Jakob R. Winther

Why is DsbA such an oxidizing disulfide catalyst

DsbA, a member of the thioredoxin family of disulfide oxidoreductases, acts in catalyzing disulfide bond formation by donating its disulfide to newly translocated proteins. We have found that the two central residues within the active site Cys-30-Pro-31-His-32-Cys-33 motif are critical in determining the exceptional oxidizing power of DsbA. Mutations that change these two residues can alter the equilibrium oxidation potential of DsbA by more than 1000-fold. A quantitative explanation for the very high redox potential of DsbA was found by measuring the pKa of a single residue, Cys-30. The pKa of Cys-30 varied dramatically from mutant to mutant and could accurately predict the oxidizing power of each DsbA mutant protein.

Quantifying the global cellular thiol–disulfide status

It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been determined. In this study, we have assembled a global picture of the cellular thiol–disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data were combined with quantification of reduced and oxidized glutathione in the same cells. Of the total protein cysteines, 6% and 9.6% are engaged in disulfide bond formation in HEK and HeLa cells, respectively. Furthermore, the steady-state level of PSSG is <0.1% of the total protein cysteines in both cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the “cellular redox buffer”; nevertheless, our data show that protein thiols represent a larger active redox pool than glutathione. Accordingly, protein thiols are likely to be directly involved in the cellular defense against oxidative stress.

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

To visualize the formation of disulfide bonds in living cells, a pair of redox‐active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2‐fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non‐invasive fluorimetric measurements. The 1.5 Å crystal structure of the oxidized protein revealed a disulfide bond‐induced distortion of the β‐barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state‐dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox‐active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations.

The majority of the thiols (SH) and disulfides (SS) in cells are found as the amino acid cysteine and its disulfide, cystine (Fig. 1A). The thiolate anion is intrinsically one of the strongest biological nucleophiles; thus, the thiol group of cysteine is one of the most reactive functional groups found in proteins [1]. Protein disulfide bonds are typically introduced and removed through a thiol– disulfide exchange reaction (Fig. 1B). This mechanism of transferring reducing equivalents between thiol and disulfide pairs is central in redox biology and is, for example, applied by cytosolic thioredoxin with its active site in the reduced form to reduce protein disulfides and in the endoplasmic reticulum (ER) by protein disulfide isomerases in their oxidized form to generate disulfide bonds. The reaction is initiated by a nucleophilic attack of a thiolate on an existing disulfide bond, leading to oxidation of the nucleophilic thiol and reduction of the leaving group sulfur [2]. In thiol–disulfide exchange reactions, it is important to consider reaction rate and the equilibrium constants between various thiol and disulfide species. Because the thiolate anion is the reactive species, these properties are particularly sensitive to thiol pKa values. In addition, the kinetics and thermodynamics of thiol–disulfide exchange reactions are affected by electrostatic factors from neighboring charged groups as well as strain and entropy (for detailed reviews, see Refs. [3,4]). Cellular SH groups are implicated in the coordination of metal ions and the defense against oxidants, and the reversible formation of disulfide bonds is involved in regulation of enzyme activity, sig-

Review: Biosynthesis and function of yeast vacuolar proteases

The yeast vacuole, which is equivalent to the lysosome of higher eukaryotes, is one of the best characterized degradative organelles. This review describes the biosynthesis and function of yeast vacuolar proteases. Most of these enzymes are delivered to the vacuole via the early compartments of the secretory pathway and the endosome, while one of them is directly imported from the cytoplasm. The proteases are synthesized as precursors which undergo many post‐translational modifications before the final active form is generated. Proteolytic activation by removal of propeptides is performed by proteinase A and/or proteinase B in an intricate cascade‐like fashion. Recent developments in the analysis of the functions of vacuolar proteolysis are described. Substrates of the vacuolar proteases are mostly imported via endocytosis or autophagocytosis, and vacuolar proteolysis appears to be mainly important under nutritional stress conditions and sporulation.

Surprisingly high stability of barley lipid transfer protein, LTP1, towards denaturant, heat and proteases.

