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  • 1
    Language: English
    In: Nature, May 2018, Vol.557(7707), pp.734-738
    Description: Conventional ubiquitination regulates key cellular processes by catalysing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins . Recently, the SidE family of bacterial effector proteins (SdeA, SdeB, SdeC and SidE) from pathogenic Legionella pneumophila were shown to use NAD to mediate phosphoribosyl-linked ubiquitination of serine residues in host proteins. However, the molecular architecture of the catalytic platform that enables this complex multistep process remains unknown. Here we describe the structure of the catalytic core of SdeA, comprising mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains, and shed light on the activity of two distinct catalytic sites for serine ubiquitination. The mART catalytic site is composed of an α-helical lobe (AHL) that, together with the mART core, creates a chamber for NAD binding and ADP-ribosylation of ubiquitin. The catalytic site in the PDE domain cleaves ADP-ribosylated ubiquitin to phosphoribosyl ubiquitin (PR-Ub) and mediates a two-step PR-Ub transfer reaction: first to a catalytic histidine 277 (forming a transient SdeA H277-PR-Ub intermediate) and subsequently to a serine residue in host proteins. Structural analysis revealed a substrate binding cleft in the PDE domain, juxtaposed with the catalytic site, that is essential for positioning serines for ubiquitination. Using degenerate substrate peptides and newly identified ubiquitination sites in RTN4B, we show that disordered polypeptides with hydrophobic residues surrounding the target serine residues are preferred substrates for SdeA ubiquitination. Infection studies with L. pneumophila expressing substrate-binding mutants of SdeA revealed that substrate ubiquitination, rather than modification of the cellular ubiquitin pool, determines the pathophysiological effect of SdeA during acute bacterial infection.
    Keywords: Biocatalysis ; Ubiquitination ; Legionella Pneumophila -- Enzymology ; Membrane Proteins -- Chemistry ; Serine -- Metabolism
    ISSN: 00280836
    E-ISSN: 1476-4687
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  • 2
    Language: English
    In: Analytical chemistry, 05 August 2014, Vol.86(15), pp.7421-7
    Description: Successful proteome analyses of highly dilute samples are strongly dependent on optimized workflows considering especially sample preparation prior to highly sensitive mass spectrometric analysis. Various methods are available for enrichment of proteome samples, each characterized by specific advantages and disadvantages limiting their general application as a method of choice. Here we suggest an optimized universal protocol ensuring reproducibility and effective enrichment of dilute samples by commercial affinity beads. By comparably assessing the performance of the new protocol with selected standard enrichment techniques, we show the seamless application of the enrichment in common mass spectrometry based proteomic workflows. Further, novel applications are suggested including a facile storage and shipping of desiccated, trapped proteome samples at ambient temperatures and usage of the affinity beads for gel-free proteomic approaches.
    Keywords: Proteins -- Chemistry
    ISSN: 00032700
    E-ISSN: 1520-6882
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  • 3
    In: EMBO Journal, 06 July 2011, Vol.30(13), pp.2545-2556
    Description: ‐AAA proteases exert dual functions in the mitochondrial inner membrane: they mediate the processing of specific regulatory proteins and ensure protein quality control degrading misfolded polypeptides to peptides. Loss of these activities leads to neuronal cell death in several neurodegenerative disorders. However, it is unclear how the ‐AAA protease chooses between specific processing and complete degradation. A central and conserved function of the ‐AAA protease is the processing of the ribosomal subunit MrpL32, which regulates ribosome biogenesis and the formation of respiratory complexes. Here, we demonstrate that the formation of a tightly folded domain harbouring a conserved CxxC‐X‐CxxC sequence motif halts degradation initiated from the N‐terminus and triggers the release of mature MrpL32. Oxidative stress impairs folding of MrpL32, resulting in its degradation by the ‐AAA protease and decreased mitochondrial translation. Surprisingly, MrpL32 folding depends on its mitochondrial targeting sequence. Presequence‐assisted folding of MrpL32 requires the complete import of the MrpL32 precursor before maturation occurs and therefore explains the need for post‐translocational processing by the ‐AAA protease rather than co‐translocational cleavage by the general mitochondrial processing peptidase. The mitochondrial ribosomal subunit Mrp32L requires its presequence for proper folding, so cannot be processed via co‐translocational cleavage. Instead, the AAA protease begins to degrade it from the N‐terminus. Its activity is halted by a redox‐sensitive tightly folded domain, releasing the mature protein.
