Biology subjects

Schittenhelm, R. B.

Publications and source records attributed to Schittenhelm, R. B..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Atmospheric hydrogen oxidation extends to the domain archaea

Diverse aerobic bacteria use atmospheric hydrogen (H2) and carbon monoxide (CO) as energy sources to support growth and survival. Though recently discovered, trace gas oxidation is now recognised as a globally significant process that serves as the main sink in the biogeochemical H2 cycle and sustains microbial biodiversity in oligotrophic ecosystems. While trace gas oxidation has been reported in nine phyla of bacteria, it was not known whether archaea also use atmospheric H2. Here we show that a thermoacidophilic archaeon, Acidianus brierleyi (Thermoproteota), constitutively consumes H2 and CO to sub-atmospheric levels. Oxidation occurred during both growth and survival across a wide range of temperatures (10 to 70{degrees}C). Genomic analysis demonstrated that A. brierleyi encodes a canonical carbon monoxide dehydrogenase and, unexpectedly, four distinct [NiFe]-hydrogenases from subgroups not known to mediate aerobic H2 uptake. Quantitative proteomic analyses showed that A. brierleyi differentially produced these enzymes in response to electron donor and acceptor availability. A previously unidentified group 1 [NiFe]-hydrogenase, with a unique genetic arrangement, is constitutively expressed and upregulated during stationary phase and aerobic hydrogenotrophic growth. Another archaeon, Metallosphaera sedula, was also found to oxidize atmospheric H2. These results suggest that trace gas oxidation is a common trait of aerobic archaea, which likely plays a role in their survival and niche expansion, including during dispersal through temperate environments. These findings also demonstrate that atmospheric H2 consumption is a cross-domain phenomenon, suggesting an ancient origin of this trait, and identify previously unknown microbial and enzymatic sinks of atmospheric H2 and CO.

microbiology↗

bioRxiv2022

Temporal landscape of mitochondrial proteostasis governed by the UPRmt

Breakdown of mitochondrial proteostasis activates quality control pathways including the mitochondrial unfolded protein response (UPRmt) and PINK1/Parkin mitophagy. However, beyond the upregulation of chaperones and proteases, we have a limited understanding of how the UPRmt remodels and restores damaged mito-proteomes. Here, we have developed a functional proteomics framework, termed MitoPQ (Mitochondrial Proteostasis Quantification), to dissect the UPRmts role in maintaining proteostasis during stress. We discover essential roles for the UPRmt in both protecting and repairing proteostasis, with oxidative phosphorylation metabolism being a central target of the UPRmt. Transcriptome analyses together with MitoPQ reveal that UPRmt transcription factors drive independent signaling arms that act in concert to maintain proteostasis. Unidirectional interplay between the UPRmt and PINK1/Parkin mitophagy was found to promote oxidative phosphorylation recovery when the UPRmt failed. Collectively, this study defines the network of proteostasis mediated by the UPRmt and highlights the value of functional proteomics in decoding stressed proteomes.

cell biology↗

bioRxiv2022

Male mammalian meiosis and spermiogenesis is critically dependent on the shared functions of the katanins KATNA1 and KATNAL1

Katanin microtubule severing enzymes are potent M-phase regulators in oocytes and somatic cells. How the complex, and evolutionarily critical, male mammalian meiotic spindle is sculpted remains unknown. Here, using multiple single and double gene knockout mice, we reveal that the canonical katanin A-subunit, KATNA1, and its close paralogue, KATNAL1, together execute multiple aspects of meiosis. We show KATNA1 and KATNAL1 collectively regulate the male meiotic spindle, cytokinesis and midbody abscission, in addition to diverse spermatid remodelling events, including Golgi organisation, and acrosome and manchette formation. We also define KATNAL1-specific roles in sperm flagella development, manchette regulation, and sperm-epithelial disengagement. Finally, using proteomic approaches we define the KATNA1, KATNAL1, and KATNB1 mammalian testis interactome, which includes a network of cytoskeletal and vesicle trafficking proteins. Collectively, we reveal the presence of multiple katanin A-subunit paralogs in mammalian spermatogenesis allows for customized cutting via neofunctionalization and protective buffering via gene redundancy.

cell biology↗

bioRxiv2022

Single-fibre proteomics reveals a greater fibre-type specific mitochondrial remodelling with moderate than high-intensity training.

