Polysaccharide sulfotransferases: the identification of putative sequences and respective functional characterisation.

The vast structural diversity of sulfated polysaccharides demands an equally diverse array of enzymes known as polysaccharide sulfotransferases (PSTs). PSTs are present across all kingdoms of life, including algae, fungi and archaea, and their sulfation pathways are relatively unexplored. Sulfated polysaccharides possess anti-inflammatory, anticoagulant and anti-cancer properties and have great therapeutic potential. Current identification of PSTs using Pfam has been predominantly focused on the identification of glycosaminoglycan (GAG) sulfotransferases because of their pivotal roles in cell communication, extracellular matrix formation and coagulation. As a result, our knowledge of non-GAG PSTs structure and function remains limited. The major sulfotransferase families, Sulfotransfer_1 and Sulfotransfer_2, display broad homology and should enable the capture of a wide assortment of sulfotransferases but are limited in non-GAG PST sequence annotation. In addition, sequence annotation is further restricted by the paucity of biochemical analyses of PSTs. There are now high-throughput and robust assays for sulfotransferases such as colorimetric PAPS (3'-phosphoadenosine 5'-phosphosulfate) coupled assays, Europium-based fluorescent probes for ratiometric PAP (3'-phosphoadenosine-5'-phosphate) detection, and NMR methods for activity and product analysis. These techniques provide real-time and direct measurements to enhance the functional annotation and subsequent analysis of sulfated polysaccharides across the tree of life to improve putative PST identification and characterisation of function. Improved annotation and biochemical analysis of PST sequences will enhance the utility of PSTs across biomedical and biotechnological sectors.

Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms.

In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications (PTMs), including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study, we investigate redox regulation of the AMPK-related Brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intramolecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.

Discovery of a Cushing's syndrome protein kinase A mutant that biases signaling through type I AKAPs.

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 PKAcW196G 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 PKAcW196G 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 PKAcW196G 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.

Custom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides.

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 Zr4+ immobilized metal-ion affinity chromatography (IMAC) and TiO2 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.

Mechanistic and evolutionary insights into isoform-specific 'supercharging' in DCLK family kinases.

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.

A peroxiredoxin-P38 MAPK scaffold increases MAPK activity by MAP3K-independent mechanisms.

Peroxiredoxins (Prdxs) utilize reversibly oxidized cysteine residues to reduce peroxides and promote H2O2 signal transduction, including H2O2-induced activation of P38 MAPK. Prdxs form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here, we show that a genetically encoded fusion between a Prdx and P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. We demonstrate that a P38-Prdx fusion protein compensates for loss of the yeast scaffold protein Mcs4 and MAP3K activity, driving yeast into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides an alternative scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx is sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.

Classification of Cushing's syndrome PKAc mutants based upon their ability to bind PKI.

Cushing's syndrome is an endocrine disorder caused by excess production of the stress hormone cortisol. Precision medicine strategies have identified single allele mutations within the PRKACA gene that drive adrenal Cushing's syndrome. These mutations promote perturbations in the catalytic core of protein kinase A (PKAc) that impair autoinhibition by regulatory subunits and compartmentalization via recruitment into AKAP signaling islands. PKAcL205R is found in ∼45% of patients, whereas PKAcE31V, PKAcW196R, and L198insW and C199insV insertion mutants are less prevalent. Mass spectrometry, cellular, and biochemical data indicate that Cushing's PKAc variants fall into two categories: those that interact with the heat-stable protein kinase inhibitor PKI, and those that do not. In vitro activity measurements show that wild-type PKAc and W196R activities are strongly inhibited by PKI (IC50 < 1 nM). In contrast, PKAcL205R activity is not blocked by the inhibitor. Immunofluorescent analyses show that the PKI-binding variants wild-type PKAc, E31V, and W196R are excluded from the nucleus and protected against proteolytic processing. Thermal stability measurements reveal that upon co-incubation with PKI and metal-bound nucleotide, the W196R variant tolerates melting temperatures 10°C higher than PKAcL205. Structural modeling maps PKI-interfering mutations to a ∼20 Šdiameter area at the active site of the catalytic domain that interfaces with the pseudosubstrate of PKI. Thus, Cushing's kinases are individually controlled, compartmentalized, and processed through their differential association with PKI.

Investigating the Genetic Diversity of H5 Avian Influenza Viruses in the United Kingdom from 2020-2022.

