Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 195
Filter
1.
Nature ; 580(7805): E20, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32350466

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

2.
Mol Phylogenet Evol ; 141: 106632, 2019 12.
Article in English | MEDLINE | ID: mdl-31560986

ABSTRACT

Database surveys in the vertebrate model organisms: chicken (Gallus gallus), western clawed frog (Xenopus tropicalis), anole lizard (Anolis carolinensis) and zebrafish (Danio rerio) indicate that in some of these species the number of actin paralogues differs from the well-established six paralogues in mouse (Mus musculus). To investigate differential functions of actins and for establishing disease models it is important to know how actins in the different model organisms relate to each other and whether the vertebrate actin family is truly limited to six groups. Primarily through synteny analyses we discovered that the vertebrate actin family consists of eight instead of six orthologous actin groups for which we propose improved gene nomenclature. We also established that α-skeletal muscle, γ-enteric smooth muscle and γ-cytoplasmic actin genes originated prior to tetrapods contradicting an earlier and widely accepted model of actin evolution. Our findings allow a more reliable predictive classification of actin paralogues in (non-mammalian) vertebrates and contribute to a better understanding of actin evolution as basis for biomedical research on actin-related diseases.


Subject(s)
Actins/genetics , Evolution, Molecular , Models, Genetic , Vertebrates/genetics , Animals , Exons/genetics , Likelihood Functions , Muscle, Smooth/metabolism , Phylogeny , Species Specificity , Synteny/genetics
3.
Trends Biochem Sci ; 44(6): 502-516, 2019 06.
Article in English | MEDLINE | ID: mdl-30611609

ABSTRACT

Actin is one of the most abundant proteins in eukaryotic cells and the main component of the microfilament system. It plays essential roles in numerous cellular activities, including muscle contraction, maintenance of cell integrity, and motility, as well as transcriptional regulation. Besides interacting with various actin-binding proteins (ABPs), proper actin function is regulated by post-translational modifications (PTMs), such as acetylation, arginylation, oxidation, and others. Here, we explain how actin PTMs can contribute to filament formation and stability, and may have additional actin regulatory functions, which potentially contribute to disease development.


Subject(s)
Actins/chemistry , Actins/metabolism , Cytoskeleton/metabolism , Protein Processing, Post-Translational , Animals , Humans , Microfilament Proteins/metabolism
4.
Mol Ther ; 22(10): 1768-78, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25023329

ABSTRACT

Gelsolin amyloidosis is an autosomal dominant incurable disease caused by a point mutation in the GSN gene (G654A/T), specifically affecting secreted plasma gelsolin. Incorrect folding of the mutant (D187N/Y) second gelsolin domain leads to a pathological proteolytic cascade. D187N/Y gelsolin is first cleaved by furin in the trans-Golgi network, generating a 68 kDa fragment (C68). Upon secretion, C68 is cleaved by MT1-MMP-like proteases in the extracellular matrix, releasing 8 kDa and 5 kDa amyloidogenic peptides which aggregate in multiple tissues and cause disease-associated symptoms. We developed nanobodies that recognize the C68 fragment, but not native wild type gelsolin, and used these as molecular chaperones to mitigate gelsolin amyloid buildup in a mouse model that recapitulates the proteolytic cascade. We identified gelsolin nanobodies that potently reduce C68 proteolysis by MT1-MMP in vitro. Converting these nanobodies into an albumin-binding format drastically increased their serum half-life in mice, rendering them suitable for intraperitoneal injection. A 12-week treatment schedule of heterozygote D187N gelsolin transgenic mice with recombinant bispecific gelsolin-albumin nanobody significantly decreased gelsolin buildup in the endomysium and concomitantly improved muscle contractile properties. These findings demonstrate that nanobodies may be of considerable value in the treatment of gelsolin amyloidosis and related diseases.


