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Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO 2 -fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire β-carboxysome. This study and recent findings on the β-carboxysome structure provide insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications., Competing Interests: Competing Interests The authors declare no conflict of interest.

Adrenal Cushing's syndrome is a disease of cortisol hypersecretion often caused by mutations in protein kinase A catalytic subunit (PKAc). Using a personalized medicine screening platform, we discovered a Cushing's driver mutation, PKAc-W196G, in ~20% of patient samples analyzed. Proximity proteomics and photokinetic imaging reveal that PKAc W196G is unexpectedly distinct from other described Cushing's variants, exhibiting retained association with type I regulatory subunits (RI) and their corresponding A kinase anchoring proteins (AKAPs). Molecular dynamics simulations predict that substitution of tryptophan-196 with glycine creates a 653-cubic angstrom cleft between the catalytic core of PKAc W196G and type II regulatory subunits (RII), but only a 395-cubic angstrom cleft with RI. Endocrine measurements show that overexpression of RIα or redistribution of PKAc W196G via AKAP recruitment counteracts stress hormone overproduction. We conclude that a W196G mutation in the kinase catalytic core skews R subunit selectivity and biases AKAP association to drive Cushing's syndrome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O 6 -methylguanine O 6 -methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O 6 -alkylguanines ( O 6 -alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O 6 -alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O 6 -alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O 6 -MethylG ( O 6 -MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S -methyl, S -ethyl, S -propyl, S -hydroxyethyl, S -carboxymethyl, S -benzyl, and S -pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O 6 -alkGs. The LOQ of ASPs containing S -methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O 6 -MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S -methylcysteine at levels that correlated with those of O 6 -MeG determined previously by HPLC-radioimmunoassay ( r 2 = 0.74; p = 0.014). O 6 -CMG, a putative O 6 -hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O 6 -alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O 6 -alkG adductome and, given the mutagenic potential of O 6 -alkGs, can provide mechanistic information about cancer pathogenesis.

Top-down proteomics (TDP) aims to identify and profile intact protein forms (proteoforms) extracted from biological samples. True proteoform characterization requires that both the base protein sequence be defined and any mass shifts identified, ideally localizing their positions within the protein sequence. Being able to fully elucidate proteoform profiles lends insight into characterizing proteoform-unique roles, and is a crucial aspect of defining protein structure-function relationships and the specific roles of different (combinations of) protein modifications. However, defining and pinpointing protein post-translational modifications (PTMs) on intact proteins remains a challenge. Characterization of (heavily) modified proteins (>∼30 kDa) remains problematic, especially when they exist in a population of similarly modified, or kindred, proteoforms. This issue is compounded as the number of modifications increases, and thus the number of theoretical combinations. Here, we present our perspective on the challenges of analyzing kindred proteoform populations, focusing on annotation of protein modifications on an "average" protein. Furthermore, we discuss the technical requirements to obtain high quality fragmentation spectral data to robustly define site-specific PTMs, and the fact that this is tempered by the time requirements necessary to separate proteoforms in advance of mass spectrometry analysis.

Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalyzed by Golgi-resident tyrosyl protein sulfo transferases (TPSTs). Information on sY in humans is currently limited to ∼50 proteins, with only a handful having verified sites of sulfation. As such, the contribution of sulfation to the regulation of biological processes remains poorly defined. Mass spectrometry (MS)-based proteomics is the method of choice for PTM analysis but has yet to be applied for systematic investigation of the "sulfome", primarily due to issues associated with discrimination of sY-containing from phosphotyrosine (pY)-containing peptides. In this study, we developed an MS-based workflow for sY-peptide characterization, incorporating optimized Zr 4+ immobilized metal-ion affinity chromatography (IMAC) and TiO 2 enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of fragmentation regimes (CID, HCD, EThcD, ETciD, UVPD) highlighted differences in the generation of site-determining product ions and allowed us to develop a strategy for differentiating sulfated peptides from nominally isobaric phosphopeptides based on low collision energy-induced neutral loss. Application of our "sulfomics" workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically, and reveals new interplay between enzymes relevant to both protein and glycan sulfation.

Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The doublecortin-like kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory 'tail' segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations, and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to 'supercharge' the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other calcium calmodulin kinases (CAMKs), and a 'Swiss Army' assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for autoregulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome-wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically divergent DCLK1 modulators, stabilizers, or degraders., Competing Interests: AV, GW, DB, BO, SS, NG, EF, LD, CB, WY, IA, SK, CE, PE, NK No competing interests declared, (© 2023, Venkat, Watterson, Byrne et al.)

