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Berlin Brandenburg

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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 16 April 2013, Vol.110(16), pp.6601-6
    Description: Large-scale proteomic approaches have identified numerous mitochondrial acetylated proteins; however in most cases, their regulation by acetyltransferases and deacetylases remains unclear. Sirtuin 3 (SIRT3) is an NAD(+)-dependent mitochondrial protein deacetylase that has been shown to regulate a limited number of enzymes in key metabolic pathways. Here, we use a rigorous label-free quantitative MS approach (called MS1 Filtering) to analyze changes in lysine acetylation from mouse liver mitochondria in the absence of SIRT3. Among 483 proteins, a total of 2,187 unique sites of lysine acetylation were identified after affinity enrichment. MS1 Filtering revealed that lysine acetylation of 283 sites in 136 proteins was significantly increased in the absence of SIRT3 (at least twofold). A subset of these sites was independently validated using selected reaction monitoring MS. These data show that SIRT3 regulates acetylation on multiple proteins, often at multiple sites, across several metabolic pathways including fatty acid oxidation, ketogenesis, amino acid catabolism, and the urea and tricarboxylic acid cycles, as well as mitochondrial regulatory proteins. The widespread modification of key metabolic pathways greatly expands the number of known substrates and sites that are targeted by SIRT3 and establishes SIRT3 as a global regulator of mitochondrial protein acetylation with the capability of coordinating cellular responses to nutrient status and energy homeostasis.
    Keywords: Lysine -- Metabolism ; Metabolic Networks and Pathways -- Physiology ; Mitochondria, Liver -- Metabolism ; Mitochondrial Proteins -- Metabolism ; Proteomics -- Methods ; Sirtuin 3 -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    In: Nature, 2011, Vol.473(7346), p.226
    Description: Dietary restriction is a robust means of extending adult lifespan and postponing age-related disease in many species, including yeast, nematode worms, flies and rodents (1,2). Studies of the genetic requirements for lifespan extension by dietary restriction in the nematode Caenorhabditis elegans have implicated a number of key molecules in this process (3-5), including the nutrient-sensing target of rapamycin (TOR) pathway (6) and the Foxa transcription factor PHA-4 (ref. 7). However, little is known about the metabolic signals that coordinate the organismal response to dietary restriction and maintain homeostasis when nutrients are limited. The endocannabinoid system is an excellent candidate for such a role given its involvement in regulating nutrient intake and energy balance (8). Despite this, a direct role for endocannabinoid signalling in dietary restriction or lifespan determination has yet to be demonstrated, in part due to the apparent absence of endocannabinoid signalling pathways in model organisms that are amenable to lifespan analysis (9). N-acylethanolamines (NAEs) are lipid-derived signalling molecules, which include the mammalian endocannabinoid arachidonoyl ethanolamide. Here we identify NAEs in C. elegans, show that NAE abundance is reduced under dietary restriction and that NAE deficiency is sufficient to extend lifespan through a dietary restriction mechanism requiring PHA-4. Conversely, dietary supplementation with the nematode NAE eicosapentaenoyl ethanolamide not only inhibits dietary-restriction-induced lifespan extension in wild-type worms, but also suppresses lifespan extension in a TOR pathway mutant. This demonstrates a role for NAE signalling in ageing and indicates that NAEs represent a signal that coordinates nutrient status with metabolic changes that ultimately determine lifespan.