Subject(s)
Amyloid/metabolism , Amyloidosis/metabolism , Gelsolin/metabolism , Matrix Metalloproteinase 14/metabolism , Molecular Chaperones/metabolism , Single-Domain Antibodies/metabolism , Amyloidosis, Familial/metabolism , Animals , Antibodies, Bispecific/immunology , Antibodies, Bispecific/metabolism , Antibody Specificity/immunology , Disease Models, Animal , Gelsolin/chemistry , Gelsolin/immunology , Humans , Mice , Molecular Chaperones/chemistry , Molecular Chaperones/immunology , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Peptides/immunology , Peptides/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Proteolysis , Single-Domain Antibodies/immunology
5.
FASEB J ; 28(4): 1805-18, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24414419

ABSTRACT

Invadopodia are actin-rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen-binding domains of Camelid heavy-chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA-MB-231 breast and PC-3 prostate cancer cells. A nanobody (K(d)~35 nM, 1:1 stoichiometry) that disrupts fascin F-actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion. Cortactin-SH3 dependent WIP recruitment toward the plasma membrane was specifically inhibited by a cortactin nanobody (K(d)~75 nM, 1:1 stoichiometry). This functional domain is shown to be important for formation of properly organized invadopodia, MMP-9 secretion, matrix degradation, and cancer cell invasion. Notably, using a subcellular delocalization strategy to trigger protein loss of function, we uncovered a fascin-bundling-independent role in MMP-9 secretion. Hence, we demonstrate that nanobodies enable high resolution protein function mapping in cells.


Subject(s)
Carrier Proteins/metabolism , Cell Surface Extensions/metabolism , Cortactin/metabolism , Microfilament Proteins/metabolism , Single-Domain Antibodies/metabolism , Actins/metabolism , Blotting, Western , Carrier Proteins/genetics , Carrier Proteins/immunology , Cell Line, Tumor , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Movement , Cell Surface Extensions/ultrastructure , Cortactin/genetics , Cortactin/immunology , Cytoskeletal Proteins/metabolism , Epitopes/genetics , Epitopes/immunology , Epitopes/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Matrix Metalloproteinase 9/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/immunology , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Neoplasms/metabolism , Neoplasms/pathology , Protein Binding , Pseudopodia/metabolism , Pseudopodia/ultrastructure , Single-Domain Antibodies/genetics , Single-Domain Antibodies/immunology , Thermodynamics , src Homology Domains
6.
Biochim Biophys Acta ; 1843(2): 352-65, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24315858

ABSTRACT

Alpha-synuclein is a small protein implicated in the pathophysiology of Parkinson's disease (PD). We have investigated the mechanism of cleavage of alpha-synuclein by the 20S proteasome. Alpha-synuclein interacts with the C8 (α7) subunit of the proteasome. The N-terminal part of alpha-synuclein (amino acids 1-60) is essential for its proteasomal degradation and analysis of peptides released from proteasomal digestion allows concluding that initial cleavages occur within the N-terminal region of the molecule. Aggregated alpha-synucleins are also degraded by the proteasome with a reduced rate, likely due to Met oxidation. In fact, mild oxidation of alpha-synuclein with H2O2 resulted in the inhibition of its degradation by the proteasome, mainly due to oxidation of Met 1 and 5 of alpha-synuclein. The inhibition was reversed by treatment of the oxidized protein with methionine sulfoxide reductases (MsrA plus MsrB). Similarly, treatment with H2O2 of N2A cells transfected with alpha-synuclein resulted in the inhibition of its degradation that was also reverted by co-transfection of MsrA plus MsrB. These results clearly indicate that oxidative stress, a common feature of PD and other synucleinopathies, promotes a RedOx change in the proteostasis of alpha-synuclein due to Met oxidation and reduced proteasomal degradation; compromised reversion of those oxidative changes would result in the accumulation of oxidative damaged alpha-synuclein likely contributing to the pathogenesis of PD.


Subject(s)
Methionine/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , alpha-Synuclein/metabolism , Amino Acid Sequence , Animals , Humans , Hydrogen Peroxide/pharmacology , Immunoblotting , Methionine Sulfoxide Reductases/metabolism , Mice , Molecular Sequence Data , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Peptides/chemistry , Peptides/metabolism , Protein Binding/drug effects , Protein Interaction Mapping , Protein Structure, Quaternary , Protein Subunits/metabolism , Proteolysis/drug effects , Rats , Silver Staining , alpha-Synuclein/chemistry
7.
Breast Cancer Res ; 15(6): R116, 2013 Dec 13.
Article in English | MEDLINE | ID: mdl-24330716

ABSTRACT

INTRODUCTION: Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit. METHODS: We raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading. RESULTS: With G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS). CONCLUSIONS: CapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers a fast track for gauging the therapeutic merit of drug targets. Mapping of the nanobody-CapG interface may provide a platform for rational design of pharmacologic compounds.


