Wilson B. Muse

Chaperone Activity with a Redox Switch

Hsp33, a member of a newly discovered heat shock protein family, was found to be a very potent molecular chaperone. Hsp33 is distinguished from all other known molecular chaperones by its mode of functional regulation. Its activity is redox regulated. Hsp33 is a cytoplasmically localized protein with highly reactive cysteines that respond quickly to changes in the redox environment. Oxidizing conditions like H2O2 cause disulfide bonds to form in Hsp33, a process that leads to the activation of its chaperone function. In vitro and in vivo experiments suggest that Hsp33 protects cells from oxidants, leading us to conclude that we have found a protein family that plays an important role in the bacterial defense system toward oxidative stress.

Oxidative Protein Folding Is Driven by the Electron Transport System

Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidative folding system using purified components. We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism. We find that disulfide bond formation and the electron transport chain are directly coupled. DsbB uses quinones as electron acceptors, allowing various choices for electron transport to support disulfide bond formation. Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions. Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron acceptors such as fumarate. This flexibility reflects the vital nature of the disulfide catalytic system.

A chemical approach for detecting sulfenic acid-modified proteins in living cells

Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H) and sulfonic acids (Cys-SO3H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acid-modified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues.

Drug Targets in Mycobacterial Sulfur Metabolism

The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress in the development of inhibitors of sulfur metabolism enzymes.

RECONSTITUTION OF A PROTEIN DISULFIDE CATALYTIC SYSTEM

Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes their formation is catalyzed by the Dsb proteins. The DsbA protein acts as a direct donor of disulfides to newly synthesized periplasmic proteins. Genetic evidence suggests that a second protein called DsbB acts to specifically reoxidize DsbA. Here we demonstrate the direct reoxidation of DsbA by DsbB. We have developed a fluorescence assay that allows us to directly follow the reoxidation of DsbA. We show that membranes containing catalytic amounts of DsbB can rapidly reoxidize DsbA to completion. The reaction strongly depends on the presence of oxygen, implying that oxygen serves as the final electron acceptor for this disulfide bond formation reaction. Membranes from a dsbBnull mutant display no DsbA reoxidation activity. The ability of DsbB to reoxidize DsbA fits Michaelis-Menten behavior with DsbA acting as a high affinity substrate for DsbB with a K m = 10 μm. The in vitro reconstitution described here is the first biochemical analysis of DsbB and allows us to study the major pathway of disulfide bond formation in Escherichia coli.

Nitrogen Regulation of the codBA (Cytosine Deaminase) Operon from Escherichia coli by the Nitrogen Assimilation Control Protein, NAC

Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. Beta-galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position -59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions -120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions -83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.