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Results for “Protein Modification, Translational”

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PubMed2005

Production of mouse adiponectin, an anti-diabetic protein, in transgenic sweet potato plants.

Adiponectin is a 30kDa protein exclusively produced and secreted from adipocytes and as a cytokine has been found to link obesity, insulin resistance, and type 2 diabetes. Production of biologically active adiponectin in large scale is desirable for pharmaceutical applications. Mouse adiponectin cDNA was used for developing transgenic sweet potato plants via Agrobacterium-mediated transformation. The presence of the transgene was verified by PCR and DNA gel blot analysis. Further investigated were five independent transgenic lines, all of which expressed high levels of adiponectin mRNA. Immuno blot analysis with a mouse adiponectin antiserum revealed that, in addition to a 29 kDa-protein which co-migrates with the adiponectin protein produced in Escherichia coli cells, a 31 kDa-protein was produced, indicative of a post-translational modification of the protein. The transgenic plants did not show obvious differences in growth rate and morphology in response to adiponectin production.

Adiponectin↗

PubMed1994

A novel 105-kDa lamina lucida autoantigen: association with bullous pemphigoid.

Several cases have been reported of patients with immunemediated subepidermal blistering disorders whose autoantibodies react to antigens present on both the dermal and epidermal side of 1 M NaCl-split skin. In this report, we identify, localize, and characterize the basement membrane zone antigen corresponding to the dermal staining in a patient whose serum stains both the dermal and epidermal side of 1 M NaCl-split skin. This patient's serum contains autoantibodies directed against a 105-kilodalton(kDa) dermal antigen and the 230-kDa epidermal (bullous pemphigoid) antigen. This novel 105-kDa protein was previously identified as the sole antigen in another patient with a unique bullous disease whose autoantibodies were directed against only the dermal side of 1 M NaCl-split skin. This 105-kDa antigen was identical by one- and two-dimensional immunoblot analysis in these two patients. By immunoblot analysis, autoantibodies from our patient labeled a 105-kDa protein within various extracts of human skin basement membrane. Immunoblot analyses using epitope-selected autoantibodies directed against the 105-kDa protein demonstrated that this antigen is independent and distinct from other known basement membrane antigens. The 105-kDa antigen is an extracellular matrix component of the basement membrane, which is synthesized and secreted by both keratinocytes and fibroblasts. Identical electrophoretic migration of cellular and secreted forms of the protein suggested there is no major post-translational modification of the protein. Immunomapping of normal human skin fractured through the dermal-epidermal junction by incubation in 1 M NaCl or by suction blistering demonstrated that the location of the 105-kDa antigen within the basement membrane zone is between the bullous pemphigoid antigens and two other lamina lucida components, laminin and nicein. These data demonstrate clearly that a subepidermal autoimmune bullous disease may have autoantibodies directed against two distinct components of the dermal-epidermal junction.

Aged↗

PubMed2004

Proteomics approaches to identify tumor antigen directed autoantibodies as cancer biomarkers.

The identification of autoantibodies to tumor cell proteins by proteomics approaches has great potential impact on cancer biomarker discovery. The humoral immune response represents a form of biological amplification of signals that are otherwise weak due to very low concentrations of antigen, especially in the early stages of cancers. In addition, proteomics can detect immunoreactivity directed against protein post-translational modifications. Two-dimensional gel based Western blots, protein antigen microarrays, and multiplex ELISA reactions have been applied by our group to antigen based biomarker detection and validation. The latter two are based on liquid-phase separations that are suitable for automation. This work has resulted in the identification of numerous cancer biomarker candidates. Large clinical studies are currently planned to establish their value in early cancer diagnosis.

Antibodies, Neoplasm↗

PubMed2001

[Proteic marker of hypoxic-ischemic damage].

Perinatal hypoxic injury is the major cause of normal neural developmental alterations. Recent studies concerning animal models show that an hypoxic/ischaemic event triggers a process taking to a synaptic architecture reorganization which induces a transient change in the synaptic (synapsin 1, SNAP 25, APP) and neuronal (MAP2, N-CAM, GAP-43 and presenilins) protein expression. Here we review the post-translational modifications of some proteins after hypoxic-ischaemic events. A deeper study on synaptic proteins plasticity could give an important key for the understanding of the recovery mechanisms of the nervous system.

Amyloid beta-Peptides↗

PubMed2002

Managing sarcopenia with progressive resistance exercise training.

