Biology subjects

Skehel, M.

Publications and source records attributed to Skehel, M..

32 records · Page 2Linked to original sources

bioRxiv2024

Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites.

The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.

microbiology↗

bioRxiv2024

Mechanism of chaperone coordination during cotranslational protein folding in bacteria

Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally-distinct classes of chaperone promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavoured close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor subsequently recognises compact folding intermediates exposing extensive non-native surface and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates, and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to create a protected space for protein maturation that extends well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

biochemistry↗

bioRxiv2023

The V-ATPase/ATG16L1 axis is controlled by the V1H subunit

Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically de-acidified compartments targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to pH gradient disruption is unclear. Here, we reveal a direct role for the V1H subunit of the V-ATPase proton pump in recruiting ATG16L1. The interaction between V1H and ATG16L1 occurs within assembled V-ATPases, but not dissociated V1 complexes. This selectivity allows recruitment to be coupled to changes in V-ATPase assembly that follow pH dissipation. Cells lacking V1H undergo canonical macroautophagy but are unable to recruit ATG16L1 in response to influenza infection or ionophore drugs. We identify a loop within V1H that mediates ATG16L1 binding, which is absent in a neuronal isoform of V1H. Thus, V1H controls ATG16L1 recruitment in response to proton gradient dissipation, suggesting that the V-ATPase acts autonomously as a cell-intrinsic damage sensor.

molecular biology↗

bioRxiv2023

Multi-signal regulation of the GSK-3β homolog Rim11 governs meiosis entry in yeast

Starvation of budding yeast diploid cells induces the cell-fate program that drives meiosis and spore formation. Transcription activation of early meiotic genes (EMGs) requires the transcription activator Ime1, its DNA-binding partner Ume6, and GSK-3{beta} kinase Rim11. Phosphorylation of Ume6 by Rim11 is key for EMG activation. We report that Rim11 integrates multiple input signals to control Ume6 phosphorylation and EMG transcription. Under nutrient-rich conditions PKA represses Rim11 to low levels while TORC1 keeps Rim11 localized to the cytoplasm. Inhibiting PKA and TORC1 induces Rim11 expression and nuclear localization. Remarkably, nuclear Rim11 is required, but not sufficient, for Rim11-dependent Ume6 phosphorylation. Additionally, Ime1 is an essential anchor protein for phosphorylating Ume6. Subsequently, Ume6-Ime1 coactivator complexes form that drive EMG transcription. Our results demonstrate how varied signalling inputs (PKA/TORC1/Ime1) integrated by Rim11 determine EMG expression and entry into meiosis. We propose that the signalling-regulatory network described here generates robustness in cell-fate control.

molecular biology↗

bioRxiv2023

The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles

N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited in mammals/insects/plants by m6A methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast MTC is critical for meiosis and was known to comprise three proteins, of which two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologs. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The mRNA interacting module, comprising Ime4, Mum2, Vir1, and Kar4, exerts the MTCs m6A-independent function, while Slz1 enables the MTC catalytic function in m6A deposition. Both functions are critical for meiotic progression. Kar4 also has a mechanistically separate role from the MTC during mating. The yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent and MTC-independent functions, highlighting their complexity and paving the path towards dissecting multi-layered MTC functions in mammals.

molecular biology↗

bioRxiv2023

A chemoproteomic platform for reactive fragment profiling against the deubiquitinases

Chemoproteomics is a powerful method capable of detecting interactions between small molecules and the proteome, however its use as a high-throughput screening method for chemical libraries has so far been limited. To address this need, we have further developed a chemoproteomics workflow to screen cysteine reactive covalent fragments in cell lysates against the deubiquitinating (DUB) enzymes using activity-based protein profiling. By using targeted ubiquitin probes, we have addressed sensitivity and affinity limitations, enabling target identification and covalent fragment library profiling in a 96-well plate format. The use of data independent acquisition (DIA) methods for MS analysis combined with automated Evosep liquid chromatography (LC) reduced instrument runtimes to 21 minutes per sample and simplified the workflow. In this proof-of-concept study, we have profiled 138 covalent fragments against 57 DUB proteins and validated four hit fragments against OTUD7B and UCHL3 through site identification experiments and orthogonal biochemical activity assays. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/526632v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@2e036borg.highwire.dtl.DTLVardef@e27962org.highwire.dtl.DTLVardef@8f1caaorg.highwire.dtl.DTLVardef@1a3159a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Contribute to the Mutational Trajectory in Variants of Concern