Post-translational modifications (PTMs) are ubiquitous and key to regulating protein function. Understanding the dynamics of individual PTMs and their biological roles requires robust characterisation. Mass spectrometry (MS) is the method of choice for the identification and quantification of protein modifications. This article focusses on the MS-based analysis of those covalent modifications that induce a mass shift of +80 Da, notably phosphorylation and sulfation, given the challenges associated with their discrimination and pinpointing the sites of modification on a polypeptide chain. Phosphorylation in particular is highly abundant, dynamic and can occur on numerous residues to invoke specific functions, hence robust characterisation is crucial to understanding biological relevance. Showcasing our work in the context of other developments in the field, we highlight approaches for enrichment and site localisation of phosphorylated (canonical and non-canonical) and sulfated peptides, as well as modification analysis in the context of intact proteins (top down proteomics) to explore combinatorial roles. Finally, we discuss the application of native ion-mobility MS to explore the effect of these PTMs on protein structure and ligand binding.

The novel coronavirus pandemic (COVID-19) has necessitated a global increase in the use of face masks to limit the airborne spread of the virus. The global demand for personal protective equipment has at times led to shortages of face masks for the public, therefore makeshift masks have become commonplace. The severe acute respiratory syndrome caused by coronavirus-2 (SARS-CoV-2) has a spherical particle size of ~97 nm. However, the airborne transmission of this virus requires the expulsion of droplets, typically ~0.6-500 µm in diameter (by coughing, sneezing, breathing, and talking). In this paper, we propose a face covering that has been designed to effectively capture SARS-CoV-2 whilst providing uncompromised comfort and breathability for the wearer. Herein, we describe a material approach that uses amorphous silica microspheres attached to cotton fibres to capture bioaerosols, including SARS CoV-2. This has been demonstrated for the capture of aerosolised proteins (cytochrome c, myoglobin, ubiquitin, bovine serum albumin) and aerosolised inactivated SARS CoV-2, showing average filtration efficiencies of ~93% with minimal impact on breathability., (© 2023. Springer Nature Limited.)

Schistosomes are blood flukes with specialised tissues and organs, each one playing a pivotal role in perpetuating the parasite life cycle. Herein, we describe a detailed methodology for preserving the proteome of adult Schistosoma mansoni worms during manual dissection for enrichment of tissues associated with the parasite's alimentary tract. We provide step-by-step directions for specimen storage and dissection while in preservative solution, tissue homogenisation, protein extraction and digestion using a methodology fully compatible with downstream quantitative liquid chromatography-mass spectrometry analysis. Our methodology uses label-free and QconCAT-based absolute quantification for detection of S. mansoni oesophageal gland products proposed as vaccine candidates. Through stabilisation of the proteome and minimising sample degradation during dissection our approach has allowed us to access the hidden proteome of target tissues not readily available from total lysates because of their small volume. This protocol can be replicated or adapted to other Schistosoma species lacking quantitative proteomics characterisation of specialised tissues for discovery of proteins with potential diagnostic and therapeutic utility., (© 2023 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Pseudokinases, so named because they lack one or more conserved canonical amino acids that define their catalytically active relatives, have evolved a variety of biological functions in both prokaryotic and eukaryotic organisms. Human PSKH2 is closely related to the canonical kinase PSKH1, which maps to the CAMK family of protein kinases. Primates encode PSKH2 in the form of a pseudokinase, which is predicted to be catalytically inactive due to loss of the invariant catalytic Asp residue. Although the biological role(s) of vertebrate PSKH2 proteins remains unclear, we previously identified species-level adaptions in PSKH2 that have led to the appearance of kinase or pseudokinase variants in vertebrate genomes alongside a canonical PSKH1 paralog. In this paper we confirm that, as predicted, PSKH2 lacks detectable protein phosphotransferase activity, and exploit structural informatics, biochemistry and cellular proteomics to begin to characterise vertebrate PSKH2 orthologues. AlphaFold 2-based structural analysis predicts functional roles for both the PSKH2 N- and C-regions that flank the pseudokinase domain core, and cellular truncation analysis confirms that the N-terminal domain, which contains a conserved myristoylation site, is required for both stable human PSKH2 expression and localisation to a membrane-rich subcellular fraction containing mitochondrial proteins. Using mass spectrometry-based proteomics, we confirm that human PSKH2 is part of a cellular mitochondrial protein network, and that its expression is regulated through client-status within the HSP90/Cdc37 molecular chaperone system. HSP90 interactions are mediated through binding to the PSKH2 C-terminal tail, leading us to predict that this region might act as both a cis and trans regulatory element, driving outputs linked to the PSKH2 pseudokinase domain that are important for functional signalling., (© 2023 The Author(s).)