Advancing age appears to alter the chemical and physical properties of skeletal muscle proteins. Alterations include: reduced contractile, mitochondrial, and enzyme protein synthesis rates, altered expression and post-translational modifications to muscle proteins, reduced maximum voluntary muscle strength, reduced muscle strength per unit muscle mass and muscle power. These age-associated impairments in muscle protein quantity and quality contribute to physical disability and frailty, a loss of independent function, the risk of falling and fractures, and contribute to escalating health care costs. Progressive resistance exercise training is a potent, non-pharmacologic, effective therapy that opposes the impairments in muscle protein quantity and quality in middle age and physically frail adults. In the absence of contraindications to exercise, muscle proteins adapt to an exercise training stimulus despite the depredation of age. The proposed pathogenesis for some of these impairments is briefly reviewed. Evidence that supports the use of progressive resistance exercise training to restore muscle quality and quantity in elderly adults is reviewed.

Journal Article↗

PubMed2004

Post-translational modifications in the survival motor neuron protein.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by a progressive loss of the spinal motoneurons. The SMA-determining gene has been termed survival motor neuron (SMN) and is deleted or mutated in over 98% of patients. The encoded gene product is a protein expressed as different isoforms. In particular, we showed that the rat SMN cDNA produces two isoforms with M(r) of 32 and 35kDa, both localized in nuclear coiled bodies, but the 32kDa form is also cytoplasmic, whereas the 35kDa form is also microsomal. To determine the molecular relationship between these two isoforms and potential post-translational modifications, we performed transfection experiments with a double-tagged rat SMN. Immunoblot and immunostaining studies demonstrated that the 32kDa SMN isoform derives from the full length 35kDa, through a proteolytic cleavage at the C-terminal. Furthermore, the 35kDa SMN isoform is physiologically phosphorylated in vivo. This may modulate its interaction with molecular partners, either proteins or nucleic acids.

3T3 Cells↗

PubMed1993

Regulation of zygotic gene activation in the mouse.

Zygotic gene activation (ZGA) is the critical event that governs the transition from maternal to embryonic control of development. In the mouse, ZGA occurs during the 2-cell stage and appears to be regulated by the time following fertilization, i.e. a zygotic clock, rather than by progression through the first cell cycle. The onset of ZGA must depend on maternally inherited proteins, and post-translational modification of these maternally derived proteins is likely to play a role in ZGA. Consistent with this prediction is that protein phosphorylation catalyzed by the cAMP-dependent protein kinase is involved in ZGA and that protein synthesis is not required for ZGA. Recent results suggest that ZGA may occur earlier than previously thought, i.e. not during the 2-cell stage, but rather in G2 of the 1-cell embryo. Thus ZGA may comprise a period of minor gene activation in the 1-cell embryo that is followed by a period of major gene activation in the 2-cell embryo. Following ZGA, the expression of constitutively activated genes may require an enhancer.

Animals↗

PubMed1991

Post-translational modifications of the C-terminal region of the rho protein are important for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins.

We have recently found, by use of the rhoA p21 purified from bovine aortic smooth muscle, that it is similarly post-translationally processed as described for ras p21s: it is first geranylgeranylated at the cysteine residue in the C-terminal region followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the revealed C-terminal cysteine residue. In the present study, we investigated the function(s) of these post-translational modifications of the C-terminal region of rhoA p21 by use of the rhoA p21s purified from bovine aortic smooth muscle and rhoA p21-overexpressing Escherichia coli since the bacterial protein was not modified with a geranylgeranyl moiety. Bovine rhoA p21 bound to plasma membranes and phosphatidylserine-linked Affigel, but bacterial rhoA p21 did not bind to them. The inhibitory GDP/GTP exchange protein for rhoA p21, named GDP dissociation inhibitor (GDI), made a complex with the GDP-bound form of bovine rhoA p21 and thereby inhibited the dissociation of GDP from and the subsequent binding of GTP to it. However, rho GDI neither made a complex with the GDP-bound form of bacterial rhoA p21 nor affected these reactions of the bacterial protein. The stimulatory GDP/GTP exchange protein for rhoA p21, named GDP dissociation stimulator (GDS), stimulated the dissociation of GDP from bovine rhoA p21, but was inactive for the bacterial protein. In contrast, the GTPase activating protein for rhoA p21 is active not only for bovine rhoA p21 but also for the bacterial protein. These results suggest that the post-translational modifications of the C-terminal region of bovine rhoA p21, most presumably the geranylgeranylation, which are absent in bacterial rhoA p21, play important roles in its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins but not with the GAP.

Animals↗

PubMed2006

Protein function microarrays based on self-immobilizing and self-labeling fusion proteins.