Since 2020, the United Kingdom and Europe have experienced annual epizootics of high-pathogenicity avian influenza virus (HPAIV). The first epizootic, during the autumn/winter of 2020-2021, involved six H5Nx subtypes, although H5N8 HPAIV dominated in the United Kingdom. While genetic assessments of the H5N8 HPAIVs within the United Kingdom demonstrated relative homogeneity, there was a background of other genotypes circulating at a lower degree with different neuraminidase and internal genes.  Following a small number of detections of H5N1 in wild birds over the summer of 2021, the autumn/winter of 2021-2022 saw another European H5 HPAIV epizootic that dwarfed the prior epizootic. This second epizootic was dominated almost exclusively by H5N1 HPAIV, although six distinct genotypes were defined. We have used genetic analysis to evaluate the emergence of different genotypes and proposed reassortment events that have been observed. The existing data suggest that the H5N1 viruses circulating in Europe during late 2020 continued to circulate in wild birds throughout 2021, with minimal adaptation, but then went on to reassort with AIVs in the wild bird population. We have undertaken an in-depth genetic assessment of H5 HPAIVs detected in the United Kingdom over two winter seasons and demonstrate the utility of in-depth genetic analyses in defining the diversity of H5 HPAIVs circulating in avian species, the potential for zoonotic risk, and whether incidents of lateral spread can be defined over independent incursions of infections from wild birds. This provides key supporting data for mitigation activities. IMPORTANCE High-pathogenicity avian influenza virus (HPAIV) outbreaks devastate avian species across all sectors, having both economic and ecological impacts through mortalities in poultry and wild birds, respectively. These viruses can also represent a significant zoonotic risk. Since 2020, the United Kingdom has experienced two successive outbreaks of H5 HPAIV. While H5N8 HPAIV was predominant during the 2020-2021 outbreak, other H5 subtypes were also detected. The following year, there was a shift in the subtype dominance to H5N1 HPAIV, but multiple H5N1 genotypes were detected. Through the thorough utilization of whole-genome sequencing, it was possible to track and characterize the genetic evolution of these H5 HPAIVs in United Kingdom poultry and wild birds. This enabled us to assess the risk posed by these viruses at the poultry-wild bird and the avian-human interfaces and to investigate the potential lateral spread between infected premises, a key factor in understanding the threat to the commercial sector.

Effectiveness of laparoscopic removal of isolated superficial peritoneal endometriosis for the management of chronic pelvic pain in women (ESPriT2): protocol for a multi-centre randomised controlled trial.

BACKGROUND: Endometriosis affects 190 million women and those assigned female at birth worldwide. For some, it is associated with debilitating chronic pelvic pain. Diagnosis of endometriosis is often achieved through diagnostic laparoscopy. However, when isolated superficial peritoneal endometriosis (SPE), the most common endometriosis subtype, is identified during laparoscopy, limited evidence exists to support the common decision to surgically remove it via excision or ablation. Improved understanding of the impact of surgical removal of isolated SPE for the management of chronic pelvic pain in women is required. Here, we describe our protocol for a multi-centre trial to determine the effectiveness of surgical removal of isolated SPE for the management of endometriosis-associated pain. METHODS: We plan to undertake a multi-centre participant-blind parallel-group randomised controlled clinical and cost-effectiveness trial with internal pilot. We plan to randomise 400 participants from up to 70 National Health Service Hospitals in the UK. Participants with chronic pelvic pain awaiting diagnostic laparoscopy for suspected endometriosis will be consented by the clinical research team. If isolated SPE is identified at laparoscopy, and deep or ovarian endometriosis is not seen, participants will be randomised intraoperatively (1:1) to surgical removal (by excision or ablation or both, according to surgeons' preference) versus diagnostic laparoscopy alone. Randomisation with block-stratification will be used. Participants will be given a diagnosis but will not be informed of the procedure they received until 12 months post-randomisation, unless required. Post-operative medical treatment will be according to participants' preference. Participants will be asked to complete validated pain and quality of life questionnaires at 3, 6 and 12 months after randomisation. Our primary outcome is the pain domain of the Endometriosis Health Profile-30 (EHP-30), via a between randomised group comparison of adjusted means at 12 months. Assuming a standard deviation of 22 points around the pain score, 90% power, 5% significance and 20% missing data, 400 participants are required to be randomised to detect an 8-point pain score difference. DISCUSSION: This trial aims to provide high quality evidence of the clinical and cost-effectiveness of surgical removal of isolated SPE. TRIAL REGISTRATION: ISRCTN registry ISRCTN27244948. Registered 6 April 2021.

Mechanistic and evolutionary insights into isoform-specific 'supercharging' in DCLK family kinases.

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 auto-regulation 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.

ERK1/2 inhibitors act as monovalent degraders inducing ubiquitylation and proteasome-dependent turnover of ERK2, but not ERK1.