Analyses of mitochondrial adaptations in human skeletal muscle have mostly used whole-muscle samples, where results may be confounded by the presence of a mixture of type I and II muscle fibres. Using our adapted mass spectrometry-based proteomics workflow, we provide new insights into fibre-specific mitochondrial differences in human skeletal muscle before and after training. Our findings challenge previous conclusions regarding the extent of fibre-type-specific remodelling of the mitochondrial proteome and highlight that most baseline differences in mitochondrial protein abundances between fibre types reported by us, and others, might be due to differences in total mitochondrial content or a consequence of adaptations to habitual physical activity (or inactivity). Most training-induced changes in different mitochondrial functional groups, in both fibre types, were stoichiometrically linked to changes in markers of mitochondrial content.

physiology↗

bioRxiv2022

Energy extraction from air: structural basis of atmospheric hydrogen oxidation

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival. This recently discovered yet globally significant process regulates the composition of the atmosphere, enhances soil biodiversity, and drives primary production in certain extreme environments. Atmospheric H2 oxidation has been attributed to still uncharacterised members of the [NiFe]-hydrogenase superfamily. However, it is unresolved how these enzymes overcome the extraordinary catalytic challenge of selectively oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2, and how the derived electrons are transferred to the respiratory chain. Here we determined the 1.52 [A] resolution CryoEM structure of the mycobacterial hydrogenase Huc and investigated its mechanism by integrating kinetics, electrochemistry, spectroscopy, mass spectrometry, and molecular dynamics simulations. Purified Huc is an oxygen-insensitive enzyme that couples the oxidation of atmospheric H2 at its large subunit to the hydrogenation of the respiratory electron carrier menaquinone at its small subunit. The enzyme uses a narrow hydrophobic gas channel to selectively bind atmospheric H2 at the expense of O2, while three [3Fe-4S] clusters and their unusual ligation by a D-histidine modulate the electrochemical properties of the enzyme such that atmospheric H2 oxidation is energetically feasible. Huc forms an 833 kDa complex composed of an octamer of catalytic subunits around a membrane-associated central stalk, which extracts and transports menaquinone a remarkable 94 [A] from the membrane, enabling its reduction. These findings provide a mechanistic basis for the biogeochemically and ecologically critical process of atmospheric H2 oxidation. Through the first characterisation of a group 2 [NiFe]-hydrogenase, we also uncover a novel mode of energy coupling dependent on long-range quinone transport and pave way for the development of biocatalysts that oxidize H2 in ambient air.

biochemistry↗

bioRxiv2022

Systems-level investigation of mucopolysaccharidosis IIIA identifies deficient synaptic activity as a key driver of disease progression

Mucopolysaccharidoses are lysosomal storage diseases that collectively represent a major cause of lethal, treatment-refractory childhood dementias 1-7 Clinically-useful interventions are hampered due to an incomplete understanding of their neuropathological origins. Using the zebrafish sgsh model of mucopolysaccharidosis IIIA 8 (MPS IIIA, Sanfilippo syndrome A), we conducted several omics-based analyses, and developed and benchmarked a novel bioinformatic feature classification and ranking model for high-throughput datasets - ExIR - to prioritise important features in the progression of neurological manifestations of the disease. We find that the massive endolysosomal burden resulting from increased lysosomal storage of heparan sulfate and other secondarily accumulating substrates, such as sphingolipids, induces abnormal microtubule organisation and vesicle trafficking in neurons. This results in a gradual impairment of synaptic vesicle localisation at the presynaptic terminal and consequently impaired neuronal activity. Importantly, the endolysosomal phenotype in MPS IIIA zebrafish well-precedes the onset of neural pathology, though the larval MPS IIIA brain was found to be more susceptible to perturbation than wild type siblings. Collectively, these analyses demonstrate the presence of a progressive functional neurodegenerative phenotype underpinning neurological disease in MPS IIIA. Our findings provide direct mechanistic evidence linking the well-described lysosomal storage basis for MPS IIIA to its disproportionately severe neural clinical involvement, enabling development and refinement of future therapeutic interventions for this currently untreatable disorder. HighlightsO_LIMPS IIIA represents one of the most common causes of broadly fatal childhood dementia, but the mechanisms underlying disease progression are poorly understood. C_LIO_LIThe first systems-level analyses of disease state and progression in the CNS of an MPS IIIA animal model were performed. C_LIO_LIExperimental data-based Integrative Ranking (ExIR) was developed to provide unbiased prioritisation and classification of biological data as drivers, biomarkers and mediators of biological processes from high-throughput data at a systems level. C_LIO_LIApplication of ExIR to a transcriptomic and proteomic analyses of a zebrafish model of MPS IIIA implies progressive deficiencies in synaptic activity as a key driver of disease progression correlating with progressive neuronal endolysosomal burden and secondary storage diseases. C_LIO_LIA novel unifying explanation of pathobiology and progression of MPS IIIA facilitates identification of clinically targetable features and may be generalised to other neuronopathic storage disorders. C_LI