Subject(s)
Actins/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Microfilament Proteins/immunology , Molecular Targeted Therapy/methods , Nuclear Proteins/immunology , Single-Domain Antibodies/pharmacology , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Escherichia coli/genetics , Female , Humans , Mice, SCID , Microfilament Proteins/genetics , Nuclear Proteins/genetics , Protein Structure, Tertiary
8.
PLoS One ; 8(11): e78108, 2013.
Article in English | MEDLINE | ID: mdl-24236012

ABSTRACT

Podosomes are cellular structures acting as degradation 'hot-spots' in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages.


Subject(s)
Extracellular Matrix/metabolism , Macrophages/metabolism , Membrane Glycoproteins/metabolism , Microfilament Proteins/metabolism , Organelles/metabolism , Single-Domain Antibodies/pharmacology , Actin Cytoskeleton/metabolism , Cell Line, Tumor , Gelatin/metabolism , Gelsolin/metabolism , Humans , Macrophages/drug effects , Macrophages/ultrastructure , Matrix Metalloproteinases, Secreted/metabolism , Membrane Glycoproteins/immunology , Microfilament Proteins/immunology , Phosphoproteins/immunology , Phosphoproteins/metabolism , Protein Transport , Proteolysis
9.
J Proteomics ; 91: 164-71, 2013 Oct 08.
Article in English | MEDLINE | ID: mdl-23851314

ABSTRACT

Multidimensional peptide fractionation is widely used in proteomics to reduce the complexity of peptide mixtures prior to mass spectrometric analysis. Here, we describe the sequential use of strong cation exchange and reversed phase liquid chromatography in both basic and acidic pH buffers for separating tryptic peptides from complex mixtures of proteins. Strong cation exchange exclusively separates peptide by their charge state into neutral, singly and multi-charged species. To further reduce complexity, each peptide group was separated by reversed phase liquid chromatography at basic pH and the resultant fractions were analyzed by LC-MS/MS. This workflow was applied to a soluble protein lysate from mouse embryonic fibroblast cells, and more than 5000 proteins from 29,843 peptides were identified. The high selectivity displayed during the SCX step (93% to 100%) and the overlaps between proteins identified from the SCX-separated peptide groups, are interesting assets of the procedure. BIOLOGICAL SIGNIFICANCE: The present work shows how complex mixture of peptides can be selectively separated by SCX based essentially on the net charge of peptides. The proposed workflow results in three well-defined subset of peptides of specific amino acid composition, which are representative of the constituent proteins. The very high selectivity obtained (93% to 99%) on the peptide side, underscores for the first time the possibility of SCX chromatography to aid in validating identified peptides.


Subject(s)
Chromatography, High Pressure Liquid/methods , Proteomics/methods , Trypsin/chemistry , Animals , Cations , Computational Biology , Fibroblasts/metabolism , Humans , Lysine/chemistry , Mice , Peptide Mapping/methods , Peptides/chemistry
10.
Cell Mol Life Sci ; 70(5): 909-22, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23001012

ABSTRACT

The T cell integrin receptor LFA-1 orchestrates adhesion between T cells and antigen-presenting cells (APCs), resulting in formation of a contact zone known as the immune synapse (IS) which is supported by the cytoskeleton. L-plastin is a leukocyte-specific actin bundling protein that rapidly redistributes to the immune synapse following T cell-APC engagement. We used single domain antibodies (nanobodies, derived from camelid heavy-chain only antibodies) directed against functional and structural modules of L-plastin to investigate its contribution to formation of an immune synapse between Raji cells and human peripheral blood mononuclear cells or Jurkat T cells. Nanobodies that interact either with the EF hands or the actin binding domains of L-plastin both trapped L-plastin in an inactive conformation, causing perturbation of IS formation, MTOC docking towards the plasma membrane, T cell proliferation and IL-2 secretion. Both nanobodies delayed Ser(5) phosphorylation of L-plastin which is required for enhanced bundling activity. Moreover, one nanobody delayed LFA-1 phosphorylation, reduced the association between LFA-1 and L-plastin and prevented LFA-1 enrichment at the IS. Our findings reveal subtle mechanistic details that are difficult to attain by conventional means and show that L-plastin contributes to immune synapse formation at distinct echelons.