    Keywords: Aaa Protease ; Mitochondria ; Oxidative Stress ; Presequence‐Assisted Folding ; Ribosome Biogenesis
    ISSN: 0261-4189
    E-ISSN: 1460-2075
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  • 4
    In: The Journal of Bacteriology, 2010, Vol. 192(3), p.870
    Description: In its natural habitats, Bacillus subtilis is exposed to changing osmolarity, necessitating adaptive stress responses. Transcriptomic and proteomic approaches can provide a picture of the dynamic changes occurring in salt-stressed B. subtilis cultures because these studies provide an unbiased view of cells coping with high salinity. We applied whole-genome microarray technology and metabolic labeling, combined with state-of-theart proteomic techniques, to provide a global and time-resolved picture of the physiological response of B. subtilis cells exposed to a severe and sudden osmotic upshift. This combined experimental approach provided quantitative data for 3,961 mRNA transcription profiles, 590 expression profiles of proteins detected in the cytosol, and 383 expression profiles of proteins detected in the membrane fraction. Our study uncovered a well-coordinated induction of gene expression subsequent to an osmotic upshift that involves large parts of the SigB, SigW, SigM, and SigX regulons. Additionally osmotic upregulation of a large number of genes that do not belong to these regulons was observed. In total, osmotic upregulation of about 500 B. subtilis genes was detected. Our data provide an unprecedented rich basis for further in-depth investigation of the physiological and genetic responses of B. subtilis to hyperosmotic stress. doi: 10.1128/JB.01106-09
    Keywords: Bacillus Subtilis -- Genetic Aspects ; Bacillus Subtilis -- Physiological Aspects ; Bacillus Subtilis -- Research ; Salt Stress (Botany) -- Research ; Transcription (Genetics) -- Research ; Evolutionary Adaptation -- Research;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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  • 5
    Language: English
    In: Cell, 01 December 2016, Vol.167(6), pp.1636-1649.e13
    Description: Conventional ubiquitination involves the ATP-dependent formation of amide bonds between the ubiquitin C terminus and primary amines in substrate proteins. Recently, SdeA, an effector protein of pathogenic , was shown to mediate NAD-dependent and ATP-independent ubiquitin transfer to host proteins. Here, we identify a phosphodiesterase domain in SdeA that efficiently catalyzes phosphoribosylation of ubiquitin on a specific arginine via an ADP-ribose-ubiquitin intermediate. SdeA also catalyzes a chemically and structurally distinct type of substrate ubiquitination by conjugating phosphoribosylated ubiquitin to serine residues of protein substrates via a phosphodiester bond. Furthermore, phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes of the conventional ubiquitination cascade, thereby impairing numerous cellular processes including mitophagy, TNF signaling, and proteasomal degradation. We propose that phosphoribosylation of ubiquitin potently modulates ubiquitin functions in mammalian cells. Proteins can be ubiquitinated through a serine-linked phosphodiester bond independently of conventional E1 and E2 enzymes.
    Keywords: Biology
    ISSN: 0092-8674
    E-ISSN: 1097-4172
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  • 6
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2018, Vol.1841, pp.11-18
    Description: Efficient and reproducible protein enrichment is a prerequisite for the proteomic analysis of highly dilute solutions like culture supernatant or body fluids. Traditionally several precipitation strategies were used for this purpose, but they have certain drawbacks. Here, a protocol for StrataClean enrichment-a very efficient and easy-to-use method for the enrichment of proteins from highly dilute samples-which is compatible to gel-free proteomics, is presented.
    Keywords: Highly Dilute Samples ; Protein Enrichment ; Solid-Phase Enrichment ; Strataclean
    E-ISSN: 1940-6029
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 7
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2017, Vol.1520, pp.281-289
    Description: Absolute protein quantification is an essential tool for system biology approaches and elucidation of stoichiometry of multi-protein complexes. In this chapter, a universal protocol for gel free absolute protein quantification in bacterial systems is described, which can be used for sample preparation prior to miscellaneous mass-spectrometry-based quantification workflows like AQUA, Hi3, and emPAI. In addition, a focus has been set to the specific challenges in antibiotic stress research.
