HUMMR, a hypoxia- and HIF-1α–inducible protein, alters mitochondrial distribution and transport

Mitochondrial transport is critical for maintenance of normal neuronal function. Here, we identify a novel mitochondria protein, hypoxia up-regulated mitochondrial movement regulator (HUMMR), which is expressed in neurons and is markedly induced by hypoxia-inducible factor 1 α (HIF-1α). Interestingly, HUMMR interacts with Miro-1 and Miro-2, mitochondrial proteins that are critical for mediating mitochondrial transport. Interestingly, knockdown of HUMMR or HIF-1 function in neurons exposed to hypoxia markedly reduces mitochondrial content in axons. Because mitochondrial transport and distribution are inextricably linked, the impact of reduced HUMMR function on the direction of mitochondrial transport was also explored. Loss of HUMMR function in hypoxia diminished the percentage of motile mitochondria moving in the anterograde direction and enhanced the percentage moving in the retrograde direction. Thus, HUMMR, a novel mitochondrial protein induced by HIF-1 and hypoxia, biases mitochondria transport in the anterograde direction. These findings have broad implications for maintenance of neuronal viability and function during physiological and pathological states

The phosphomimetic mutation of syndecan-4 binds and inhibits Tiam1 modulating Rac1 activity in PDZ interaction-dependent manner

The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1-RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways

Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach

Many protein interaction domains bind short peptides based on canonical sequence consensus motifs. Here we report the development of a peptide array-based proteomics tool to identify proteins directly interacting with ligand peptides from cell lysates. Array-formatted bait peptides containing an amino acid-derived cross-linker are photo-induced to crosslink with interacting proteins from lysates of interest. Indirect associations are removed by high stringency washes under denaturing conditions. Covalently trapped proteins are subsequently identified by LC-MS/MS and screened by cluster analysis and domain scanning. We apply this methodology to peptides with different proline-containing consensus sequences and show successful identifications from brain lysates of known and novel proteins containing polyproline motif-binding domains such as EH, EVH1, SH3, WW domains. These results suggest the capacity of arrayed peptide ligands to capture and subsequently identify proteins by mass spectrometry is relatively broad and robust. Additionally, the approach is rapid and applicable to cell or tissue fractions from any source, making the approach a flexible tool for initial protein-protein interaction discovery.National Institutes of Health (U.S.) (Grant R21-CA-140030-01

Atypical Rho GTPases the RhoBTB family: role as adaptors of ubiquitin ligase complexes

RhoBTB proteins, a subfamily of atypical Rho GTPases, seem to have distinct function from that of the classical RhoGTPases. They consist of a .GTPase domain, a proline-rich region and two BTB domains. Using Y2H and Co-IP studies we found that RhoBTB2 and 3 interact with Cul3 through both BTB domains. RhoBTB3 might also be degraded in the proteasome. GTPbinding assay revealed that the GTPase domain of RhoBTB3 does not bind GTP. In our working model RhoBTB proteins play a role in targeting of substrates for ubiquitinylation and degradation via Cul3-dependent ubiquitin ligase complexes. Y2H screening identified nuclear protein MUFI as a potential interaction partner of RhoBTB3. MUFI interacts with RhoBTB3 in vivo and seems to be recruited by RhoBTB3 from the nucleus to the cytoplasm. MUFl serves as a linker protein in Cul5-based ubiquitin ligases. Our aim is to answer the question, whether MUF 1 is in volved in a multiprotein complex that involves Cul3 and Cul5. and e!ucidate the role of RhoBTB in the regulation of this complex.Peer Reviewe

Characterization of RhoBTB-dependent Cul3 ubiquitin ligase complexes. Evidence for an autoregulatory mechanism

S. R. was a recipient of a fellowship from Fundación Ramón Areces. F.R was supported by grants of the Center for Molecular Medicine Cologne (CMMC) and the Deutsche Forschungsgemeinschaft. Support by the Köln Fortune program of the Medical Faculty, University of Cologne to F.R. is also acknowledged. P.A. was supported by funds from the Ludwig Institute for Cancer Research, the Swedish Research Council and the Swedish Science Foundation. M. F. was supported by RR018759 from the National Center for Research Resources, a component of the National Institutes of Health and CA036727 from National Cancer Institute.Peer Reviewe

HUMMR, a hypoxia- and HIF-1alpha-inducible protein, alters mitochondrial distribution and transport.

Mitochondrial transport is critical for maintenance of normal neuronal function. Here, we identify a novel mitochondria protein, hypoxia up-regulated mitochondrial movement regulator (HUMMR), which is expressed in neurons and is markedly induced by hypoxia-inducible factor 1 alpha (HIF-1alpha). Interestingly, HUMMR interacts with Miro-1 and Miro-2, mitochondrial proteins that are critical for mediating mitochondrial transport. Interestingly, knockdown of HUMMR or HIF-1 function in neurons exposed to hypoxia markedly reduces mitochondrial content in axons. Because mitochondrial transport and distribution are inextricably linked, the impact of reduced HUMMR function on the direction of mitochondrial transport was also explored. Loss of HUMMR function in hypoxia diminished the percentage of motile mitochondria moving in the anterograde direction and enhanced the percentage moving in the retrograde direction. Thus, HUMMR, a novel mitochondrial protein induced by HIF-1 and hypoxia, biases mitochondria transport in the anterograde direction. These findings have broad implications for maintenance of neuronal viability and function during physiological and pathological states

HUMMR, a hypoxia- and HIF-1alpha-inducible protein, alters mitochondrial distribution and transport.