    Keywords: Cellular Signal Transduction -- Research ; Transcription Factors -- Genetic Aspects ; Transcription Factors -- Research ; Caenorhabditis Elegans -- Research ; Caenorhabditis Elegans -- Genetic Aspects;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 3
    Language: English
    In: Analytical chemistry, 17 July 2012, Vol.84(14), pp.5845-50
    Description: LC-MS/MS-based proteomics studies rely on stable analytical system performance that can be evaluated by objective criteria. The National Institute of Standards and Technology (NIST) introduced the MSQC software to compute diverse metrics from experimental LC-MS/MS data, enabling quality analysis and quality control (QA/QC) of proteomics instrumentation. In practice, however, several attributes of the MSQC software prevent its use for routine instrument monitoring. Here, we present QuaMeter, an open-source tool that improves MSQC in several aspects. QuaMeter can directly read raw data from instruments manufactured by different vendors. The software can work with a wide variety of peptide identification software for improved reliability and flexibility. Finally, QC metrics implemented in QuaMeter are rigorously defined and tested. The source code and binary versions of QuaMeter are available under Apache 2.0 License at http://fenchurch.mc.vanderbilt.edu.
    Keywords: Chromatography, Liquid -- Instrumentation ; Proteomics -- Instrumentation ; Tandem Mass Spectrometry -- Instrumentation
    ISSN: 00032700
    E-ISSN: 1520-6882
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  • 4
    Language: English
    In: The Journal of biological chemistry, 04 March 2011, Vol.286(9), pp.7601-8
    Description: Differential cysteine oxidation within mitochondrial Complex I has been quantified in an in vivo oxidative stress model of Parkinson disease. We developed a strategy that incorporates rapid and efficient immunoaffinity purification of Complex I followed by differential alkylation and quantitative detection using sensitive mass spectrometry techniques. This method allowed us to quantify the reversible cysteine oxidation status of 34 distinct cysteine residues out of a total 130 present in murine Complex I. Six Complex I cysteine residues were found to display an increase in oxidation relative to controls in brains from mice undergoing in vivo glutathione depletion. Three of these residues were found to reside within iron-sulfur clusters of Complex I, suggesting that their redox state may affect electron transport function.
    Keywords: Electron Transport Complex I -- Chemistry ; Mass Spectrometry -- Methods ; Mitochondria -- Enzymology ; Parkinson Disease -- Metabolism
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 5
    Language: English
    In: The Journal of biological chemistry, 19 August 2016, Vol.291(34), pp.17496-17509
    Description: Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level.
    Keywords: Gadd45a ; Mekk4 ; Aging ; Mass Spectrometry (MS) ; Muscle ; Muscle Atrophy ; Skeletal Muscle ; Skeletal Muscle Metabolism ; Cell Cycle Proteins -- Metabolism ; MAP Kinase Kinase Kinase 4 -- Metabolism ; Multiprotein Complexes -- Metabolism ; Muscle Fibers, Skeletal -- Metabolism ; Muscular Atrophy -- Metabolism ; Nuclear Proteins -- Metabolism
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 6
    Language: English
    In: The Journal of biological chemistry, 16 June 2017, Vol.292(24), pp.10239-10249
    Description: SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. , succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5 HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5 liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5 mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function.
    Keywords: Cardiolipin ; Electron Transport System (Ets) ; Mitochondria ; Protein Acylation ; Sirtuin ; Models, Molecular ; Protein Processing, Post-Translational ; Cardiolipins -- Metabolism ; Electron Transport Chain Complex Proteins -- Metabolism ; Hepatocytes -- Enzymology ; Sirtuins -- Metabolism
    E-ISSN: 1083-351X
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  • 7
    Language: English
    In: PLoS ONE, 01 January 2015, Vol.10(3), p.e0122297
    Description: SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 8
    Language: English
    In: PLoS ONE, 2012, Vol.7(6), p.e38303
    Description: Neisseria gonorrhoeae , the causative agent of gonorrhea, can form biofilms in vitro and in vivo . In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with 13 C 6 -arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values 〈0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related to this shift may have other functions.