The emergence of a polybasic cleavage motif for the protease furin in the SARS-CoV-2 spike protein has been established as a major factor for enhanced viral transmission in humans. The peptide region N-terminal to that motif is extensively mutated in major variants of concern including Alpha, Delta and Omicron. Besides furin, spike proteins from these variants appear to rely on other proteases for maturation, including TMPRSS2 that may share the same cleavage motif. Glycans found near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, with a suite of chemical tools, we establish O-linked glycosylation as a major determinant of SARS-CoV-2 spike cleavage by the host proteases furin and TMPRSS2, and as a likely driving force for the emergence of common mutations in variants of concern. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, and this glycosylation event is suppressed by many, but not all variant mutations. A novel strategy for rapid bioorthogonal modification of Thr678-containing glycopeptides revealed that introduction of a negative charge completely abrogates furin activity. In a panel of synthetic glycopeptides containing elaborated O-glycans, we found that the sole incorporation of N-acetylgalactosamine did not substantially impact furin activity, but the presence of sialic acid in elaborated O-glycans reduced furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on spike cleavage by TMPRSS2. With a chemistry-centered approach, we firmly establish O-glycosylation as a major determinant of spike maturation and propose that a disruption of O-GalNAc glycosylation is a substantial driving force for the evolution of variants of concern. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/508093v3_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@71b54eorg.highwire.dtl.DTLVardef@1361b4aorg.highwire.dtl.DTLVardef@139bdd9org.highwire.dtl.DTLVardef@1df192b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

NDR1/2 kinases regulate membrane trafficking, enable efficient autophagy and prevent neurodegeneration

Autophagy is essential for neuronal development and its deregulation contributes to neurodegenerative diseases. NDR1 and NDR2 are highly conserved kinases implicated in neuronal development, mitochondrial health and autophagy, but how they affect mammalian brain development in vivo is not known. Using single and double Ndr1/2 knockout mouse models we show that, dual, but not individual loss of Ndr1/2 in neurons causes neurodegeneration during brain development, but also in adult mice. Proteomic and phosphoproteomic comparisons between Ndr1/2 knockout and control brains revealed novel kinase substrates and indicated that endocytosis is significantly affected in the absence of NDR1/2. We validated the endocytic protein, Raph1/Lpd1 as a novel NDR1/2 substrate and showed that both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. In NDR1/2 knockout brains, we observed prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment of protein homeostasis. Furthermore, the levels of LC3-positive autophagosomes were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation and neurotoxicity. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health. HighlightsO_LIDual neuronal Ndr1 and Ndr2 knockout during development or in adult mice causes neurodegeneration. C_LIO_LIPhosphoproteomics comparison of Ndr1/2 knockouts with control littermates shows endocytosis and membrane trafficking to be affected and reveals novel substrates. C_LIO_LIRaph1/Lamellipodin is a novel NDR1/2 substrate that is required for TfR endocytosis. C_LIO_LINdr1/2 knockout brains exhibit a severe defect in ubiquitinated protein clearance and reduced autophagy. C_LIO_LINDR1/2 and Raph1 are required for the trafficking of the only transmembrane autophagy protein, ATG9A. C_LI

neuroscience↗

bioRxiv2021

Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace

mTORC1 is a kinase complex regulating cell growth, proliferation and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTORs PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. Here we show, in contrast to previous cellular studies, that both the PDZ and linker regions are essential for inhibition, and it is likely that interaction with the FRB is crucial to the unique partial inhibition.

