The SLC25 Carrier Family: Important Transport Proteins in Mitochondrial Physiology and Pathology.

Members of the mitochondrial carrier family (SLC25) transport a variety of compounds across the inner membrane of mitochondria. These transport steps provide building blocks for the cell and link the pathways of the mitochondrial matrix and cytosol. An increasing number of diseases and pathologies has been associated with their dysfunction. In this review, the molecular basis of these diseases is explained based on our current understanding of their transport mechanism

Lipid, detergent, and coomassie blue G-250 affect the migration of small membrane proteins in blue native gels:Mitochondrial carriers migrate as monomers not dimers

Background: Mitochondrial carriers were thought to be dimeric based on their migration in blue native gels.  Results: The high molecular mass species observed in blue native gels are composed of protein monomers, detergent, lipid, and Coomassie stain.  Conclusion: The mitochondrial carriers are monomeric not dimeric.  Significance: The apparent mass of small membrane proteins in blue native gels requires significant correction

The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier.

Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT

Defense-in-depth by mucosally administered anti-HIV dimeric IgA2 and systemic IgG1 mAbs: complete protection of rhesus monkeys from mucosal SHIV challenge.

Although IgA is the most abundantly produced immunoglobulin in humans, its role in preventing HIV-1 acquisition, which occurs mostly via mucosal routes, remains unclear. In our passive mucosal immunizations of rhesus macaques (RMs), the anti-HIV-1 neutralizing monoclonal antibody (nmAb) HGN194, given either as dimeric IgA1 (dIgA1) or dIgA2 intrarectally (i.r.), protected 83% or 17% of the RMs against i.r. simian-human immunodeficiency virus (SHIV) challenge, respectively. Data from the RV144 trial implied that vaccine-induced plasma IgA counteracted the protective effector mechanisms of IgG1 with the same epitope specificity. We thus hypothesized that mucosal dIgA2 might diminish the protection provided by IgG1 mAbs targeting the same epitope. To test our hypothesis, we administered HGN194 IgG1 intravenously (i.v.) either alone or combined with i.r. HGN194 dIgA2. We enrolled SHIV-exposed, persistently aviremic RMs protected by previously administered nmAbs; RM anti-human IgG responses were undetectable. However, low-level SIV Gag-specific proliferative T-cell responses were found. These animals resemble HIV-exposed, uninfected humans, in which local and systemic cellular immune responses have been observed. HGN194 IgG1 and dIgA2 used alone and the combination of the two neutralized the challenge virus equally well in vitro. All RMs given only i.v. HGN194 IgG1 became infected. In contrast, all RMs given HGN194 IgG1+dIgA2 were completely protected against high-dose i.r. SHIV-1157ipEL-p challenge. These data imply that combining suboptimal defenses at the mucosal and systemic levels can completely prevent virus acquisition. Consequently, active vaccination should focus on defense-in-depth, a strategy that seeks to build up defensive fall-back positions well behind the fortified frontline.We thank Dr. J. Mascola for providing mAb VRC01, Dr. S.-L. Hu for providing SHIV-1157ip Env proteins, and Dr. W. Marasco for providing mAb Fm-6. We thank Dr. K. Rogers and K. Kinsley for TRIM5α genotype analysis, Dr. S. Lee for assistance in statistical analysis, V. Shanmuganathan for technical assistance, and Juan Esquivel for assistance with the preparation of the manuscript. This was work supported by the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery (CAVD) UCL-VDC Grant 38637 (R.A.W.). This project was also funded in part by NIH grants P01 AI048240, R01 AI100703 and R37 AI034266 to RMR. Base grant P51 OD011132 provided support to the Yerkes National Primate Research Center. The Southwest National Primate Research Center is supported by an NIH primate center base grant (previously NCRR grant P51 RR013986; currently Office of Research Infrastructure Programs/OD P51 OD011133).This is the accepted manuscript of a paper published in Vaccine (Sholukh AM, et al., Vaccine, 2015, 33, 2086-2095, doi:10.1016/j.vaccine.2015.02.020). The final version is available at http://dx.doi.org/10.1016/j.vaccine.2015.02.02

Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers

Background: Methods for rapid assessment of interactions of small molecules with membrane proteins in detergent are lacking.  Results: Thermostability measurements of mitochondrial transporters display informative trends about detergent, lipid, substrate, and inhibitor interactions.  Conclusion: Mechanistic insights are obtained by studying the thermostability of mitochondrial transporters.  Significance: Information about the nature of compound interactions with membrane proteins can be obtained rapidly

An Anti-HIV-1 V3 Loop Antibody Fully Protects Cross-Clade and Elicits T-Cell Immunity in Macaques Mucosally Challenged with an R5 Clade C SHIV

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization-sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high-dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient “Hit and Run” infection or cross priming via Ag-Ab-mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target

Structural basis of purine nucleotide inhibition of human uncoupling protein 1

Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo–electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier–like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak

The transport mechanism of the mitochondrial ADP/ATP carrier

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou

S-glutathionylation activates STIM1 and alters mitochondrial homeostasis

Oxidant stress induces constitutive calcium entry by tacking glutathiones onto the Orai CRAC channel activator STIM1

An anti-HIV-1 V3 loop antibody fully protects cross-clade and elicits T-cell immunity in macaques mucosally challenged with an R5 clade C SHIV

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization- sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high- dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient “Hit and Run” infection or cross priming via Ag-Ab- mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target