Mitochondrial Biology Unit

Cambridge, United Kingdom

Mitochondrial Biology Unit

Cambridge, United Kingdom
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Harris A.K.,U.S. National Cancer Institute | Meyerson J.R.,U.S. National Cancer Institute | Meyerson J.R.,Mitochondrial Biology Unit | Matsuoka Y.,Laboratories of Infectious Diseases | And 10 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2013

Rapid antigenic variation of HA, the major virion surface protein of influenza A virus, remains the principal challenge to the development of broader and more effective vaccines. Some regions of HA, such as the stem region proximal to the viral membrane, are nevertheless highly conserved across strains and among most subtypes. A fundamental question in vaccine design is the extent to which HA stem regions on the surface of the virus are accessible to broadly neutralizing antibodies. Here we report 3D structures derived from cryoelectron tomography of HA on intact 2009 H1N1 pandemic virions in the presence and absence of the antibody C179, which neutralizes viruses expressing a broad range of HA subtypes, including H1, H2, H5, H6, and H9. By fitting previously derived crystallographic structures of trimeric HA into the density maps, we deduced the locations of the molecular surfaces of HA involved in interaction with C179. Using computational methods to distinguish individual unliganded HA trimers from those that have bound C179 antibody, we demonstrate that ~75% of HA trimers on the surface of the virus have C179 bound to the stem domain. Thus, despite their close packing on the viral membrane, the majority of HA trimers on intact virions are available to bind anti-stem antibodies that target conserved HA epitopes, establishing the feasibility of universal influenza vaccines that elicit such antibodies.

Ruprecht J.J.,Mitochondrial Biology Unit | Hellawell A.M.,Mitochondrial Biology Unit | Harding M.,Mitochondrial Biology Unit | Crichton P.G.,Mitochondrial Biology Unit | And 2 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2014

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix. The carrier cycles by an unresolved mechanism between the cytoplasmic state, in which the carrier accepts ADP from the cytoplasm, and the matrix state, in which it accepts ATP from the mitochondrial matrix. Here we present the structures of the yeast ADP/ATP carriers Aac2p and Aac3p in the cytoplasmic state. The carriers have three domains and are closed at the matrix side by three interdomain salt-bridge interactions, one of which is braced by a glutamine residue. Glutamine braces are conserved in mitochondrial carriers and contribute to an energy barrier, preventing the conversion to the matrix state unless substrate binding occurs. At the cytoplasmic side a second salt-bridge network forms during the transport cycle, as demonstrated by functional analysis of mutants with charge-reversed networks. Analyses of the domain structures and properties of the interdomain interfaces indicate that interconversion between states involves movement of the even-numbered ?-helices across the surfaces of the odd-numbered ?-helices by rotation of the domains. The odd-numbered ?-helices have an L-shape, with proline or serine residues at the kinks, which functions as a lever-arm, coupling the substrate-induced disruption of the matrix network to the formation of the cytoplasmic network. The simultaneous movement of three domains around a central translocation pathway constitutes a unique mechanism among transport proteins. These findings provide a structural description of transport by mitochondrial carrier proteins, consistent with an alternating-access mechanism.

Lee Y.,Mitochondrial Biology Unit | Lee Y.,Medical Research Council | Willers C.,University of Cambridge | Kunji E.R.S.,Mitochondrial Biology Unit | Crichton P.G.,Mitochondrial Biology Unit
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein. Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria. © 2015, National Academy of Sciences. All rights reserved.

