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1.
Blood ; 119(18): 4152-61, 2012 May 03.
Article in English | MEDLINE | ID: mdl-22411870

ABSTRACT

The transcription factor runt-related transcription factor 1 (Runx1) is essential for the establishment of definitive hematopoiesis during embryonic development. In adult blood homeostasis, Runx1 plays a pivotal role in the maturation of lymphocytes and megakaryocytes. Furthermore, Runx1 is required for the regulation of hematopoietic stem and progenitor cells. However, how Runx1 orchestrates self-renewal and lineage choices in combination with other factors is not well understood. In the present study, we describe a genome-scale RNA interference screen to detect genes that cooperate with Runx1 in regulating hematopoietic stem and progenitor cells. We identify the polycomb group protein Pcgf1 as an epigenetic regulator involved in hematopoietic cell differentiation and show that simultaneous depletion of Runx1 and Pcgf1 allows sustained self-renewal while blocking differentiation of lineage marker-negative cells in vitro. We found an up-regulation of HoxA cluster genes on Pcgf1 knock-down that possibly accounts for the increase in self-renewal. Moreover, our data suggest that cells lacking both Runx1 and Pcgf1 are blocked at an early progenitor stage, indicating that a concerted action of the transcription factor Runx1, together with the epigenetic repressor Pcgf1, is necessary for terminal differentiation. The results of the present study uncover a link between transcriptional and epigenetic regulation that is required for hematopoietic differentiation.


Subject(s)
Core Binding Factor Alpha 2 Subunit/physiology , DNA-Binding Proteins/physiology , Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Animals , Bone Marrow Transplantation , Cell Division , Cells, Cultured/cytology , Chromatin Immunoprecipitation , Colony-Forming Units Assay , Core Binding Factor Alpha 2 Subunit/deficiency , Core Binding Factor Alpha 2 Subunit/genetics , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Epigenesis, Genetic , Hematopoietic Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Polycomb Repressive Complex 1 , RNA, Small Interfering/pharmacology , Radiation Chimera , Real-Time Polymerase Chain Reaction , Recombinant Fusion Proteins/physiology , Specific Pathogen-Free Organisms , Transduction, Genetic
2.
Mol Cell Biol ; 32(6): 1173-88, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22252321

ABSTRACT

Mitochondria possess an outer membrane (OMM) and an inner membrane (IMM), which folds into invaginations called cristae. Lipid composition, membrane potential, and proteins in the IMM influence organization of cristae. Here we show an essential role of the OMM protein Sam50 in the maintenance of the structure of cristae. Sam50 is a part of the sorting and assembly machinery (SAM) necessary for the assembly of ß-barrel proteins in the OMM. We provide evidence that the SAM components exist in a large protein complex together with the IMM proteins mitofilin and CHCHD3, which we term the mitochondrial intermembrane space bridging (MIB) complex. Interactions between OMM and IMM components of the MIB complex are crucial for the preservation of cristae. After destabilization of the MIB complex, we observed deficiency in the assembly of respiratory chain complexes. Long-term depletion of Sam50 influences the amounts of proteins from all large respiratory complexes that contain mitochondrially encoded subunits, pointing to a connection between the structural integrity of cristae, assembly of respiratory complexes, and/or the maintenance of mitochondrial DNA (mtDNA).


Subject(s)
Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Proteins/metabolism , Cell Respiration , Gene Knockdown Techniques , HeLa Cells , Humans , Membrane Proteins/genetics , Mitochondrial Membrane Transport Proteins , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Muscle Proteins/metabolism , Proteins/metabolism
3.
J Mol Biol ; 397(1): 219-32, 2010 Mar 19.
Article in English | MEDLINE | ID: mdl-20117113

ABSTRACT

beta-Barrel proteins are found in the outer membranes of bacteria, chloroplasts and mitochondria. The evolutionary conserved sorting and assembly machinery (SAM complex) assembles mitochondrial beta-barrel proteins, such as voltage-dependent anion-selective channel 1 (VDAC1), into complexes in the outer membrane by recognizing a sorting beta-signal in the carboxy-terminal part of the protein. Here we show that in mammalian mitochondria, masking of the C-terminus of beta-barrel proteins by a tag leads to accumulation of soluble misassembled protein in the intermembrane space, which causes mitochondrial fragmentation and loss of membrane potential. A similar phenotype is observed if the beta-signal is shortened, removed or when the conserved hydrophobic residues in the beta-signal are mutated. The length of the tag at the C-terminus is critical for the assembly of VDAC1, as well as the amino acid residues at positions 130, 222, 225 and 251 of the protein. We propose that if the recognition of the beta-signal or the folding of the beta-barrel proteins is inhibited, the nonassembled protein will accumulate in the intermembrane space, aggregate and damage mitochondria. This effect offers easy tools for studying the requirements for the membrane assembly of beta-barrel proteins, but also advises caution when interpreting the outcome of the beta-barrel protein overexpression experiments.