Previously, we discovered that deletion of c-Rel in the Eµ-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that Eµ-Myc/cRel-/- cells have an unexpected and major defect in the CHK1 pathway. Total and phospho proteomic analysis revealed that Eµ-Myc/cRel-/- lymphomas highly resemble wild-type (WT) Eµ-Myc lymphomas treated with an acute dose of the CHK1 inhibitor (CHK1i) CCT244747. Further analysis demonstrated that this is a consequence of Eµ-Myc/cRel-/- lymphomas having lost expression of CHK1 protein itself, an effect that also results in resistance to CCT244747 treatment in vivo. Similar down-regulation of CHK1 protein levels was also seen in CHK1i resistant U2OS osteosarcoma and Huh7 hepatocellular carcinoma cells. Further investigation revealed that the deubiquitinase USP1 regulates CHK1 proteolytic degradation and that its down-regulation in our model systems is responsible, at least in part, for these effects. We demonstrate that treating WT Eµ-Myc lymphoma cells with the USP1 inhibitor ML323 was highly effective at reducing tumour burden in vivo. Targeting USP1 activity may thus be an alternative therapeutic strategy in MYC-driven tumours., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The development of resistance and the activation of bypass pathway signalling represents a major problem for the clinical application of protein kinase inhibitors. While investigating the effect of either a c-Rel deletion or RelAT505A phosphosite knockin on the Eµ-Myc mouse model of B-cell lymphoma, we discovered that both NF-κB subunit mutations resulted in CHK1 inhibitor resistance, arising from either loss or alteration of CHK1 activity, respectively. However, since Eµ-Myc lymphomas depend on CHK1 activity to cope with high levels of DNA replication stress and consequent genomic instability, it was not clear how these mutant NF-κB subunit lymphomas were able to survive. To understand these survival mechanisms and to identify potential compensatory bypass signalling pathways in these lymphomas, we applied a multi-omics strategy. With c-Rel-/- Eµ-Myc lymphomas we observed high levels of Phosphatidyl-inositol 3-kinase (PI3K) and AKT pathway activation. Moreover, treatment with the PI3K inhibitor Pictilisib (GDC-0941) selectively inhibited the growth of reimplanted c-Rel-/- and RelAT505A, but not wild type (WT) Eµ-Myc lymphomas. We also observed up-regulation of a RHO/RAC pathway gene expression signature in both Eµ-Myc NF-κB subunit mutation models. Further investigation demonstrated activation of the RHO/RAC effector p21-activated kinase (PAK) 2. Here, the PAK inhibitor, PF-3758309 successfully overcame resistance of RelAT505A but not WT lymphomas. These findings demonstrate that up-regulation of multiple bypass pathways occurs in CHK1 inhibitor resistant Eµ-Myc lymphomas. Consequently, drugs targeting these pathways could potentially be used as either second line or combinatorial therapies to aid the successful clinical application of CHK1 inhibitors., (© 2022 The Author(s).)

Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing's syndrome. We define mechanisms of action for the PKAc-L205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing's mutants are excluded from A kinase-anchoring protein (AKAP)-signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knockin mice and Cushing's syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing's mutant engages a different mitogenic-signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing's variant promotes the transmission of distinct downstream pathogenic signals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glycogen is the major glucose reserve in eukaryotes, and defects in glycogen metabolism and structure lead to disease. Glycogenesis involves interaction of glycogenin (GN) with glycogen synthase (GS), where GS is activated by glucose-6-phosphate (G6P) and inactivated by phosphorylation. We describe the 2.6 Å resolution cryo-EM structure of phosphorylated human GS revealing an autoinhibited GS tetramer flanked by two GN dimers. Phosphorylated N- and C-termini from two GS protomers converge near the G6P-binding pocket and buttress against GS regulatory helices. This keeps GS in an inactive conformation mediated by phospho-Ser641 interactions with a composite "arginine cradle". Structure-guided mutagenesis perturbing interactions with phosphorylated tails led to increased basal/unstimulated GS activity. We propose that multivalent phosphorylation supports GS autoinhibition through interactions from a dynamic "spike" region, allowing a tuneable rheostat for regulating GS activity. This work therefore provides insights into glycogen synthesis regulation and facilitates studies of glycogen-related diseases., (© 2022. The Author(s).)