neuroscience↗

bioRxiv2022

DNA methylation and proteomics integration uncover dose-dependent group and individual responses to exercise in human skeletal muscle

ObjectiveExercise is a major regulator of muscle metabolism, and health benefits acquired by exercise are a result of molecular shifts occurring across multiple OMIC levels (i.e. epigenome, transcriptome, proteome). Identifying robust targets associated with exercise response, at both group and individual levels, is therefore important to develop health guidelines and targeted health interventions. MethodsTwenty, apparently healthy, moderately trained (VO2 max= 51.0{+/-}10.6 mL{middle dot}min-1{middle dot}kg-1) males (age range= 18-45yrs) from the Gene SMART (Skeletal Muscle Adaptive Responses to Training) study completed a 12-week High-Intensity Interval Training (HIIT) intervention. Muscle biopsies were collected at baseline and after 4, 8, and 12 weeks of HIIT. High throughput DNA methylation ([~]850 CpG sites), and proteomic ([~]3000 proteins) analyses were conducted at all-time points. Mixed-models were applied to estimate group and individual changes, and methylome and proteome integration was conducted using a holistic multilevel approach with the mixOmics package. ResultsSignificant shifts in the methylome (residual analysis) and proteome profiles were observed after 12 weeks of HIIT. 461 proteins significantly changed over time (at 4, 8, and 12 weeks), whilst only one differentially methylated position (DMP) was changed (adj.p-value <0.05). K-means analysis revealed clear protein clustering exhibiting similar changes over time. Individual responses to training were observed in 101 proteins. Seven proteins had a large effect-sizes >0.5, among them are two novel exercise-related proteins, LYRM7 and EPN1. Integration analysis uncovered bidirectional relationships between the methylome and proteome. ConclusionsWe showed a significant influence of HIIT on the epigenome and proteome in human muscle, and uncovered groups of proteins clustering according to similar patterns across the exercise intervention. Individual responses to exercise were observed in the proteome with novel mitochondrial and metabolic proteins consistently changed across individuals. Future work is required to elucidate the role of such proteins in response to exercise as well as to investigate the mechanisms associating genes and proteins in response to exercise.

molecular biology↗

bioRxiv2022

Disease-modifying effects of sodium selenate in a model of drug-resistant, temporal lobe epilepsy.