Barley LTP1 belongs to a large family of plant proteins termed non‐specific lipid transfer proteins. The in vivo function of these proteins is unknown, but it has been suggested that they are involved in responses towards stresses such as pathogens, drought, heat, cold and salt. Also, the proteins have been suggested as transporters of monomers for cutin synthesis. We have analysed the stability of LTP1 towards denaturant, heat and proteases and found it to be a highly stable protein, which apparently does not denature at temperatures up to 100°C. This high stability may be important for the biological function of LTP1.

Quantification of Thiols and Disulfides

BACKGROUND Disulfide bond formation is a key posttranslational modification, with implications for structure, function and stability of numerous proteins. While disulfide bond formation is a necessary and essential process for many proteins, it is deleterious and disruptive for others. Cells go to great lengths to regulate thiol-disulfide bond homeostasis, typically with several, apparently redundant, systems working in parallel. Dissecting the extent of oxidation and reduction of disulfides is an ongoing challenge due, in part, to the facility of thiol/disulfide exchange reactions. SCOPE OF REVIEW In the present account, we briefly survey the toolbox available to the experimentalist for the chemical determination of thiols and disulfides. We have chosen to focus on the key chemical aspects of current methodology, together with identifying potential difficulties inherent in their experimental implementation. MAJOR CONCLUSIONS While many reagents have been described for the measurement and manipulation of the redox status of thiols and disulfides, a number of these methods remain underutilized. The ability to effectively quantify changes in redox conditions in living cells presents a continuing challenge. GENERAL SIGNIFICANCE Many unresolved questions in the metabolic interconversion of thiols and disulfides remain. For example, while pool sizes of redox pairs and their intracellular distribution are being uncovered, very little is known about the flux in thiol-disulfide exchange pathways. New tools are needed to address this important aspect of cellular metabolism. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Competition between folding and glycosylation in the endoplasmic reticulum.

Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N‐glycosylation was studied. Seven new sites for N‐glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new acceptor sites was analysed by pulse‐labelling under two sets of conditions that are known to reduce the rate of folding: (i) addition of dithiothreitol to the growth medium and (ii) introduction of deletions in the propeptide. A variety of effects was observed, depending on the position of the new acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation reactions can compete in vivo and that glycosylation does not necessarily precede folding. The approach described may be generally applicable for the analysis of protein folding in vivo.

The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix.

Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae . Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2.2 and 1.8 Å, respectively, for complexes of proteinase A with full-length IA3 and with a truncated form consisting only of residues 2–34, reveal an unprecedented mode of inhibitor–enzyme interactions. Neither form of the free inhibitor has detectable intrinsic secondary structure in solution. However, upon contact with the enzyme, residues 2–32 become ordered and adopt a near-perfect α-helical conformation. Thus, the proteinase acts as a folding template, stabilizing the helical conformation in the inhibitor, which results in the potent and specific blockage of the proteolytic activity.

Evidence That Translation Reinitiation Leads to a Partially Functional Menkes Protein Containing Two Copper-Binding Sites

Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1-CBS6). Most patients die in early childhood. We investigated the functional effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7A(Delta ex3+ex4), contains a premature termination codon after 46 codons. Although such transcripts are generally degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7A(Delta ex3+ex4) transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7A(Delta ex3+ex4) transcript was protected from degradation because of reinitiation of protein translation. Our findings suggest that reinitiation takes place at two downstream internal codons. The putative N-terminally truncated proteins contain only CBS5 and CBS6. Cellular localization and copper-dependent trafficking of the major part of endogenous and recombinant ATP7A(Delta ex3+ex4) proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7A(Delta ex3+ex4) cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD-resistant ATP7A(Delta ex3+ex4) transcript leads to the synthesis of N-terminally truncated and at-least-partially functional Menkes proteins missing CBS1-CBS4. This finding--that a mutation that would have been assumed to be null is not--highlights the need to examine the biochemical phenotype of patients to deduce the efficacy of copper therapy.