Public phosphorylation databases such as PhosphoSitePlus (PSP) and PeptideAtlas (PA) compile results from published papers or openly available mass spectrometry (MS) data. However, there is no database-level control for false discovery of sites, likely leading to the overestimation of true phosphosites. By profiling the human phosphoproteome, we estimate the false discovery rate (FDR) of phosphosites and predict a more realistic count of true identifications. We rank sites into phosphorylation likelihood sets and analyze them in terms of conservation across 100 species, sequence properties, and functional annotations. We demonstrate significant differences between the sets and develop a method for independent phosphosite FDR estimation. Remarkably, we report estimated FDRs of 84, 98, and 82% within sets of phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr) sites, respectively, that are supported by only a single piece of identification evidence─the majority of sites in PSP. We estimate that around 62 000 Ser, 8000 Thr, and 12 000 Tyr phosphosites in the human proteome are likely to be true, which is lower than most published estimates. Furthermore, our analysis estimates that 86 000 Ser, 50 000 Thr, and 26 000 Tyr phosphosites are likely false-positive identifications, highlighting the significant potential of false-positive data to be present in phosphorylation databases.

Protein kinase inhibitors are highly effective in treating diseases driven by aberrant kinase signaling and as chemical tools to help dissect the cellular roles of kinase signaling complexes. Evaluating the effects of binding of small molecule inhibitors on kinase conformational dynamics can assist in understanding both inhibition and resistance mechanisms. Using gas-phase ion-mobility mass spectrometry (IM-MS), we characterize changes in the conformational landscape and stability of the protein kinase Aurora A (Aur A) driven by binding of the physiological activator TPX2 or small molecule inhibition. Aided by molecular modeling, we establish three major conformations, the relative abundances of which were dependent on the Aur A activation status: one highly populated compact conformer similar to that observed in most crystal structures, a second highly populated conformer possessing a more open structure infrequently found in crystal structures, and an additional low-abundance conformer not currently represented in the protein databank. Notably, inhibitor binding induces more compact configurations of Aur A, as adopted by the unbound enzyme, with both IM-MS and modeling revealing inhibitor-mediated stabilization of active Aur A.

Dietary restriction (DR) has been shown to increase lifespan in organisms ranging from yeast to mammals. This suggests that the underlying mechanisms may be evolutionarily conserved. Indeed, upstream signalling pathways, such as TOR, are strongly linked to DR-induced longevity in various organisms. However, the downstream effector proteins that ultimately mediate lifespan extension are less clear. To shed light on this, we used a proteomic approach on budding yeast. Our reasoning was that analysis of proteome-wide changes in response to DR might enable the identification of proteins that mediate its physiological effects, including replicative lifespan extension. Of over 2500 proteins we identified by liquid chromatography-mass spectrometry, 183 were significantly altered in expression by at least 3-fold in response to DR. Most of these proteins were mitochondrial and/or had clear links to respiration and metabolism. Indeed, direct analysis of oxygen consumption confirmed that mitochondrial respiration was increased several-fold in response to DR. In addition, several key proteins involved in mating, including Ste2 and Ste6, were down-regulated by DR. Consistent with this, shmoo formation in response to α-factor pheromone was reduced by DR, thus confirming the inhibitory effect of DR on yeast mating. Finally, we found that Hsp26, a member of the conserved small heat shock protein (sHSP) family, was up-regulated by DR and that overexpression of Hsp26 extended yeast replicative lifespan. As overexpression of sHSPs in Caenorhabditis elegans and Drosophila has previously been shown to extend lifespan, our data on yeast Hsp26 suggest that sHSPs may be universally conserved effectors of longevity., (© 2021 The Author(s).)

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19., (© 2021. The Author(s).)