Subject(s)
Antigen-Presenting Cells/immunology , Leukocytes, Mononuclear/immunology , Lymphocyte Function-Associated Antigen-1/immunology , Membrane Glycoproteins/immunology , Microfilament Proteins/immunology , Microtubule-Organizing Center/immunology , Single-Domain Antibodies/immunology , T-Lymphocytes/immunology , Actins/metabolism , Animals , Antigen-Presenting Cells/cytology , Antigen-Presenting Cells/metabolism , Calmodulin/immunology , Calmodulin/metabolism , Cell Communication , Cell Line , Cells, Cultured , EF Hand Motifs , Humans , Interleukin-2/immunology , Jurkat Cells , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation , Lymphocyte Function-Associated Antigen-1/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Microtubule-Organizing Center/metabolism , Microtubule-Organizing Center/ultrastructure , Models, Molecular , Phosphorylation , Protein Interaction Mapping , T-Lymphocytes/cytology , T-Lymphocytes/metabolism
11.
Nucleic Acids Res ; 41(Database issue): D333-7, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23093603

ABSTRACT

We here present The Online Protein Processing Resource (TOPPR; http://iomics.ugent.be/toppr/), an online database that contains thousands of published proteolytically processed sites in human and mouse proteins. These cleavage events were identified with COmbinded FRActional DIagonal Chromatography proteomics technologies, and the resulting database is provided with full data provenance. Indeed, TOPPR provides an interactive visual display of the actual fragmentation mass spectrum that led to each identification of a reported processed site, complete with fragment ion annotations and search engine scores. Apart from warehousing and disseminating these data in an intuitive manner, TOPPR also provides an online analysis platform, including methods to analyze protease specificity and substrate-centric analyses. Concretely, TOPPR supports three ways to retrieve data: (i) the retrieval of all substrates for one or more cellular stimuli or assays; (ii) a substrate search by UniProtKB/Swiss-Prot accession number, entry name or description; and (iii) a motif search that retrieves substrates matching a user-defined protease specificity profile. The analysis of the substrates is supported through the presence of a variety of annotations, including predicted secondary structure, known domains and experimentally obtained 3D structure where available. Across substrates, substrate orthologs and conserved sequence stretches can also be shown, with iceLogo visualization provided for the latter.


Subject(s)
Databases, Protein , Peptide Hydrolases/metabolism , Protein Processing, Post-Translational , Proteolysis , Animals , Humans , Internet , Mice , Proteins/metabolism , Substrate Specificity
12.
Biol Chem ; 393(9): 915-31, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22944692

ABSTRACT

Processing of human immunodeficiency virus (HIV) proteins by the HIV-1 protease is essential for HIV infectivity. In addition, several studies have revealed cleavage of human proteins by this viral protease during infection; however, no large-scale HIV-1 protease degradomics study has yet been performed. To identify putative host substrates in an unbiased manner and on a proteome-wide scale, we used positional proteomics to identify peptides reporting protein processing by the HIV-1 protease, and a catalogue of over 120 cellular HIV-1 protease substrates processed in vitro was generated. This catalogue includes previously reported substrates as well as recently described interaction partners of HIV-1 proteins. Cleavage site alignments revealed a specificity profile in good correlation with previous studies, even though the ELLE consensus motif was not cleaved efficiently when incorporated into peptide substrates due to subsite cooperativity. Our results are further discussed in the context of HIV-1 infection and the complex substrate recognition by the viral protease.


Subject(s)
HIV Protease/metabolism , HIV-1/enzymology , Amino Acid Sequence , Humans , Jurkat Cells , Substrate Specificity
13.
Mol Cell Proteomics ; 11(8): 255-71, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22448045

ABSTRACT

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic ß- and γ-actin. Because of the presence and localized translation of ß-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates ß-actin in gene regulation. Cell migration without ß-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking ß-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, ß-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of ß-actin knockout cells. This also explains why reintroducing ß-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in ß-actin knockout cells based on increased Rho-ROCK signaling and increased TGFß production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of ß-actin knockout cells indicating that other actins compensate for ß-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but ß-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation.


