David Tooth

Role of Tyr84 in controlling the reactivity of Cys34 of human albumin.

Cys34 in domain I of the three‐domain serum protein albumin is the binding site for a wide variety of biologically and clinically important small molecules, provides antioxidant activity, and constitutes the largest portion of free thiol in blood. Analysis of X‐ray structures of albumin reveals that the loop containing Tyr84 occurs in multiple conformations. In structures where the loop is well defined, there appears to be an H‐bond between the OH of Tyr84 and the sulfur of Cys34. We show that the reaction of 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) with Tyr84Phe mutant albumin is approximately four times faster than with the wild‐type protein between pH 6 and pH 8. In contrast, the His39Leu mutant reacts with DTNB more slowly than the wild‐type protein at pH < 8, but at a similar rate at pH 8. Above pH 8 there is a dramatic increase in reactivity for the Tyr84Phe mutant. We also report 1H NMR studies of disulfide interchange reactions with cysteine. The tethering of the two loops containing Tyr84 and Cys34 not only appears to control the redox potential and accessibility of Cys34, but also triggers the transmission of information about the state of Cys34 throughout domain I, and to the domainI/II interface.

Purification of poly‐ubiquitinated proteins by S5a‐affinity chromatography

Poly‐ubiquitination, the post‐translational covalent conjugation of isopeptide‐linked chains of ubiquitin to other target proteins, is the central signal for proteolytic degradation by the 26S proteasome complex. The S5a subunit of the 26S proteasome binds poly‐ubiquitin chains containing four or more ubiquitins. We have used an immobilised glutathione‐S‐transferase (GST)‐S5a fusion protein to purify poly‐ubiquitinated proteins from mammalian tissues, with the intention of expanding the repertoire of known substrates of the ubiquitin pathway. A complex mixture of poly‐ubiquitinated proteins was successfully purified from normal pig brain extract following induction of in vitro ubiquitination. Western blots of two‐dimensional gels of this mixture showed at least two diagonal series of ubiquitin‐positive spots. Individual spots in each series were separated by approximately 9 kDa suggesting that they represent poly‐ubiquitinated proteins with increasing numbers of ubiquitins in the chains. S5a‐binding proteins purified from ubiquitination‐induced human placental extracts, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualised by Coomassie staining, contained a single major species with an apparent denatured molecular mass of approximately 60 kDa. Edman degradation identified this protein as hHR23B, a human homologue of the Saccharomyces cerevisiae DNA repair protein Rad23p. In this case hHR23B is not ubiquitinated but instead contains an intrinsic ubiquitin‐like domain at its N‐terminus, through which it interacts with S5a (Hiyama, H., et al., J Biol. Chem. 1999, 274, 28 019–28 025).

The production, characterisation and enhanced pharmacokinetics of scFv–albumin fusions expressed in Saccharomyces cerevisiae

An expression system is described for the production of monomeric scFvs and scFv antibody fragments genetically fused to human albumin (at either the N- or C-terminus or both). Based upon strains of Saccharomyces cerevisiae originally developed for the production of a recombinant human albumin (Recombumin) this system has delivered high levels of secreted product into the supernatant of shake flask and high cell density fed-batch fermentations. Specific binding to the corresponding ligand was demonstrated for each of the scFvs and scFv-albumin fusions and pharmacokinetic studies showed that the fusion products had greatly extended circulatory half-lives. The system described provides an attractive alternative to other microbial systems for the manufacture of this type of product.

Characterisation of faecal protease activity in irritable bowel syndrome with diarrhoea: origin and effect of gut transit

Objectives Faecal serine proteases (FSPs) may play a role in irritable bowel syndrome with diarrhoea (IBS-D), but their origin is unclear. We aimed to structurally characterise them and define the impact of colonic cleansing and transit time. Design Faecal samples were obtained from 30 healthy volunteers (HV) and 79 patients with IBS-D participating in a trial of ondansetron versus placebo. Colonic transit was measured using radio-opaque markers. Samples were also obtained from 24 HV before and after colonic cleansing with the osmotic laxative MoviPrep. FSPs were purified from faecal extracts using benzamidine-Sepharose affinity chromatography. SDS-PAGE profiled components were identified using trypsinolysis and tandem mass spectrometry. Functional protease activity in faecal extracts was measured using a colorimetric assay based on the proteolysis of azo-casein. Results Protein analysis identified the most abundant FSPs as being of human origin and probably derived from pancreatic juice. Functional assays showed increased faecal protease (FP) and amylase in patients with IBS-D compared with HV. Those with higher amylase had significantly higher FP and greater anxiety. FP activity correlated negatively with whole gut transit in patients with IBS-D (Spearman r=−0.32, p=0.005) and HV (r=−0.55, p=0.014). Colon cleansing caused a significant rise in FP activity in HV from a baseline of median (IQR) 253 (140–426) to 1031 (435–2296), levels similar to those seen in patients with IBS-D. FSP activity correlated positively with days/week with urgency. Conclusions The most abundant FSPs are of human origin. Rapid transit through the colon and/or decreased (possibly bacterial) proteolytic degradation increases their faecal concentration and could contribute to visceral hypersensitivity in patients with IBS-D. ClinicalTrials.gov NCT00745004.

Insights into the Molecular Composition of Endogenous Unanchored Polyubiquitin Chains

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.

Myosin-Va and Dynamic Actin Oppose Microtubules to Drive Long-Range Organelle Transport

Summary In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively [1–8]. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the “highways and local roads” model for transport along microtubule and actin tracks [2]. The “cooperative capture” model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering [5, 9]. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning [10, 11].

Alcohol-related brain damage in humans

Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann’s area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin β II, and α- and β-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in α-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in β-spectrin protein levels, and a significant increase in transmembranous α3 (catalytic) subunit of the Na+,K+-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of α-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic α- and β-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics.

Framework for a systems approach to proteomic biomarker profiling in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in females of reproductive age, and its prevalence ranges between 6 and 8%. Associated problems include infertility, menstrual disorders, hirsutism and obesity. In addition, individuals with PCOS may be at increased risk of diabetes, endometrial cancer and, possibly, cardiovascular disease and breast cancer in later life. Biomarkers identified from proteomic analyses may help to improve the clinical management of PCOS, provided that new proteomic data can be integrated with existing knowledge and/or pathways implicated in disease etiology. In this study, a database of identity, descriptions and functions/pathways has been developed from 148 published proteomic biomarkers in PCOS. From analysis of the database, a variety of pathways possibly implicated in PCOS were determined, including those related to fibrinolysis, thrombosis, the antioxidant pathway and the immune system. This database, if developed further, will provide a framework for a systems approach to profiling biomarkers in the future.

Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury.

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and (3)H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ∼ 75-80 kDa, ∼ 95-100 kDa, and ∼ 155-160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ∼ 160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (∼ 2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.

Mass spectrometry insights into a tandem ubiquitin‐binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin

Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate‐conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker‐optimized ubiquitin‐binding domain hybrid (t‐UBD) containing two UBDs, a ZnF‐UBP domain in tandem with a linkage‐selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48‐linked polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48‐linked ubiquitin dimers. Furthermore, MS/MS analyses indicate that the t‐UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48‐linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain‐selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the ‘ubiquitome’. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 (http://proteomecentral.proteomexchange.org/dataset/PXD004059).