spectrometry, x-ray crystallography and small-angle x-ray scattering experiments provided insight into the CTT-mediated conformational changes in SecA. Finally, photocrosslinking experiments indicated that binding of SecA to substrate protein affected its interaction with the ribosome. Taken together, our results suggest a mechanism for substrate protein recognition.\n\nImpact StatementSecA is an evolutionarily conserved ATPase that is required for the translocation of a subset of proteins across the cytoplasmic membrane in bacteria. We investigated how SecA recognises its substrate proteins at the ribosome as they are still being synthesised (i.e. cotranslationally).

biochemistry↗

bioRxiv2018

Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting {beta}-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

biophysics↗

bioRxiv2017

A genetic screen suggests an alternative mechanism for inhibition of SecA by azide

Sodium azide prevents bacterial growth by inhibiting the activity of SecA, which is required for translocation of proteins across the cytoplasmic membrane. Azide inhibits ATP turnover in vitro, but its mechanism of action in vivo is unclear. To investigate how azide inhibits SecA in cells, we used transposon directed insertion-site sequencing (TraDIS) to screen a library of transposon insertion mutants for mutations that affect the susceptibility of E. coli to azide. Insertions disrupting components of the Sec machinery generally increased susceptibility to azide, but insertions truncating the C-terminal tail (CTT) of SecA decreased susceptibility of E. coli to azide. Treatment of cells with azide caused increased aggregation of the CTT, suggesting that azide disrupts its structure. Analysis of the metal-ion content of the CTT indicated that SecA binds to iron and the azide disrupts the interaction of the CTT with iron. Azide also disrupted binding of SecA to membrane phospholipids, as did alanine substitutions in the metal-coordinating amino acids. Furthermore, treating purified phospholipid-bound SecA with azide in the absence of added nucleotide disrupted binding of SecA to phospholipids. Our results suggest that azide does not inhibit SecA by inhibiting the rate of ATP turnover in vivo. Rather, azide inhibits SecA by causing it to \"backtrack\" from the ADP-bound to the ATP-bound conformation, which disrupts the interaction of SecA with the cytoplasmic membrane.\n\nSignificance statementSecA is a bacterial ATPase that is required for the translocation of a subset of secreted proteins across the cytoplasmic membrane. Sodium azide is a well-known inhibitor of SecA, but its mechanism of action in vivo is poorly understood. To investigate this mechanism, we examined the effect of azide on the growth of a library of [~]1 million transposon insertion mutations. Our results suggest that azide causes SecA to backtrack in its ATPase cycle, which disrupts binding of SecA to the membrane and to its metal cofactor, which is iron. Our results provide insight into the molecular mechanism by which SecA drives protein translocation and how this essential biological process can be disrupted.

microbiology↗

bioRxiv2017

MCE domain proteins: conserved inner membrane lipid-binding proteins required for outer membrane homeostasis

Bacterial proteins with MCE domains were first described as being important for Mammalian Cell Entry. More recent evidence suggests they are components of lipid ABC transporters. In Escherichia coli, the single-domain protein MlaD is known to be part of an inner membrane transporter that is important for maintenance of outer membrane lipid asymmetry. Here we describe two multi MCE domain-containing proteins in Escherichia coli, PqiB and YebT, the latter of which is an orthologue of MAM-7 that was previously reported to be an outer membrane protein. We show that all three MCE domain-containing proteins localise to the inner membrane. Bioinformatic analyses revealed that MCE domains are widely distributed across bacterial phyla but multi MCE domain-containing proteins evolved in Proteobacteria from single-domain proteins. Mutants defective in mlaD, pqiAB and yebST were shown to have distinct but partially overlapping phenotypes, but the primary functions of PqiB and YebT differ from MlaD. Complementing our previous findings that all three proteins bind phospholipids, results presented here indicate that multi-domain proteins evolved in Proteobacteria for specific functions in maintaining cell envelope homeostasis.

microbiology↗