Innate or acquired resistance to small molecule BRAF or MEK1/2 inhibitors (BRAFi or MEKi) typically arises through mechanisms that sustain or reinstate ERK1/2 activation. This has led to the development of a range of ERK1/2 inhibitors (ERKi) that either inhibit kinase catalytic activity (catERKi) or additionally prevent the activating pT-E-pY dual phosphorylation of ERK1/2 by MEK1/2 (dual-mechanism or dmERKi). Here, we show that eight different ERKi (both catERKi or dmERKi) drive the turnover of ERK2, the most abundant ERK isoform, with little or no effect on ERK1. Thermal stability assays show that ERKi do not destabilise ERK2 (or ERK1) in vitro, suggesting that ERK2 turnover is a cellular consequence of ERKi binding. ERK2 turnover is not observed upon treatment with MEKi alone, suggesting it is ERKi binding to ERK2 that drives ERK2 turnover. However, MEKi pre-treatment, which blocks ERK2 pT-E-pY phosphorylation and dissociation from MEK1/2, prevents ERK2 turnover. ERKi treatment of cells drives the poly-ubiquitylation and proteasome-dependent turnover of ERK2 and pharmacological or genetic inhibition of Cullin-RING E3 ligases prevents this. Our results suggest that ERKi, including current clinical candidates, act as 'kinase degraders', driving the proteasome-dependent turnover of their major target, ERK2. This may be relevant to the suggestion of kinase-independent effects of ERK1/2 and the therapeutic use of ERKi.

Spillover effects of retirement: Does health vulnerability matter?

The current literature investigating the impact of retirement and the associated spousal spillover effects overlooks the unintended effects of retirement on spouses in vulnerable health, namely spouses with long-term health conditions (LTHCs). In this paper, we fill this gap in the literature and investigate the impact of an individual's retirement on their partner's health outcomes when their partner has LTHCs. Given the inherent identification challenges associated with entry into retirement, we use the pension-qualifying age in Australia as an instrument. Based on data from the Household Income and Labour Dynamics in Australia survey, we find that the husband's retirement has a positive impact on the wife's quality-adjusted life years (QALY) and other physical and mental health outcomes. We also identify redistribution of domestic workload as a key transmission mechanism of the spousal spillover effects. Women with LTHCs will see their QALY and health improve only if their husband devotes more time to domestic tasks after retirement.

Evolutionary and cellular analysis of the 'dark' pseudokinase PSKH2.

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.

Impact of fluoroquinolones and aminoglycosides on P. aeruginosa virulence factor production and cytotoxicity.

The opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments. ExoU is a phospholipase which causes destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosa in vitrothan other antimicrobial classes. Their overuse over the past decade, however, has resulted in the emergence of antibiotic resistance. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered that tobramycin disrupted T3SS expression and reduced both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-t cells at concentrations below the minimal inhibitory concentration (MIC). The fluoroquinolones moxifloxacin and ciprofloxacin, however, up-regulated the T3SS and did not inhibit and may have increased the cytotoxic effects of ExoS and ExoU.

Significantly increased risk of chronic obstructive pulmonary disease amongst adults with predominantly mild congenital heart disease.

Adults with congenital heart disease (CHD) face increased risk of various comorbid diseases. Previous work on lung dysfunction in this population has mainly focused on restrictive lung disease, in patients with severe CHD phenotypes. We examined the association of mild CHD with chronic obstructive pulmonary disease (COPD) in the UK Biobank (UKB). Electronic health records (EHR) were used to identify 3385 CHD cases and 479,765 healthy controls in UKB, before performing a case-control analysis over a 20-year study period for a total of > 9.5 M person-years of follow-up. Our analysis showed that UKB participants with CHD are at substantially greater risk of developing COPD than healthy controls (8.7% vs 3.1% prevalence, unadjusted OR 2.98, 95% CI 2.63, 3.36, P = 1.40e-53). Slightly increased rates of smoking were observed amongst CHD cases, however the association with COPD was shown to be robust to adjustment for smoking and other factors known to modulate COPD risk within a multivariable-adjusted Cox regression framework (fully adjusted HR 2.21, 95% CI 1.97, 2.48, P = 5.5e-41). Care for adults with CHD should aim to mitigate their increased risk of COPD, possibly via increased smoking cessation support.

Mislocalization of protein kinase A drives pathology in Cushing's syndrome.

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.

Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota.

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.

Analysis of human Tribbles 2 (TRIB2) pseudokinase.

Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a broad human protein interactome, including the well-studied AKT, C/EBPα and MAPK modules. Several lines of evidence indicate that human TRIB2 promotes cell survival and drug-resistance in solid tumors and blood cancers and is therefore of interest as a potential therapeutic target, although its physiological functions remain relatively poorly understood. The unique TRIB2 pseudokinase domain lacks the canonical 'DFG' motif, and subsequently possesses very low affinity for ATP in both the presence and absence of metal ions. However, TRIB2 also contains a unique cysteine-rich αC-helix, which interacts with a conserved peptide motif in its own carboxyl-terminal tail. This regulatory flanking region drives regulated interactions with distinct E3 ubiquitin ligases that serve to control the stability and turnover of TRIB2 client proteins. TRIB2 is also a low-affinity target of several known small-molecule protein kinase inhibitors, which were originally identified using purified recombinant TRIB2 proteins and a thermal shift assay. In this chapter, we discuss laboratory-based procedures for purification, stabilization and analysis of human TRIB2, including screening procedures that can be used for the identification of both reversible and covalent small molecule ligands.