biochemistry↗

bioRxiv2021

Interactome analysis of C. elegans synapses by TurboID-based proximity labeling

Proximity labelling provides a powerful in vivo tool to characterize the proteome of sub-cellular structures and the interactome of specific proteins. Using the highly active biotin ligase TurboID, we optimize a proximity labelling protocol for C. elegans. We use this protocol to characterise the proteomes of the worms gut, muscle, skin, and nervous system. We express TurboID exclusively in the pair of AFD neurons and show we can identify known and previously unknown proteins expressed selectively in AFD. We knock TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identify many known ELKS-1 interacting proteins as well as previously uncharacterised synaptic proteins. Versatile vectors, and the inherent advantage of C. elegans for biochemistry, make proximity labelling a valuable addition to the nematodes armory. TeaserWe optimize a TurboID proximity labeling protocol for C. elegans and use it to characterize tissue and synaptic proteomes

biochemistry↗

bioRxiv2020

Shulin packages axonemal outer dynein arms for ciliary targeting

The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During cilio-genesis, these [~]1.8 MDa complexes are assembled in the cytoplasm and targeted to cilia via an unknown mechanism. Here we use the ciliate Tetrahymena to identify two novel factors (Q22YU3 and Q22MS1) which bind ODAs in the cytoplasm and are required for their delivery to cilia. We show that Q22YU3, which we name Shulin, locks the ODA motor domains into a closed conformation and inhibits motor activity. Cryo-EM reveals how Shulin stabilizes this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.

molecular biology↗

bioRxiv2019

Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork

The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), that have important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3-3.5 [A] resolution cryo-EM structures comprising CMG, Ctf4, Csm3/Tof1 and Mrc1 at a replication fork. The structures provide high-resolution views of CMG:DNA interactions, revealing the mechanism of strand separation. Furthermore, they illustrate the topology of Mrc1 in the replisome and show Csm3/Tof1 grips duplex DNA ahead of CMG via a network of interactions that are important for efficient replication fork pausing. Our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.

biochemistry↗

bioRxiv2019

MALT1 mediates IL-17 Neural Signaling to regulate C. elegans behavior, immunity and longevity

Besides well-known immune roles, the evolutionarily ancient cytokine interleukin-17 (IL-17) modulates neural circuit function. We investigate how IL-17 signals in neurons, and the extent to which this signaling can alter organismal phenotypes. We combine immunoprecipitation and mass spectrometry to biochemically characterize endogenous signaling complexes that function downstream of IL-17 receptors in C. elegans (Ce) neurons. We identify the Ce ortholog of MALT1 as a critical output of the pathway. MALT1 was not previously implicated in IL-17 signaling or in nervous system function. MALT1 forms a complex with homologs of Act1 and IRAK and functions both as a scaffold for I{kappa}B recruitment, and as a protease. MALT1 is expressed broadly in the Ce nervous system, and neuronal IL-17-MALT1 signaling regulates many phenotypes, including escape behavior, associative learning, immunity and longevity. Our data suggest MALT1 has an ancient role modulating neural function downstream of IL-17 to remodel physiological and behavioral state.

neuroscience↗

bioRxiv2018

Covalent Aurora A regulation by the metabolic integrator coenzyme A

Aurora A is a cell cycle protein kinase implicated in multiple human cancers, and several Aurora A-specific kinase inhibitors have progressed into clinical trials. In this study, we report structural and cellular analysis of a novel biochemical mode of Aurora A inhibition, which occurs through reversible covalent interaction with the universal metabolic integrator coenzyme A (CoA). Mechanistically, the CoA 3-phospho ADP moiety interacts with Thr 217, an Aurora A selectivity filter, which permits the formation of an unprecedented covalent bond with Cys 290 in the kinase activation segment, lying some 15 [A] away. CoA modification (CoAlation) of endogenous Aurora A is rapidly induced by oxidative stresses at Cys 290 in human cells, and microinjection of CoA into mouse embryos perturbs meitoic spindle formation and chromosome alignment. Aurora A regulation by CoA reveals how targeting of Aurora A might be accomplished in the future by development of a double-anchored covalent inhibitor.

biochemistry↗