Arieti F.,University of Geneva | Gabus C.,University of Geneva | Tambalo M.,University of Geneva | Tambalo M.,Mitochondrial Biology Unit | And 3 more authors.
Nucleic Acids Research | Year: 2014

The Split Ends (SPEN) protein was originally discovered in Drosophila in the late 1990s. Since then, homologous proteins have been identified in eukaryotic species ranging from plants to humans. Every family member contains three predicted RNA recognition motifs (RRMs) in the N-terminal region of the protein. We have determined the crystal structure of the region of the human SPEN homolog that contains these RRMs - the SMRT/HDAC1 Associated Repressor Protein (SHARP), at 2.0 Å resolution. SHARP is a co-regulator of the nuclear receptors. We demonstrate that two of the three RRMs, namely RRM3 and RRM4, interact via a highly conserved interface. Furthermore, we show that the RRM3-RRM4 block is the main platform mediating the stable association with the H12-H13 substructure found in the steroid receptor RNA activator (SRA), a long, non-coding RNA previously shown to play a crucial role in nuclear receptor transcriptional regulation. We determine that SHARP association with SRA relies on both single- and double-stranded RNA sequences. The crystal structure of the SHARP-RRM fragment, together with the associated RNA-binding studies, extend the repertoire of nucleic acid binding properties of RRM domains suggesting a new hypothesis for a better understanding of SPEN protein functions. © 2014 The Author(s) 2014.

Vanderperre B.,University of Geneva | Bender T.,University of Geneva | Kunji E.R.S.,Mitochondrial Biology Unit | Martinou J.-C.,University of Geneva
Current Opinion in Cell Biology | Year: 2015

Pyruvate metabolism plays a pivotal role in cell homeostasis and energy production. Pyruvate, the end product of glycolysis, is either catabolized in the cytosol, or enters into mitochondria to promote oxidative phosphorylation. The import of pyruvate into mitochondria requires a specific carrier in the inner mitochondrial membrane, the mitochondrial pyruvate carrier (MPC), whose identity was only recently discovered. Here we report our current knowledge of the structure and function of the MPC and we describe how dysfunction of the MPC could participate in various pathologies, including type 2 diabetes and cancer. © 2014 Elsevier Ltd.

Palmer S.M.,Mitochondrial Biology Unit | Kunji E.R.S.,Mitochondrial Biology Unit
Methods in Molecular Biology | Year: 2012

Methods of biomass monitoring have increasingly been developed towards online, in situ techniques in order to advance process analysis and control. Off-line, ex situ methods, such as dry cell mass determination and direct cell counts, remain the reference for determining cell mass and number, respectively, but this type of analysis is time consuming. Absorbance measurement, which is used extensively as an off-line, ex situ, or online, in situ technique, is fast and straightforward, as the absorbance can be correlated to cell mass and number by a simple calibration. The downside is that absorbance measurements provide no estimation of viability and in situ applications can suffer from interference, such as aeration. Impedance spectroscopy is widely available and provides a quick measure of viable cell concentration, but does not give an estimation of total cell concentration and, hence, potential product. Sensitivity of impedance spectroscopy remains an issue at low cell concentration, and interference during in situ measurements is also a significant factor. In this chapter, a range of protocols is presented for online determination of biomass yields of recombinant yeast cultures. © 2012 Springer Science+business Media, LLC.

Minczuk M.,Mitochondrial Biology Unit | Kolasinska-Zwierz P.,University of Cambridge | Murphy M.P.,Mitochondrial Biology Unit | Papworth M.A.,University of Cambridge
Nature Protocols | Year: 2010

Engineered zinc-finger proteins (ZFPs) are hybrid proteins developed to direct various effector domains (EDs) of choice to predetermined DNA sequences. They are used to alter gene expression and to modify DNA in a sequence-specific manner in vivo and in vitro. Until now, ZFPs have mostly been used to target DNA sites in nuclear genomes. This protocol describes how to adapt engineered ZFP technology to specifically modify the mammalian mitochondrial genome. The first step describes how to construct mitochondrially targeted ZFPs (mtZFPs) so that they are efficiently imported into mammalian mitochondria. In the second step, methods to test the basic properties of mtZFPs in vitro are described. Finally, we outline how the mtZFPs can be transiently transfected into mammalian cells and their mitochondrial import tested by both immunofluorescence and biochemical methods. The protocol can be completed within a week, although time-consuming DNA cloning steps may extend this. © 2010 Nature Publishing Group.