    Keywords: Aqua ; Absolute Protein Quantification ; Antibiotic Stress Response ; Gel Free Proteomics ; Hi3 ; In-Solution Digestion ; Qconcat ; Sample Preparation ; Empai ; Stress, Physiological ; Anti-Bacterial Agents -- Pharmacology ; Bacterial Proteins -- Analysis ; Mass Spectrometry -- Methods
    E-ISSN: 1940-6029
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 8
    In: Nature Genetics, 2010, Vol.42(4), p.313
    Description: Autosomal dominant spinocerebellar ataxias (SCAs) are genetically heterogeneous neurological disorders characterized by cerebellar dysfunction mostly due to Purkinje cell degeneration. Here we show that AFG3L2 mutations cause SCA type 28. Along with paraplegin, which causes recessive spastic paraplegia, AFG3L2 is a component of the conserved m-AAA metalloprotease complex involved in the maintenance of the mitochondrial proteome. We identified heterozygous missense mutations in five unrelated SCA families and found that AFG3L2 is highly and selectively expressed in human cerebellar Purkinje cells. m-AAA-deficient yeast cells expressing human mutated AFG3L2 homocomplex show respiratory deficiency, proteolytic impairment and deficiency of respiratory chain complex IV. Structure homology modeling indicates that the mutations may affect AFG3L2 substrate handling. This work identifies AFG3L2 as a novel cause of dominant neurodegenerative disease and indicates a previously unknown role for this component of the mitochondrial protein quality control machinery in protecting the human cerebellum against neurodegeneration. [PUBLICATION ]
    Keywords: ATP-Dependent Proteases–Genetics ; Adenosine Triphosphatases–Metabolism ; Adenosine Triphosphatases–Metabolism ; Base Sequence–Metabolism ; Cell Respiration–Metabolism ; Cerebellum–Genetics ; Electron Transport Complex IV–Genetics ; Genetic Complementation Test–Genetics ; Humans–Genetics ; Molecular Sequence Data–Genetics ; Mutation, Missense–Genetics ; Purkinje Cells–Genetics ; Saccharomyces Cerevisiae–Genetics ; Spinocerebellar Degenerations–Genetics ; Proteins ; Mutation ; Genes ; Genotype & Phenotype ; Ataxia ; Electron Transport Complex IV ; ATP-Dependent Proteases ; Afg3l2 Protein, Human ; Adenosine Triphosphatases;
    ISSN: 1061-4036
    E-ISSN: 15461718
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  • 9
    In: Therapeutic Apheresis and Dialysis, April 2018, Vol.22(2), pp.189-195
    Description: Technical problems during clinical lipid apheresis interfere with fractionator performance. Therefore, a large animal model was established to characterize a new plasma fractionation membrane. Four sheep were randomized, controlled, and crossover subjected to double ofiltration lipoprotein apheresis with three specimens of FractioPES having slightly different HDL sieving coefficients () (FPESa, 0.30, FPESb, 0.26, and FPESc, 0.22) versus a control fractionator (EVAL). and reduction ratios were determined for LDL, HDL, fibrinogen, IgG, and albumin. Compared to EVAL (0.42 ± 0.04 to 0.74 ± 0.08) and FPESa (0.36 ± 0.06 to 0.64 ± 0.04), for HDL were lower ( 〈 0.05) with FPESc (0.30 ± 0.04 to 0.49 ± 0.10). Fibrinogen were higher ( 〈 0.05) with EVAL (0.02 ± 0.01 to 0.40 ± 0.08) compared to FPESb (0.05 ± 0.02 to 0.26 ± 0.34) and FPESc (0.01 ± 0.01 to 0.21 ± 0.16). No further differences were determined. The animal model distinguished between minor differences in fractionation membrane permeability, demonstrating equivalent sieving of FPESa and EVAL and slightly inferior permeability of FPESb and FPESc.
    Keywords: High Density Lipoprotein Cholesterol ; Hypercholesterolemia ; Low Density Lipoprotein Cholesterol ; Lipid Apheresis ; Sheep
    ISSN: 1744-9979
    E-ISSN: 1744-9987
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  • 10
    Language: English
    In: International Journal of Medical Microbiology, May 2016, Vol.306(3), pp.131-140
    Description: The translation inhibitor linezolid is an antibiotic of last resort against pathogens including methicillin resistant strains of the nosocomial pathogen . Linezolid is reported to inhibit production of extracellular virulence factors, but the molecular cause is unknown. To elucidate the physiological response of to linezolid in general and the inhibition of virulence factor synthesis in particular a holistic study was performed. Linezolid was added to exponentially growing cells and the linezolid stress response was analyzed with transcriptomics and quantitative proteomics methods. In addition, scanning and transmission electron microscopy experiments as well as fluorescence microscopy analyses of the cellular DNA and membrane were performed. As previously observed in studies on other translation inhibitors, adapts its protein biosynthesis machinery to the reduced translation efficiency. For example the synthesis of ribosomal proteins was induced. Also unexpected results like a decline in the amount of extracellular and membrane proteins were obtained. In addition, cell shape and size changed after linezolid stress and cell division was diminished. Finally, the chromosome was condensed after linezolid stress and lost contact to the membrane. These morphological changes cannot be explained by established theories. A new hypothesis is discussed, which suggests that the reduced amount of membrane and extracellular proteins and observed defects in cell division are due to the disintegration of transertion complexes by linezolid.
    Keywords: Mechanism of Action ; Transertion ; Protein Translocation ; Cell Division ; Translation Inhibition ; Biology
    ISSN: 1438-4221
    E-ISSN: 1618-0607
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