    Keywords: Research Article ; Biology ; Microbiology ; Biochemistry
    E-ISSN: 1932-6203
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  • 9
    Language: English
    In: Cancer research, 01 May 2010, Vol.70(9), pp.3709-17
    Description: The overexpressed ErbB2/HER2 receptor is a clinically validated cancer target whose surface localization and internalization mechanisms remain poorly understood. Downregulation of the overexpressed 185-kDa ErbB2 receptor is rapidly (2-6 hours) induced by the HSP90 chaperone inhibitor geldanamycin (GA), whereas its downregulation and lysosomal degradation are more slowly (24 hours) induced by the proteasome inhibitor bortezomib/PS341. In PS341-treated SK-BR-3 cells, overexpressed ErbB2 coprecipitates with the E3 ubiquitin ligase c-Cbl and also with the deubiquitinating enzyme USP9x; moreover, siRNA downregulation of USP9x enhances PS341-induced ErbB2 downregulation. Because polyubiquitin linkages via lysine 48 (K48) or 63 (K63) can differentially address proteins for 26S proteasomal degradation or endosome trafficking to the lysosome, multiple reaction monitoring (MRM)/mass spectrometry (MS) and polyubiquitin linkage-specific antibodies were used to quantitatively track K48-linked and K63-linked ErbB2 polyubiquitination following either GA or PS341 treatment of SK-BR-3 cells. MRM/MS revealed that unlike the rapid, modest (4-fold to 8-fold), and synchronous GA induction of K48 and K63 polyubiquitinated ErbB2, PS341 produces a dramatic (20-fold to 40-fold) sequential increase in polyubiquitinated ErbB2 consistent with K48 polyubiquitination followed by K63 editing. Fluorescence microscopic imaging confirmed that PS341, but not GA, induces colocalization of K48-linked and K63-linked polyubiquitin with perinuclear lysosome-sequestered ErbB2. Thus, ErbB2 surface overexpression and recycling seem to depend on its polyubiquitination and deubiquitination; as well, the contrasting effects of PS341 and GA on ErbB2 receptor localization, polyubiquitination, and degradation point to alternate cytoplasmic trafficking likely regulated by different K48 and K63 polyubiquitin editing mechanisms.
    Keywords: Receptor, Erbb-2 -- Metabolism
    ISSN: 00085472
    E-ISSN: 1538-7445
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  • 10
    Language: English
    In: The Journal of biological chemistry, 11 January 2008, Vol.283(2), pp.855-65
    Description: Nontypeable Haemophilus influenzae is an opportunistic human pathogen causing otitis media in children and chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The outer membrane of nontypeable H. influenzae is dominated by lipooligosaccharides (LOS), many of which incorporate sialic acid as a terminal nonreducing sugar. Sialic acid has been demonstrated to be an important factor in the survival of the bacteria within the host environment. H. influenzae is incapable of synthesizing sialic acid and is dependent on scavenging free sialic acid from the host environment. To achieve this, H. influenzae utilizes a tripartite ATP-independent periplasmic transporter. In this study, we characterize the binding site of the extracytoplasmic solute receptor (SiaP) from nontypeable H. influenzae strain 2019. A crystal structure of N-acetyl-5-neuraminic acid (Neu5Ac)-bound SiaP was determined to 1.4A resolution. Thermodynamic characterization of Neu5Ac binding shows this interaction is enthalpically driven with a substantial unfavorable contribution from entropy. This is expected because the binding of SiaP to Neu5Ac is mediated by numerous hydrogen bonds and has several buried water molecules. Point mutations targeting specific amino acids were introduced in the putative binding site. Complementation with the mutated siaP constructs resulted either in full, partial, or no complementation, depending on the role of specific residues. Mass spectrometry analysis of the O-deacylated LOS of the R127K point mutation confirmed the observation of reduced incorporation of Neu5Ac into the LOS. The decreased ability of H. influenzae to import sialic acid had negative effects on resistance to complement-mediated killing and viability of biofilms in vitro, confirming the importance of sialic acid transport to the bacterium.
    Keywords: Cell Membrane -- Metabolism ; Haemophilus Influenzae -- Metabolism ; Receptors, Cell Surface -- Metabolism ; Sialic Acids -- Metabolism
    ISSN: 0021-9258
    E-ISSN: 1083351X
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