There are no pharmacological disease-modifying treatments that can mitigate the seizures and comorbidities associated with established chronic temporal lobe epilepsy (TLE). This study evaluated the effect of sodium selenate in the post-status epilepticus (SE) rat model of chronic drug resistant TLE. Wistar rats underwent kainic acid-induced SE or sham. Ten-weeks post-SE, rats were randomly assigned to receive either sodium selenate, levetiracetam, or vehicle treatments continuously for 4 weeks. To evaluate the effects of the treatments, 1 week of continuous video-EEG was acquired before, during, and 4, 8 weeks post-treatment, followed by behavioral tests. Targeted and untargeted proteomics and metabolomics were performed on post-mortem brain tissue to identify potential pathways associated with modified disease outcomes. Telomere length was investigated as a novel surrogate marker of disease severity. Sodium selenate treatment was able to mitigate disease severity, reducing the number of spontaneous seizures (p< 0.05), cognitive dysfunction (p< 0.05 in both novel object placement and recognition tasks), and sensorimotor deficits (p< 0.01) 8 weeks post-treatment cessation. Moreover, increased protein phosphatase 2A (PP2A) expression, reduced hyperphosphorylated tau, and reversed telomere length shortening caused by SE (p< 0.05). Network medicine integration of multi-omics/ pre-clinical outcomes identified protein-metabolite modules positively correlated with the TLE phenotype. Our results provide evidence that treatment with sodium selenate results in a sustained disease modifying effect in chronically epileptic rats in the post-KA SE model of TLE, including improved comorbid learning and memory deficits.

neuroscience↗

bioRxiv2021

The cytosolic role of EZH2-IMPDH2 complex in melanoma progression and metastasis via GTP regulation

The enhancer of zeste homolog 2 (EZH2) oncoprotein is a histone methyltransferase that functions canonically as a catalytic subunit of the polycomb repressive complex 2 (PRC2) to tri-methylate histone H3 at Lys 27 (H3K27me3). Although targeting EZH2 methyltransferase is a promising therapeutic strategy against cancer, methyltransferase-independent oncogenic functions of EZH2 are described. Moreover, pharmacological EZH2 methyltransferase inhibition was only variably effective in pre-clinical and clinical studies, suggesting that targeting EZH2 methyltransferase alone may be insufficient. Here, we demonstrate a non-canonical mechanism of EZH2s oncogenic activity characterized by interactions with inosine monophosphate dehydrogenase 2 (IMPDH2) and downstream promotion of guanosine-5-triphosphate (GTP) production. EZH2-IMPDH2 interactions identified by Liquid Chromatography-Mass Spectrometry (LC-MS) of EZH2 immunoprecipitates from melanoma cells were verified to occur between the N-terminal EED-binding domain of cytosolic EZH2 and the CBS domain of IMPDH2 in a methyltransfersase-independent manner. EZH2 silencing reduced cellular GTP, ribosome biogenesis, RhoA-mediated actomyosin contractility and melanoma cell proliferation and invasion by impeding the activity of IMPDH2. Guanosine, which replenishes GTP, reversed these effects and thereby promoted invasive and clonogenic cell states even in EZH2 silenced cells. IMPDH2 silencing antagonized the proliferative and invasive effects of EZH2, also in a guanosine-reversible manner. In human melanomas, high cytosolic EZH2 and IMPDH2 expression were associated with nucleolar enlargement, a marker of ribosome biogenesis. EZH2-IMPDH2 complexes were also observed in a range of cancers in which Sappanone A (SA), which inhibits EZH2-IMPDH2 interactions, was anti-tumorigenic, although notably non-toxic in normal cells. These findings illuminate a previously unrecognized, non-canonical, methyltransferase-independent, and GTP-dependent mechanism by which EZH2 regulates tumorigenicity in melanoma and other cancers, opening new avenues for development of anti-EZH2 therapeutics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/467024v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@153dd35org.highwire.dtl.DTLVardef@dfeb43org.highwire.dtl.DTLVardef@ce8148org.highwire.dtl.DTLVardef@189d8da_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LIEZH2 has non-canonical methyltransferase-independent and GTP-dependent tumorigenic and metastatic functions in melanoma. C_LIO_LIThe N-terminal EED-binding domain of EZH2 interacts with the CBS domain of IMPDH2 in a polycomb repressive complex 2- (PRC2-) and methylation-independent manner. C_LIO_LIEZH2 accumulates with IMPDH2 in the cytoplasm and increases IMPDH2s tetramerization-mediated activity independently of EZH2 methyltransferase. C_LIO_LIEZH2 upregulates GTP synthesis by IMPDH2 activation and thereby activates ribosome biogenesis via rRNA synthesis and actomyosin contractility via RhoA GTPase. C_LIO_LISappanone A (SA) inhibits IMPDH2-EZH2 interactions and is anti-proliferative across a range of cancers including melanoma, but not in normal cells. C_LI

cancer biology↗

bioRxiv2021

Ubr1-induced selective endo-phagy/autophagy protects against the endosomal and Ca2+-induced proteostasis disease stress