Cellular adaptation to low-oxygen environments is mediated in part by the hypoxia-inducible factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs-and consequently, the hypoxic response-are regulated by post-translational modifications (PTMs) and changes in protein-protein interactions. Our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment-based studies typically validated in fragment-expressing cells treated with hypoxia-mimicking compounds. Here, we used immunoprecipitation-based mass spectrometry to characterize the PTMs and binding partners for full-length HIF-1α and HIF-2α under normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. Hypoxia substantially altered the complexity and composition of the HIFα protein interaction networks, particularly for HIF-2α, with the hypoxic networks of both isoforms being enriched for mitochondrial proteins. Moreover, both HIFα isoforms were heavily covalently modified. We identified ~40 PTM sites composed of 13 different types of modification on both HIFα isoforms, including multiple cysteine modifications and an unusual phosphocysteine. More than 80% of the PTMs identified were not previously known and about half exhibited oxygen dependency. We further characterized an evolutionarily conserved phosphorylation of Ser 31 in HIF-1α as a regulator of its transcriptional function, and we propose functional roles for Thr 406 , Thr 528 , and Ser 581 in HIF-2α. These data will help to delineate the different physiological roles of these closely related isoforms in fine-tuning the hypoxic response., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3ε binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA., (© 2021 The Author(s).)

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely 'non-canonical' substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling., (© 2020 The Author(s).)

Reactive oxygen species (ROS) are physiological mediators of cellular signaling and play potentially damaging roles in human diseases. In this study, we found that the catalytic activity of the Ser/Thr kinase Aurora A was inhibited by the oxidation of a conserved cysteine residue (Cys 290 ) that lies adjacent to Thr 288 , a critical phosphorylation site in the activation segment. Cys is present at the equivalent position in ~100 human Ser/Thr kinases, a residue that we found was important not only for the activity of human Aurora A but also for that of fission yeast MAPK-activated kinase (Srk1) and PKA (Pka1). Moreover, the presence of this conserved Cys predicted biochemical redox sensitivity among a cohort of human CAMK, AGC, and AGC-like kinases. Thus, we predict that redox modulation of the conserved Cys 290 of Aurora A may be an underappreciated regulatory mechanism that is widespread in eukaryotic Ser/Thr kinases. Given the key biological roles of these enzymes, these findings have implications for understanding physiological and pathological responses to ROS and highlight the importance of protein kinase regulation through multivalent modification of the activation segment., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Schistosomes are blood-dwelling helminth parasites that cause schistosomiasis, a debilitating disease resulting in inflammation and, in extreme cases, multiple organ damage. Major challenges to control the transmission persist, and the discovery of protective antigens remains of critical importance for vaccine development. Rhesus macaques can self-cure following schistosome infection, generating antibodies that target proteins from the tegument, gut, and esophagus, the last of which is the least investigated. We developed a dissection technique that permitted increased sensitivity in a comparative proteomics profiling of schistosome esophagus and gut. Proteome analysis of the male schistosome esophagus identified 13 proteins encoded by microexon genes (MEGs), 11 of which were uniquely located in the esophageal glands. Based on this and transcriptome information, a QconCAT was designed for the absolute quantification of selected targets. MEGs 12, 4.2, and 4.1 and venom allergen-like protein 7 were the most abundant, spanning over 245 million to 6 million copies per cell, while aspartyl protease, palmitoyl thioesterase, and galactosyl transferase were present at <1 million copies. Antigenic variation by alternative splicing of MEG proteins was confirmed together with a specialized machinery for protein glycosylation/secretion in the esophagus. Moreover, some gastrodermal secretions were highly enriched in the gut, while others were more uniformly distributed throughout the parasite, potentially indicating lysosomal activity. Collectively, our findings provide a more rational, better-oriented selection of schistosome vaccine candidates in the context of a proven model of protective immunity.

Liquid chromatography (LC)-tandem mass spectrometry (MS/MS) is key for the characterization of phosphorylation sites in a high-throughput manner, and its application has proven essential to elucidate the phosphoproteome of many biological systems. Following proteolytic digestion of proteins extracted from tissues or cells, phosphopeptides are typically enriched by affinity chromatography using TiO 2 or metal-ions (e.g., Fe 3+ ) coupled to solid-phase materials, prior to LC-MS/MS analysis. Separation of relatively low abundance phosphopeptides from nonphosphorylated peptides in these types of extremely complex mixtures is essential to maximize coverage of the phosphoproteome. Maintaining acidic conditions during these IMAC or TiO 2 -based enrichment minimizes the concurrent unwanted binding of highly acidic peptides. However, while peptides containing phosphomonoesters, namely, phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr), are stable under these acidic binding conditions, phosphopeptides containing acid-labile phosphate group such as phosphohistidine (pHis), are not. Consequently, hydrolysis of these types of phosphopeptides occurs during standard phosphopeptide enrichment, and subsequent phosphosite identification by LC-MS/MS is severely compromised. Here we describe UPAX, unbiased phosphopeptide enrichment using strong anion exchange, for the separation of both acid-stable (pSer, pThr, pTyr) and acid-labile phosphopeptides (including those containing pHis) from nonphosphorylated peptides. We outline how implementation of UPAX prior to a minimally modified standard proteomics workflow can be used to identify sites of pHis as well as other acid-labile, as well as acid-stable phosphosites.

