Mitochondrial disulfide bond formation is driven by intersubunit electron transfer in Erv1 and proofread by glutathione.

The disulfide relay system in the intermembrane space of mitochondria is of crucial importance for mitochondrial biogenesis. Major players in this pathway are the oxidoreductase Mia40 that oxidizes substrates and the sulfhydryl oxidase Erv1 that reoxidizes Mia40. To analyze in detail the mechanism of this oxidative pathway and the interplay of its components, we reconstituted the complete process in vitro using purified cytochrome c, Erv1, Mia40, and Cox19. Here, we demonstrate that Erv1 dimerizes noncovalently and that the subunits of this homodimer cooperate in intersubunit electron exchange. Moreover, we show that Mia40 promotes complete oxidation of the substrate Cox19. The efficient formation of disulfide bonds is hampered by the formation of long-lived, partially oxidized intermediates. The generation of these side products is efficiently counteracted by reduced glutathione. Thus, our findings suggest a role for a glutathione-dependent proofreading during oxidative protein folding by the mitochondrial disulfide relay.

Glutathione redox potential in the mitochondrial intermembrane space is linked to the cytosol and impacts the Mia40 redox state.

Glutathione is an important mediator and regulator of cellular redox processes. Detailed knowledge of local glutathione redox potential (E(GSH)) dynamics is critical to understand the network of redox processes and their influence on cellular function. Using dynamic oxidant recovery assays together with E(GSH)-specific fluorescent reporters, we investigate the glutathione pools of the cytosol, mitochondrial matrix and intermembrane space (IMS). We demonstrate that the glutathione pools of IMS and cytosol are dynamically interconnected via porins. In contrast, no appreciable communication was observed between the glutathione pools of the IMS and matrix. By modulating redox pathways in the cytosol and IMS, we find that the cytosolic glutathione reductase system is the major determinant of E(GSH) in the IMS, thus explaining a steady-state E(GSH) in the IMS which is similar to the cytosol. Moreover, we show that the local E(GSH) contributes to the partially reduced redox state of the IMS oxidoreductase Mia40 in vivo. Taken together, we provide a comprehensive mechanistic picture of the IMS redox milieu and define the redox influences on Mia40 in living cells.

"I have it just like you do": voices of HIV-negative partners in serodifferent relationships receiving primary care at a public clinic in San Francisco.

HIV transmission among serodifferent couples has a significant impact on incidence of HIV worldwide. Antiretroviral interventions (i.e., preexposure prophylaxis, post-exposure prophylaxis, and treatment as prevention) are important aspects of comprehensive prevention and care for serodifferent couples. In this study, HIV-negative members of serodifferent couples were interviewed using open-ended questions to explore their health-care needs, perceptions of clinic-based prevention services, and experience of having an HIV-infected partner. Analysis of interviews with 10 HIV-negative partners revealed the following themes: (1) health needs during joint medical visits; (2) sexual risk reduction strategies; (3) relationship dynamics; and (4) strategies for coping. This study elucidated relationship, health and health care factors that might affect development and implementation of clinic-based prevention interventions for HIV serodifferent couples. The findings point to possible relationship-centered recommendations for health-care providers who serve HIV-affected couples in clinical settings.

The disulfide relay system of mitochondria is connected to the respiratory chain.

All proteins of the intermembrane space of mitochondria are encoded by nuclear genes and synthesized in the cytosol. Many of these proteins lack presequences but are imported into mitochondria in an oxidation-driven process that relies on the activity of Mia40 and Erv1. Both factors form a disulfide relay system in which Mia40 functions as a receptor that transiently interacts with incoming polypeptides via disulfide bonds. Erv1 is a sulfhydryl oxidase that oxidizes and activates Mia40, but it has remained unclear how Erv1 itself is oxidized. Here, we show that Erv1 passes its electrons on to molecular oxygen via interaction with cytochrome c and cytochrome c oxidase. This connection to the respiratory chain increases the efficient oxidation of the relay system in mitochondria and prevents the formation of toxic hydrogen peroxide. Thus, analogous to the system in the bacterial periplasm, the disulfide relay in the intermembrane space is connected to the electron transport chain of the inner membrane.

Biochemical characterization of dithiol glutaredoxin 8 from Saccharomyces cerevisiae: the catalytic redox mechanism redux.

Two dithiol glutaredoxins (Grxs), Grx1 and Grx2, from yeast have been characterized to date. A third putative dithiol glutaredoxin-encoding gene (GRX8) has been identified in silico. Here we show that deletion of GRX8 does not result in a reduced growth rate under oxidative stress conditions, nor does it enhance the defects of Deltagrx1 and Deltagrx2 single or double mutants. We furthermore compare the enzymatic properties of recombinant ScGrx8 with the monothiol glutaredoxin ScGrx7. Molecular models of ScGrx8 suggest that the protein has a canonical Grx fold, a significantly altered substrate binding site, and a Trp14-type cysteine motif at the catalytic center. ScGrx8 did not bind heavy metal ions and was exclusively monomeric. Apparent k(cat) values for ScGrx8 in the standard enzymatic assay were about 3 orders of magnitude less than for ScGrx7, whereas apparent K(m) values were comparable. Mass spectrometric analyses support a ping-pong mechanism for ScGrx7 and ScGrx8 with a glutathionylated protein as an intermediate. Reduction kinetics of ScGrx8 disulfide, glutathionylated ScGrx8(C28S), and glutathionylated ScGrx7 revealed significant differences between the proteins. Surprisingly, mutation of the more C-terminal cysteine residue in the CPDC motif of ScGrx8 also abolished the slight enzymatic activity, and thus the standard catalytic mechanism for glutathionylated substrates does not apply to the enzyme. In summary, ScGrx8 has several novel structural and mechanistic features expanding the subclasses of glutaredoxins. A refined catalytic model for monothiol and dithiol glutaredoxins is presented explaining the diversity of enzymatic activities in vitro and pointing to different functions in vivo.