Subject(s)
Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 1/metabolism , Amino Acid Sequence , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Membrane Potential, Mitochondrial , Mitochondria/ultrastructure , Mitochondrial Membranes/ultrastructure , Molecular Sequence Data , Mutation/genetics , Protein Structure, Secondary , Protein Transport , Structure-Activity Relationship , Voltage-Dependent Anion Channel 1/toxicity
4.
PLoS Pathog ; 5(10): e1000629, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19851451

ABSTRACT

The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m)). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m). The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m) loss and apoptosis, demonstrating that dissipation of DeltaPsi(m) is a requirement for cell death caused by neisserial infection.


Subject(s)
Apoptosis/drug effects , Membrane Potential, Mitochondrial/drug effects , Porins/pharmacology , Bacterial Proteins/metabolism , Bacterial Proteins/pharmacology , Bacterial Proteins/physiology , Carrier Proteins/metabolism , Caspases/metabolism , Cytochromes c/metabolism , Enzyme Activation/drug effects , HeLa Cells , Host-Pathogen Interactions/physiology , Humans , Membrane Transport Proteins/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Precursor Protein Import Complex Proteins , Models, Biological , Neisseria gonorrhoeae/chemistry , Neisseria gonorrhoeae/pathogenicity , Neisseria gonorrhoeae/physiology , Neisseriaceae Infections/metabolism , Neisseriaceae Infections/pathology , Porins/metabolism , Porins/physiology
5.
Curr Opin Microbiol ; 11(1): 9-14, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18280201

ABSTRACT

Recent research on the mechanism underlying the interaction of bacterial pathogens with their host has shifted the focus to secreted microbial proteins affecting the physiology and innate immune response of the target cell. These proteins either traverse the plasma membrane via specific entry pathways involving host cell receptors or are directly injected via bacterial secretion systems into the host cell, where they frequently target mitochondria. The import routes of bacterial proteins are mostly unknown, whereas the effect of mitochondrial targeting by these proteins has been investigated in detail. For a number of them, classical leader sequences recognized by the mitochondrial protein import machinery have been identified. Bacterial outer membrane beta-barrel proteins can also be recognized and imported by mitochondrial transporters. Besides an obvious importance in pathogenicity, understanding import of bacterial proteins into mitochondria has a highly relevant evolutionary aspect, considering the endosymbiotic, proteobacterial origin of mitochondria. The review covers the current knowledge on the mitochondrial targeting and import of bacterial pathogenicity factors.


Subject(s)
Bacterial Proteins/metabolism , Mitochondria/metabolism , Virulence Factors/metabolism , Animals , Bacterial Proteins/genetics , Humans , Models, Biological , Protein Sorting Signals , Virulence Factors/genetics
6.
EMBO Rep ; 8(6): 576-82, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17510655

ABSTRACT

Voltage-dependent anion-selective channel (VDAC) is a beta-barrel protein in the outer mitochondrial membrane that is necessary for metabolite exchange with the cytosol and is proposed to be involved in certain forms of apoptosis. We studied the biogenesis of VDAC in human mitochondria by depleting the components of the mitochondrial import machinery by using RNA interference. Here, we show the importance of the translocase of the outer mitochondrial membrane (TOM) complex in the import of the VDAC precursor. The deletion of Sam50, the central component of the sorting and assembly machinery (SAM), led to both a strong defect in the assembly of VDAC and a reduction in the steady-state level of VDAC. Metaxin 2-depleted mitochondria had reduced levels of metaxin 1 and were deficient in import and assembly of VDAC and Tom40, but not of three matrix-targeted precursors. We also observed a reduction in the levels of metaxin 1 and metaxin 2 in Sam50-depleted mitochondria, implying a connection between these three proteins, although Sam50 and metaxins seemed to be in different complexes. We conclude that the pathway of VDAC biogenesis in human mitochondria involves the TOM complex, Sam50 and metaxins, and that it is evolutionarily conserved.


Subject(s)
Evolution, Molecular , Membrane Proteins/metabolism , Proteins/metabolism , Voltage-Dependent Anion Channels/biosynthesis , Animals , Cell Cycle Proteins , HeLa Cells , Humans , Ion Channel Gating , Mice , Mitochondrial Membrane Transport Proteins , Mitochondrial Membranes/metabolism , RNA, Small Interfering/metabolism , Rats
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