Subject(s)
Actins/metabolism , Cell Movement/physiology , Fibroblasts/metabolism , Proteomics/methods , Actins/genetics , Amides/pharmacology , Animals , Blotting, Western , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cell Adhesion/physiology , Cell Movement/drug effects , Cell Movement/genetics , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Fibroblasts/cytology , Gene Expression Regulation, Developmental , Mice , Mice, Knockout , Protein Isoforms/genetics , Protein Isoforms/metabolism , Pseudopodia/genetics , Pseudopodia/metabolism , Pseudopodia/physiology , Pyridines/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolism
14.
J Proteomics ; 75(6): 1792-802, 2012 Mar 16.
Article in English | MEDLINE | ID: mdl-22227400

ABSTRACT

Quantitative proteomic comparisons require a sufficient number of samples to reach an acceptable level of significance. But 2D gel electrophoresis commonly results in incomplete data sets due to spots with missing values reducing thereby the number of parallel measurements for individual proteins. Here we investigated how many missing values per spot can be tolerated. The number of spots in common between all gels was found to decrease with the number of parallel gels in a non-linear fashion. Increasing numbers of missing values were associated with a moderate increase in the quantitative variation of spot volumes. Based on the missing value pattern in 20 gels we performed an analysis of the multiple testing power for the hypothetical scenario of a comparative 2DE study with six or twelve parallel gels. The calculation considered the statistical power of the individual spot as well as the number of spots included in the analysis. The power increased with inclusion of spots with higher number of missing values and showed an optimum at a specific minimum number of spot replicates. The results suggest that proteins with missing values can be included in a univariate analysis as long as a sufficient number of parallel gels are made.


Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Proteins/analysis , Proteomics/methods , Blood Proteins/isolation & purification , Humans , Image Processing, Computer-Assisted/methods , Reproducibility of Results , Research Design , Statistics as Topic
15.
Biochim Biophys Acta ; 1823(2): 524-33, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22173095

ABSTRACT

Parkinson's disease (PD) is characterized by dopaminergic dysfunction and degeneration. DJ-1/PARK7 mutations have been linked with a familial form of early onset PD. In this study, we found that human DJ-1 wild type and the missense mutants M26I, R98Q, A104T and D149A were stable proteins in cells, only the L166P mutant was unstable. In parallel, the former were not degraded and the L166P mutant was directly degraded in vitro by proteasome-mediated endoproteolytic cleavage. Furthermore, genetic evidence in fission yeast showed the direct involvement of proteasome in the degradation of human DJ-1 L166P and the corresponding L169P mutant of SPAC22E12.03c, the human orthologue of DJ-1 in Schizosaccharomyces Pombe, as their protein levels were increased at restrictive temperature in fission yeast (mts4 and pts1-732) harboring temperature sensitive mutations in proteasomal subunits. In total, our results provide evidence that direct proteasomal endoproteolytic cleavage of DJ-1 L166P is the mechanism of degradation contributing to the loss-of-function of the mutant protein, a property not shared by other DJ-1 missense mutants associated with PD.


Subject(s)
Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mutation, Missense , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Parkinsonian Disorders/genetics , Parkinsonian Disorders/metabolism , Proteasome Endopeptidase Complex/metabolism , Amino Acid Sequence , Animals , Cell Line , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Mice , Molecular Sequence Data , Oncogene Proteins/chemistry , Peptides/genetics , Peptides/metabolism , Proteasome Endopeptidase Complex/chemistry , Protein Deglycase DJ-1 , Protein Structure, Secondary , Protein Subunits/chemistry , Protein Subunits/metabolism , Rats , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism
16.
Biochem Biophys Res Commun ; 414(1): 240-5, 2011 Oct 14.
Article in English | MEDLINE | ID: mdl-21951856