Mitochondrial transport is critical for maintenance of normal neuronal function. Here, we identify a novel mitochondria protein, hypoxia up-regulated mitochondrial movement regulator (HUMMR), which is expressed in neurons and is markedly induced by hypoxia-inducible factor 1 alpha (HIF-1alpha). Interestingly, HUMMR interacts with Miro-1 and Miro-2, mitochondrial proteins that are critical for mediating mitochondrial transport. Interestingly, knockdown of HUMMR or HIF-1 function in neurons exposed to hypoxia markedly reduces mitochondrial content in axons. Because mitochondrial transport and distribution are inextricably linked, the impact of reduced HUMMR function on the direction of mitochondrial transport was also explored. Loss of HUMMR function in hypoxia diminished the percentage of motile mitochondria moving in the anterograde direction and enhanced the percentage moving in the retrograde direction. Thus, HUMMR, a novel mitochondrial protein induced by HIF-1 and hypoxia, biases mitochondria transport in the anterograde direction. These findings have broad implications for maintenance of neuronal viability and function during physiological and pathological states

Role of Rho-like proteins of the RhoBTB family in oncogenesis

Abstract of the 8th Joint Meeting of the Signal Transduction Society STS, Hilton Hotel, Weimar, 4–6 November 2004: Workshop E: “Interaction Domains and Signaling Complexes.Rho proteins have been implicated mainly in the regulation of the cytoskeleton, but also in vesicle trafficking,phagocytosis and transcriptional activation. They participate in tumorigenesis, where some of them are overfunc-tional. A subfamily of Rho GTPases, RhoBTB proteins, is represented by three isoforms in vertebrates: RhoBTB1,RhoBTB2 and RhoBTB3. They are around 600 amino acids long and consist of a GTPase domain, a prolin richregion, two BTB domains and a carboxyl-terminal region. The role of RhoBTB proteins is at present unknown.Tissue distribution studies by means of in situ hybridization detected the expression of RhoBTB1 and RhoBTB3specifically in vascular endothelial cells suggesting a role in angiogenesis. In addition, a role for RhoBTB2 as atumor suppressor has been proposed, and in fact we show that expression profiles of RhoBTB genes differbetween normal and tumoral human tissuesRhoBTB3 is targeted to Golgi membranes through a prenylation motif. RhoBTB1 and RhoBTB2 lack this motif,but localize at aggregates distinct from vesiclular structures like Golgi apparatus or lysosomes. We generatedtruncated contructs in order to identify the sequence responsible for targeting of RhoBTB1 and RhoBTB2 tothese aggregates.Recent studies demonstrate that BTB domain-containing proteins are involved in the formation of Cullin 3 depen-dent ubiquitin ligase complexes. Cullins are subunits of a prominent class of RING ubiquitin ligases. Using yeasttwo hybrid and coimmunoprecipitation studies we show that RhoBTB3 binds to Cullin 3 and might also be de-graded in the proteasomal apparatus. We propose a model in which RhoBTB proteins function as specific adap-tors for the degradation of unknown proteins involved in the control of cell proliferation and have identified poten-tial targets using a two-hybrid approach.Peer Reviewe

Role of Rho-like proteins of the RhoBTB family in oncogenesis

Abstract of Annual Meeting of the German Society for Cell Biology (29.3.-1.4. 2006)A subfamily of Rho GTPases, RhoBTB proteins, is represented by three isoforms in vertebrates. They consist of a GTPase domain, a proline-rich region and two BTB domains. BTB domains are involved in the formation of cullin 3-dependent ubiquitin ligase complexes. We investigate the role of RhoBTB proteins in oncogenesis and the signal transduction pathways in which these proteins are involved. Using two-hybrid and co-immunoprecipitation studies we found that RhoBTB2 and 3 interact specifi cally with cullin 3. We also show that RhoBTB2 and RhoBTB3 homo- and heterodimerize. The ring box protein Roc1 enhances the interaction between RhoBTB and Cullin 3. Furthermore, we have found that RhoBTB3 interacts with ubiquitin conjugating enzyme (E2) suggesting an important link in the ubiquitin–proteasome pathway. To address the role of RhoBTB in tumorigenesis we analyzed the expression profi le of RhoBTB genes using a cancer profi ling array. RhoBTB expression was decreased in all kidney samples and about 80% of breast samples versus their matched normal tissue. The expression change of RhoBTB3 and Cullin 3 correlate. We envision a model in which RhoBTB proteins target specifi c substrates for degradation by formation of cullin complexes and suppression of RhoBTBs results in accumulation of the target followed by cell proliferation.Peer Reviewe