The defence mechanisms against endo-lysosomal homeostasis stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) ubiquitin ligase that counteracts proteostasis stress by enhancing cargo selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations increased intracellular Ca2+ and caused endosomal compartment stress by fusion and enlargement. Endosomal protein QC pathway using ubiquitin QC ligases CHIP and Ubr1 with ESCRT-machinery was able to target only a fraction of MLC1-mutants for lysosomal degradation. As a consequence of the endosomal stress, we found an alternative QC route dependent on Ubr1, SQSTM1/p62 and arginylation to bypass MLC1-mutants to endosomal autophagy (endo-phagy). Significantly, this unfolded a general biological endo-lysosomal QC pathway for arginylated Ubr1-SQSTM1/p62 autophagy targets during Ca2+-assault. Conversely, the loss of Ubr1 with the absence of arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1-dependent endo-phagy/autophagy in constitutive and provoked endo-lysosomal proteostasis stress, and link Ubr1 to Ca2+-homeostasis and proteins implicated in various diseases including cancers and brain disorders.

biochemistry↗

bioRxiv2021

A nitrite-oxidizing bacterium constitutively consumes atmospheric hydrogen

Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) of the genus Nitrospira contribute to nitrification in diverse natural environments and engineered systems. Nitrospira are thought to be well-adapted to substrate limitation owing to their high affinity for nitrite and capacity to use alternative energy sources. Here, we demonstrate that the canonical nitrite oxidizer Nitrospira moscoviensis oxidizes hydrogen (H2) below atmospheric levels using a high-affinity group 2a nickel-iron hydrogenase [Km(app) = 32 nM]. Atmospheric H2 oxidation occurred under both nitrite-replete and nitrite-deplete conditions, suggesting low-potential electrons derived from H2 oxidation promote nitrite-dependent growth and enable survival during nitrite limitation. Proteomic analyses confirmed the hydrogenase was abundant under both conditions and indicated extensive metabolic changes occur to reduce energy expenditure and growth under nitrite-deplete conditions. Respirometry analysis indicates the hydrogenase and nitrite oxidoreductase are bona fide components of the aerobic respiratory chain of N. moscoviensis, though they transfer electrons to distinct electron carriers in accord with the contrasting redox potentials of their substrates. Collectively, this study suggests atmospheric H2 oxidation enhances the growth and survival of NOB in amid variability of nitrite supply. These findings also extend the phenomenon of atmospheric H2 oxidation to a seventh phylum (Nitrospirota) and reveal unexpected new links between the global hydrogen and nitrogen cycles.

microbiology↗

bioRxiv2021

MOV10 facilitates messenger RNA decay in an N6-methyladenosine (m6A) dependent manner to maintain the mouse embryonic stem cells state

N6-methyladenosine (m6A) is the most predominant internal mRNA modification in eukaryotes, recognised by its reader proteins (so-called m6A-readers) for regulating subsequent mRNA fates -- splicing, export, localisation, decay, stability, and translation -- to control several biological processes. Although a few m6A-readers have been identified, yet the list is incomplete. Here, we identify a new m6A-reader protein, Moloney leukaemia virus 10 homologue (MOV10), in the m6A pathway. MOV10 recognises m6A-containing mRNAs with a conserved GGm6ACU motif. Mechanistic studies uncover that MOV10 facilitates mRNA decay of its bound m6A-containing mRNAs in an m6A-dependent manner within the cytoplasmic processing bodies (P-bodies). Furthermore, MOV10 decays the Gsk-3{beta} mRNA through m6A that stabilises the {beta}-CATENIN expression of a WNT/{beta}-CATENIN signalling pathway to regulate downstream NANOG expression for maintaining the mouse embryonic stem cells (mESCs) state. Thus, our findings reveal how a newly identified m6A-reader, MOV10 mediates mRNA decay via m6A that impact embryonic stem cell biology.