ABSTRACT

DNA damage is a critical event that requires an appropriate cellular response. This is mediated by checkpoint proteins such as Cdk1 that controls S/G2 and G2/M transition. Cdk1 is required for BRCA1 transport to DNA damage sites inside the nucleus where BRCA1 functions as a scaffold to initiate a signaling cascade. BRCA1 is a multifunctional protein that also ubiquitinates γ-tubulin and, consequently, inhibits microtubule nucleation at the centrosome. Here, we report that γ-tubulin also localizes at confined areas in the microtubule network. Nocodazole-mediated microtubule depolymeration results in disappearance of this γ-tubulin fraction, while microtubule stabilization by taxol preserves this structure. Surprisingly, overexpression of Cdk1 or BRCA1 greatly expands the γ-tubulin coating of microtubules, suggesting that the microtubule-bound γ-tubulin is involved in DNA damage response. This is in accordance with numerous reports of microtubule-associated DNA damage proteins, such as p53, that are transported to the nucleus when DNA damage occurs. γ-Tubulin itself has been reported to form complexes with DNA repair proteins in the nucleus.


Subject(s)
BRCA1 Protein/metabolism , CDC2 Protein Kinase/metabolism , DNA Damage , Microtubules/metabolism , Tubulin/metabolism , Cell Line, Tumor , Humans , Nocodazole/pharmacology , Polymerization/drug effects , Protein Structure, Tertiary , Tubulin Modulators/pharmacology
17.
PLoS Genet ; 7(7): e1002169, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21750686

ABSTRACT

N-terminal acetylation (N-Ac) is a highly abundant eukaryotic protein modification. Proteomics revealed a significant increase in the occurrence of N-Ac from lower to higher eukaryotes, but evidence explaining the underlying molecular mechanism(s) is currently lacking. We first analysed protein N-termini and their acetylation degrees, suggesting that evolution of substrates is not a major cause for the evolutionary shift in N-Ac. Further, we investigated the presence of putative N-terminal acetyltransferases (NATs) in higher eukaryotes. The purified recombinant human and Drosophila homologues of a novel NAT candidate was subjected to in vitro peptide library acetylation assays. This provided evidence for its NAT activity targeting Met-Lys- and other Met-starting protein N-termini, and the enzyme was termed Naa60p and its activity NatF. Its in vivo activity was investigated by ectopically expressing human Naa60p in yeast followed by N-terminal COFRADIC analyses. hNaa60p acetylated distinct Met-starting yeast protein N-termini and increased general acetylation levels, thereby altering yeast in vivo acetylation patterns towards those of higher eukaryotes. Further, its activity in human cells was verified by overexpression and knockdown of hNAA60 followed by N-terminal COFRADIC. NatF's cellular impact was demonstrated in Drosophila cells where NAA60 knockdown induced chromosomal segregation defects. In summary, our study revealed a novel major protein modifier contributing to the evolution of N-Ac, redundancy among NATs, and an essential regulator of normal chromosome segregation. With the characterization of NatF, the co-translational N-Ac machinery appears complete since all the major substrate groups in eukaryotes are accounted for.


Subject(s)
Acetyltransferases , Chromosome Segregation/physiology , Drosophila Proteins/metabolism , Fungal Proteins/metabolism , Protein Processing, Post-Translational , Proteomics/methods , Acetylation , Acetyltransferases/genetics , Acetyltransferases/metabolism , Animals , Drosophila Proteins/genetics , Drosophila melanogaster/enzymology , Drosophila melanogaster/genetics , Evolution, Molecular , Fungal Proteins/genetics , Humans , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Substrate Specificity
18.
Nat Protoc ; 6(8): 1130-41, 2011 Jul 14.
Article in English | MEDLINE | ID: mdl-21799483

ABSTRACT

In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.


Subject(s)
Chromatography/methods , Proteins/chemistry , Butyric Acid/chemistry , Chemical Fractionation/methods , Chromatography, Liquid/methods , Esters/chemistry , Humans , Jurkat Cells , Mass Spectrometry/methods , Peptides/chemistry , Propionates/chemistry , Proteome , Proteomics/methods , Succinimides/chemistry
19.
Proteomics ; 11(14): 2867-80, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21681991