Ion mobility-mass spectrometry (IM-MS) of intact protein complexes under native conditions is a powerful tool for the analysis of protein complexes and protein-ligand interactions, permitting insight into ligand-induced changes in protein conformation. Here we describe a procedure for analyzing the effects of phosphorylation and/or inhibitor binding on protein kinase conformational flexibility using Protein Kinase A (PKA) as a model system. By calculating the protein collision cross section (CCS) before and after inhibitor binding, and additionally by performing collision-induced unfolding (CIU), we can establish the effects of protein modification or small molecule binding on protein dynamics.

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a "dual anchor" mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Protein histidine phosphorylation has largely remained unexplored due to the challenges of analyzing relatively unstable phosphohistidine-containing proteins. We describe a procedure for determining the stoichiometry of histidine phosphorylation on the human histidine kinases NME1 and NME2 by intact mass spectrometry under conditions that retain this acid-labile protein modification. By characterizing these two model histidine protein kinases in the absence and presence of a suitable phosphate donor, the stoichiometry of histidine phosphorylation can be determined. The described method can be readily adapted for the analysis of other proteins containing phosphohistidine.

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site-specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other "non-canonical" amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non-canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry-based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non-canonical phosphosites is approximately one-third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high-throughput exploration of non-canonical phosphorylation in all organisms., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Carbohydrates possess a variety of distinct features with stereochemistry playing a particularly important role in distinguishing their structure and function. Monosaccharide building blocks are defined by a high density of chiral centers. Additionally, the anomericity and regiochemistry of the glycosidic linkages carry important biological information. Any carbohydrate-sequencing method needs to be precise in determining all aspects of this stereodiversity. Recently, several advances have been made in developing fast and precise analytical techniques that have the potential to address the stereochemical complexity of carbohydrates. This perspective seeks to provide an overview of some of these emerging techniques, focusing on those that are based on NMR and MS-hybridized technologies including ion mobility spectrometry and IR spectroscopy.

The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKA c ) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKA c - and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKA c -regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κB DNA. Graphical Abstract ᅟ.

Protein phosphorylation is a universal covalent chemical modification of amino acids involved in a large number of biological processes including cell signaling, metabolism, proliferation, differentiation, survival/death, ageing, and many more. Regulation of protein phosphorylation is essential in myogenesis and indeed, when the enzymatic activity of protein kinases is distrupted in myoblasts, myogenesis is affected. In this chapter we describe a method to profile the phosphoproteome of myoblasts using mass spectrometry. Phosphate groups are labile and easily lost during the processing of samples for mass spectrometry. Thus, effective methods to enrich for phosphopeptides from protein extracts have been developed. Here, we discuss and present in detail two such methods that we routinely employ. These methods are based on a sample enrichment step performed on titanium dioxide matrices followed by label-free tandem mass spectrometry and semi-quantitation.

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein down-regulation through drug "off-targets" might be relevant for other inhibitors that serendipitously target pseudokinases., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2- O -sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 2- O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro , including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST., (© 2018 The Author(s).)

Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein-protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based inhibitors of the Ser/Thr kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors., (© 2018 The Author(s).)

Temperature fluctuation is a common environmental stress that elicits a molecular response in order to maintain intracellular protein levels. Here, for the first time, we report a comprehensive temporal and quantitative study of the proteome during a 240 minute heat stress, using label-free mass spectrometry. We report temporal expression changes of the hallmark heat stress proteins, including many molecular chaperones, tightly coupled to their protein clients. A notable lag of 30 to 120 minutes was evident between transcriptome and proteome levels for differentially expressed genes. This targeted molecular response buffers the global proteome; fewer than 15% of proteins display significant abundance change. Additionally, a parallel study in a Hsp70 chaperone mutant (ssb1Δ) demonstrated a significantly attenuated response, at odds with the modest phenotypic effects that are observed on growth rate. We cast the global changes in temporal protein expression into protein interaction and functional networks, to afford a unique, time-resolved and quantitative description of the heat shock response in an important model organism.