biochemistry↗

bioRxiv2021

EZH2 Abundance Regulated by UHRF1/UBE2L6/UBR4 Ubiquitin System is the Potential Therapeutic Target to Trigger Pigmented Phenotype in Melanoma

Cellular heterogeneity in cancer is linked to disease progression and therapy response, although the mechanisms regulating distinct cellular states within tumours are not well understood. To address this, we identified melanin pigment content as a major source of phenotypic and functional heterogeneity in melanoma and compared RNAseq data from high (HPC) and low pigmented melanoma cells (LPC), revealing the polycomb repressor complex protein, EZH2, as a master regulator of these states. EZH2 protein, but not RNA expression, was found to be upregulated in LPCs and inversely correlated with melanin in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA strategy or DZNep, MS1943 that reduces EZH2 protein levels, significantly inhibited cell growth in LPCs by hampering ribosome biogenesis. In addition, decline in EZH2 protein level induces pigmented cell phenotype by inducing melanin biosynthesis. Proteasomal inhibitor, MG132 treatment induced EZH2 protein levels in HPCs prompted us to look for differentially regulated ubiquitin system proteins in HPC vs LPCs. UBE2L6, E2 conjugating enzyme has been shown to be downregulated significantly in LPCs by UHRF1-mediated CpG methylation. Both biochemical assays and animal studies demonstrated that UBE2L6 expression decline, in turn, promotes EZH2 protein stability due to lack of ubiquitination on K381 residue in LPCs. UBR4 cooperates with UBE2L6 to facilitate this ubiquitination process. Targeting UHRF1/UBE2L6/UBR4 axis can be a better treatment option to trigger HPC state in melanoma in which conventional EZH2 inhibitors are ineffective.

cancer biology↗

bioRxiv2021

The cryo-EM structure of the neurofibromin dimer reveals the molecular basis for von Recklinghausen disease

Neurofibromin (NF1) is a tumour suppressor mutated in neurofibromatosis type 1 (von Recklinghausen disease), one of the most common human genetic diseases(1). NF1 regulates cellular growth through suppressing the Rat Sarcoma (RAS) pathway and, accordingly, mutations in this protein drive numerous cancers, including melanoma, ovarian, breast and brain cancer(2, 3). Currently, however, the molecular basis for NF1 function remains to be understood. Here we address this problem and use cryogenic Electron Microscopy (cryo-EM) to determine the structure of fulllength NF1. The 640 kDa NF1 homodimer forms an extraordinary lemniscate ({infty}) shaped molecule that is ~30 nm in length and ~ 10 nm wide. Each NF1 monomer comprises an N-terminal HEAT-repeat domain (N-HEAT), a guanosine triphosphatase activating protein (GAP)-related domain (GRD), a Sec14 homologous and pleckstrin homologous module (SEC-PH), and a C-terminal HEAT domain (C-HEAT). The core NF1 scaffold is formed via a head-to-tail dimer of the N- and C-HEAT domains. This platform, which is responsible for interacting with more than 10 regulatory binding partners, comprises an extraordinary array of over 150 -helices. Analysis of these EM data revealed that the GRD and SEC-PH domain are highly mobile with respect to the core scaffold and could not initially be accurately placed in electron density. Strikingly, however, using 3D variability analysis we were able to identify a significant subpopulation of NF1 particles and determine the complete NF1 structure to 5.6 [A] resolution. These data revealed that the catalytic GRD and lipid binding SEC-PH domain are positioned against the core scaffold in a closed, autoinhibited conformation. We postulate that interaction with the plasma membrane may release the closed conformation in order to promote RAS inactivation. Our structural data further allow us to map the location of disease-associated NF1 variants and provide a long sought-after structural explanation for the extreme susceptibility of the molecule to loss-of-function mutations. Finally, it is suggested that approaches to combat NF1-linked diseases may include release of the autoinhibited state in order to improve NF1 catalytic efficiency.