Affourtit C.,Mitochondrial Biology Unit | Affourtit C.,Buck Institute for Research on Aging | Jastroch M.,Buck Institute for Research on Aging | Brand M.D.,Mitochondrial Biology Unit | Brand M.D.,Buck Institute for Research on Aging
Free Radical Biology and Medicine | Year: 2011

Glucose-stimulated insulin secretion (GSIS) by pancreatic β cells is regulated by mitochondrial uncoupling protein-2 (UCP2), but opposing phenotypes, GSIS improvement and impairment, have been reported for different Ucp2-ablated mouse models. By measuring mitochondrial bioenergetics in attached INS-1E insulinoma cells with and without UCP2, we show that UCP2 contributes to proton leak and attenuates glucose-induced rises in both respiratory activity and the coupling efficiency of oxidative phosphorylation. Strikingly, the GSIS improvement seen upon UCP2 knockdown in INS-1E cells is annulled completely by the cell-permeative antioxidant MnTMPyP. Consistent with this observation, UCP2 lowers mitochondrial reactive oxygen species at high glucose levels. We conclude that UCP2 plays both regulatory and protective roles in β cells by acutely lowering GSIS and chronically preventing oxidative stress. Our findings thus provide a mechanistic explanation for the apparently discrepant findings in the field. © 2010 Elsevier Inc. All rights reserved.

Vidoni S.,Montpellier University | Vidoni S.,University of Bologna | Vidoni S.,Mitochondrial Biology Unit | Zanna C.,University of Bologna | And 3 more authors.
Antioxidants and Redox Signaling | Year: 2013

Significance: The maintenance of mitochondrial genome integrity is a major challenge for cells to sustain energy production by respiration. Recent Advances: Recently, mitochondrial membrane dynamics emerged as a key process contributing to prevent mitochondrial DNA (mtDNA) alterations. Indeed, both fundamental and clinical data suggest that disruption of mitochondrial fusion, related to mutations in the OPA1, MFN2, PINK1, and PARK2 genes, leads to the accumulation of mutations in the mitochondrial genome. Critical Issues: We discuss here the possibility that mitochondrial fusion acts as a direct mechanism to prevent the generation of altered mtDNA and to eliminate mutated deleterious genomes either by trans-complementation or by mitophagy. Future Directions: Finally, we conclude this review with a short evolutionary comparison between the mechanisms involved in mitochondrial and bacterial modes of genome distribution and plasticity, highlighting possible common conserved processes required for the maintenance of their genome integrity, which should inspire our future investigations. Antioxid. Redox Signal. 19, 379-388. © 2013, Mary Ann Liebert, Inc.

Fernandez-Vizarra E.,Mitochondrial Biology Unit | Fernandez-Vizarra E.,Hospital Miguel Servet | Zeviani M.,Mitochondrial Biology Unit
Frontiers in Genetics | Year: 2015

Complex III (CIII) deficiency is one of the least common oxidative phosphorylation defects associated to mitochondrial disease. CIII constitutes the center of the mitochondrial respiratory chain, as well as a crossroad for several other metabolic pathways. For more than 10 years, of all the potential candidate genes encoding structural subunits and assembly factors, only three were known to be associated to CIII defects in human pathology. Thus, leaving many of these cases unresolved. These first identified genes were MT-CYB, the only CIII subunit encoded in the mitochondrial DNA; BCS1L, encoding an assembly factor, and UQCRB, a nuclear-encoded structural subunit. Nowadays, thanks to the fast progress that has taken place in the last 3-4 years, pathological changes in seven more genes are known to be associated to these conditions. This review will focus on the strategies that have permitted the latest discovery of mutations in factors that are necessary for a correct CIII assembly and activity, in relation with their function. In addition, new data further establishing the molecular role of LYRM7/MZM1L as a chaperone involved in CIII biogenesis are provided. © 2015 Fernández-Vizarra and Zeviani.

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