In vitro import of proteins into isolated mitochondria.

Import of proteins is of vital importance for the biogenesis of mitochondria. The vast majority of mitochondrial proteins is encoded within the nuclear genome and translocated into various mitochondrial compartments after translation in the cytosol as preproteins. Even in rather primitive eukaryotes like yeasts, these are 700 to 1,000 different proteins, whereas only a handful of proteins is encoded in the mitochondrial DNA. In vitro import studies are important tools to understand import mechanisms and pathways. Using isolated mitochondria and radioactively labeled precursor proteins, it was possible to identify several import machineries and pathways consisting of a large number of components during the last decades.

The sulfhydryl oxidase Erv1 is a substrate of the Mia40-dependent protein translocation pathway.

The thiol oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin "CX(3)C" or twin "CX(9)C" motifs which are crucial for import of typical substrates of this pathway and it does not need two "CX(2)C" motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway.

Art therapy as emotional and spiritual medicine for Native Americans living with HIV/AIDS.

This article describes the intricate challenges of bringing mental health services to isolated, guarded urban HIV-positive Native Americans suffering from chronic trauma-related illnesses and imbalances, depression, anxiety, substance abuse, thought disorders and trauma-based characterological disorders. It explores the integration of art therapy, Bowen Family Systems Therapy and in-home therapy in the Family & Child Guidance Clinic's attempt to provide support to a population that has profound distrust for "services and treatment," and no historical context for psychotherapy. Changing the paradigm of thought is essential to providing services that respect culture and history as well as addressing current presenting issues. Art therapy and in-home therapy support those community members who are flooded emotionally, but have difficulty speaking about their internal processes.

Adaptation of an evidence-based HIV prevention intervention for women with incarcerated partners: expanding to community settings.

High rates of incarceration in urban, low income communities may exacerbate women's risk of HIV infection by decreasing the number of available male sexual partners and disrupting long-term partnerships. The Health Access Program for Prevention, Empowerment, and Networking for Women (HAPPEN) was established to address the HIV prevention needs of women partnered with incarcerated or recently released men in community settings. HAPPEN is an adaptation of the evidence-based HIV prevention intervention Health Options Mean Empowerment (HOME) project. HOME was designed specifically for women visiting their incarcerated male partners and was delivered at a prison visiting center. Recruitment and program implementation for HAPPEN occurred at community-based organizations serving women with histories of substance abuse, intimate partner violence and incarceration, and provided health education, HIV testing, and linkage to health care and social services. This paper describes the process of adapting HOME using input from target organization stakeholders and target population members.

Protein import and oxidative folding in the mitochondrial intermembrane space of intact mammalian cells.

Oxidation of cysteine residues to disulfides drives import of many proteins into the intermembrane space of mitochondria. Recent studies in yeast unraveled the basic principles of mitochondrial protein oxidation, but the kinetics under physiological conditions is unknown. We developed assays to follow protein oxidation in living mammalian cells, which reveal that import and oxidative folding of proteins are kinetically and functionally coupled and depend on the oxidoreductase Mia40, the sulfhydryl oxidase augmenter of liver regeneration (ALR), and the intracellular glutathione pool. Kinetics of substrate oxidation depends on the amount of Mia40 and requires tightly balanced amounts of ALR. Mia40-dependent import of Cox19 in human cells depends on the inner membrane potential. Our observations reveal considerable differences in the velocities of mitochondrial import pathways: whereas preproteins with bipartite targeting sequences are imported within seconds, substrates of Mia40 remain in the cytosol for several minutes and apparently escape premature degradation and oxidation.

Systematic analysis of the twin cx(9)c protein family.

The Mia40-Erv1 disulfide relay system is of high importance for mitochondrial biogenesis. Most so far identified substrates of this machinery contain either two cysteine-x(3)-cysteine (twin Cx(3)C) or two cysteine-x(9)-cysteine (twin Cx(9)C) motifs. While the first group is composed of well-characterized components of the mitochondrial import machinery, the molecular function of twin Cx(9)C proteins still remains unclear. To systematically characterize this protein family, we performed a database search to identify the full complement of Cx(9)C proteins in yeast. Thereby, we identified 14 potential family members, which, with one exception, are conserved among plants, fungi, and animals. Among these, three represent novel proteins, which we named Cmc2 to 4 (for Cx(9)C motif-containing protein) and which we demonstrated to be dependent for import on the Mia40-Erv1 disulfide relay. By testing deletion mutants of all 14 proteins for function of the respiratory chain, we found a critical function of most of these proteins for the assembly or stability of respiratory chain complexes. Our data suggest that already early during the evolution of eukaryotic cells, a multitude of twin Cx(9)C proteins developed, which exhibit largely nonredundant roles critical for the biogenesis of enzymes of the respiratory chain in mitochondria.