biochemistry↗

bioRxiv2021

Periplasm homeostatic regulation maintains spatial constraints essential for cell envelope processes and cell viability

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics and a synthetic lethal screen we show that lengthening Lpp to the upper limit does not change the spatial constraint, but rather impacts the load-bearing capacity across the outer membrane. Our findings demonstrate E. coli expressing elongated Lpp homeostatically counteracts periplasmic enlargement with a combination of tilting Lpp and reducing Lpp abundance. By genetic screening we identified all of the genes in E. coli that become essential in order to enact this homeostasis, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to enact this homeostasis. We observed increased levels of factors determining cell stiffness, decrease membrane integrity, increase membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling and protein translocation

cell biology↗

bioRxiv2020

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3 untranslated region in order to regulate mRNA function. Here we present a systematic analysis of 3 end formation factors, which revealed 3UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3UTRs. We show that the anti-cancer drug cordycepin, 3 deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3 end processing and controlled by the availability of 3 end factors, nucleotide levels and chromatin landscape.

cell biology↗

bioRxiv2020

Structural analysis of the PTEN:P-Rex2 signalling node reveals how cancer-associated mutations coordinate to hyperactivate Rac1

The PTEN:P-Rex2 complex is one of the most commonly mutated signaling nodes in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cell proliferation. Here, using extensive crosslinking mass spectrometry and functional studies, we provide crucial mechanistic insights into PTEN:P-Rex2 complex assembly and co-inhibition. PTEN is anchored to P-Rex2 by interactions between the PTEN PDZ-BM tail and the second PDZ domain of P-Rex2. This interaction bridges PTEN across the P-Rex2 surface, occluding PTEN membrane-binding and PI(3,4,5)P3 hydrolysis. Conversely, PTEN both allosterically promotes an autoinhibited P-Rex2 conformation and occludes G{beta}{gamma} binding and GPCR activation. These insights allow us to define a new gain-of-function class of cancer mutations within the PTEN:P-Rex2 interface that uncouples PTEN inhibition of Rac1 signaling. These findings provide a mechanistic framework to understand the dysregulation of the PTEN:P-Rex2 signaling node in metastatic cancer.

biochemistry↗

bioRxiv2020

Mycobacteria tolerate carbon monoxide by remodelling their respiratory chain

Carbon monoxide (CO) is a gas infamous for its acute toxicity. The toxicity of CO predominantly stems from its tendency to form carbonyl complexes with transition metals, thus inhibiting the heme-prosthetic groups of proteins, including the terminal oxidases of the respiratory chain. While CO has been proposed as an antibacterial agent, the evidence supporting its toxicity towards bacteria is equivocal, and its cellular targets remain poorly defined. In this work, we investigate the physiological response of mycobacteria to CO. We show that Mycobacterium smegmatis is highly resistant to the toxic effects of CO, exhibiting normal growth parameters when cultured in its presence. We profiled the proteome of M. smegmatis during growth in CO, identifying strong induction of cytochrome bd oxidase and members of the dos regulon, but relatively few other changes. We show that the activity of cytochrome bd oxidase is resistant to CO, whereas cytochrome bcc-aa3 oxidase is strongly inhibited by this gas. Consistent with these findings, growth analysis shows that M. smegmatis lacking cytochrome bd oxidase displays a significant growth defect in the presence of CO, while induction of the dos regulon appears to be unimportant for adaption to CO. Altogether, our findings suggest that M. smegmatis has considerable resistance to CO and benefits from respiratory flexibility to withstand its inhibitory effects. ImportanceCarbon monoxide has an infamous reputation as a toxic gas and it has been suggested that it has potential as an antibacterial agent. Despite this, the means by which bacteria resist its toxic effects are not well understood. In this study we determine the physiological response of Mycobacterium smegmatis to growth in CO. We show for the first time that the cytochrome bd oxidase is inherently resistant to CO and is deployed by M. smegmatis to tolerate the presence of this gas. Further, we show that aside from this remodelling of its respiratory chain, M. smegmatis makes few other functional changes to its proteome, suggesting it has a high level of inherent resistance to CO.

microbiology↗