ABSTRACT

Chlorosomes, the antenna complexes of green bacteria, are unique antenna systems in which pigments are organized in aggregates. Studies on isolated chlorosomes from Chlorobaculum tepidum based on SDS-PAGE, immunoblotting and molecular biology have revealed that they contain ten chlorosomal proteins, but no comprehensive information is available about the protein composition of the entire organelle. To extend these studies, chlorosomes were isolated from C. tepidum using three related and one independent isolation protocol and characterized by absorption spectroscopy, tricine SDS-PAGE, dynamic light scattering (DLS) and electron microscopy. Tricine SDS-PAGE showed the presence of more than 20 proteins with molecular weights ranging between 6 and 70 kDa. The chlorosomes varied in size. Their hydrodynamic radius (R(h) ) ranged from 51 to 75 nm and electron microscopy indicated that they were on average 140 nm wide and 170 nm long. Furthermore, the mass of 184 whole chlorosome organelles determined by scanning transmission electron microscopy ranged from 27 to 237 MDa being on average 88 (±28) MDa. In contrast their mass-per-area was independent of their size, indicating that there is a strict limit to chlorosome thickness. The average protein composition of the C. tepidum chlorosome organelles was obtained by MS/MS-driven proteomics and for the first time a detailed protein catalogue of the isolated chlorosomal proteome is presented. Based on the proteomics results for chlorosomes isolated by different protocols, four proteins that are involved in the electron or ion transport are proposed to be tightly associated with or incorporated into C. tepidum chlorosomes as well as the ten Csm proteins known to date.


Subject(s)
Bacterial Proteins/chemistry , Chlorobi/chemistry , Chlorobi/cytology , Mass Spectrometry/methods , Organelles/chemistry , Organelles/ultrastructure , Proteomics/methods , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Light , Microscopy, Electron/methods , Molecular Sequence Data , Proteome/analysis
20.
J Proteome Res ; 10(8): 3578-89, 2011 Aug 05.
Article in English | MEDLINE | ID: mdl-21619078

ABSTRACT

Initiation of protein translation is a well-studied fundamental process, albeit high-throughput and more comprehensive determination of the exact translation initiation sites (TIS) was only recently made possible following the introduction of positional proteomics techniques that target protein N-termini. Precise translation initiation is of crucial importance, as truncated or extended proteins might fold, function, and locate erroneously. Still, as already shown for some proteins, alternative translation initiation can also serve as a regulatory mechanism. By applying N-terminal COFRADIC (combined fractional diagonal chromatography), we here isolated N-terminal peptides of a Saccharomyces cerevisiae proteome and analyzed both annotated and alternative TIS. We analyzed this N-terminome of S. cerevisiae which resulted in the identification of 650 unique N-terminal peptides corresponding to database annotated TIS. Furthermore, 56 unique N(α)-acetylated peptides were identified that suggest alternative TIS (MS/MS-based), while MS-based evidence of N(α)-acetylation led to an additional 33 such peptides. To improve the overall sensitivity of the analysis, we also included the 5' UTR (untranslated region) in-frame translations together with the yeast protein sequences in UniProtKB/Swiss-Prot. To ensure the quality of the individual peptide identifications, peptide-to-spectrum matches were only accepted at a 99% probability threshold and were subsequently analyzed in detail by the Peptizer tool to automatically ascertain their compliance with several expert criteria. Furthermore, we have also identified 60 MS/MS-based and 117 MS-based N(α)-acetylated peptides that point to N(α)-acetylation as a post-translational modification since these peptides did not start nor were preceded (in their corresponding protein sequence) by a methionine residue. Next, we evaluated consensus sequence features of nucleic acids and amino acids across each of these groups of peptides and evaluated the results in the context of publicly available data. Taken together, we present a list of 706 annotated and alternative TIS for yeast proteins and found that under normal growth conditions alternative TIS might (co)occur in S. cerevisiae in roughly one tenth of all proteins. Furthermore, we found that the nucleic acid and amino acid features proximate to these alternative TIS favor either guanine or adenine nucleotides following the start codon or acidic amino acids following the initiator methionine. Finally, we also observed an unexpected high number of N(α)-acetylated peptides that could not be related to TIS and therefore suggest events of post-translational N(α)-acetylation.


Subject(s)
Computational Biology , Protein Biosynthesis , Protein Processing, Post-Translational , Proteome , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Acetylation , Amino Acid Sequence , Molecular Sequence Data , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Tandem Mass Spectrometry
SELECTION OF CITATIONS
SEARCH DETAIL
...