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction.

Carrier Proteins↗

PubMed2003

Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis.

Peptide mass fingerprinting (PMF) is a powerful tool for identification of proteins separated by two-dimensional electrophoresis (2-DE). With the increase in sensitivity of peptide mass determination it becomes obvious that even spots looking well separated on a 2-DE gel may consist of several proteins. As a result the number of mass peaks in PMFs increased dramatically leaving many unassigned after a first database search. A number of these are caused by experiment-specific contaminants or by neighbor spots, as well as by additional proteins or post-translational modifications. To understand the complete protein composition of a spot we suggest an iterative procedure based on large numbers of PMFs, exemplified by PMFs of 480 Helicobacter pylori protein spots. Three key iterations were applied: (1) Elimination of contaminant mass peaks determined by MS-Screener (a software developed for this purpose) followed by reanalysis; (2) neighbor spot mass peak determination by cluster analysis, elimination from the peak list and repeated search; (3) re-evaluation of contaminant peaks. The quality of the identification was improved and spots previously unidentified were assigned to proteins. Eight additional spots were identified with this procedure, increasing the total number of identified spots to 455.

Cluster Analysis↗

PubMed2004

The annexins.

Annexins are traditionally thought of as calcium-dependent phospholipid-binding proteins, but recent work suggests a more complex set of functions. More than a thousand proteins of the annexin superfamily have been identified in major eukaryotic phyla, but annexins are absent from yeasts and prokaryotes. The unique annexin core domain is made up of four similar repeats approximately 70 amino acids long, each of which usually contains a characteristic 'type 2' motif for binding calcium ions. Animal and fungal annexins also have non-homologous amino-terminal domains of varying length and sequence, which are responsible for the distinct localizations and specialized functions of the proteins through post-translational modification and binding to other proteins. Annexins interact with various cell-membrane components that are involved in the structural organization of the cell, intracellular signaling by enzyme modulation and ion fluxes, growth control, and they can act as atypical calcium channels. Analysis of site-specific conservation in the core domain suggests a role for certain buried residues in the calcium-channel activity of vertebrate annexins and in the structural stability of their core domains. Evolutionarily significant differences between subfamilies are preferentially localized to accessible sites on the protein surface that determine membrane binding and interactions with cytosolic proteins.

Animals↗

PubMed1992

Changes in sarcolemmal proteins in subacute myocardial infarction in the dog.

Previous studies have shown degradation of cardiac structural proteins and disruption of the sarcolemma as a result of acute myocardial infarction. However, there is no evidence to date on changes in sarcolemmal membrane proteins induced by experimental subacute myocardial infarction. We studied subepicardial layers overlying myocardial infarct 4 days following ligation of the left anterior descending coronary artery in 12 dog hearts. We first demonstrated that this layer provides the anatomic-electrophysiologic substrate for reentrant arrhythmias using activation mapping techniques and histologic correlations. The makeup of membrane proteins was studied using SDS polyacrylamide gel electrophoresis, peptide mapping, and laser densitometry. Sarcolemmal membrane proteins were isolated by ultracentrifugation through a sucrose gradient. We found that a sarcolemmal polypeptide (MW 126,000; n = 12) in the normal tissues has a different mobility than the corresponding protein (MW 124,000; n = 12) of the ischemic tissues although their peptide analysis appeared similar, suggesting that the protein undergoes a post-translational modification. In addition, two proteins (MW 75,000; n = 12 and MW 88,000; n = 12) were present in greater amount in the ischemic than in the control tissues suggesting either acceleration in protein synthesis or slow down of degradation turnover. These results demonstrate that specific changes occur in membrane proteins subjected to ischemic insults which might be responsible for membrane alterations following ischemia and may contribute to the abnormal electrophysiologic properties and arrhythmia seen in vivo at this stage.

Animals↗

PubMed2003

A multimeric model for murine anti-apoptotic protein Bcl-2 and structural insights for its regulation by post-translational modification.

A monomeric model for murine antiapoptotic protein Bcl-2 was constructed by comparative modeling with the software suite MPACK (EXDIS/DIAMOD/FANTOM) using human Bcl-xL as a template. The monomeric model shows that murine Bcl-2 is an all alpha-helical protein with a central (helix 5) hydrophobic helix surrounded by amphipathic helices and an unstructured loop of 30 residues connecting helices 1 and 2. It has been previously shown that phosphorylation of Ser 70 located in this loop region regulates the anti-apoptotic activity of Bcl-2. Based on our current model, we propose that this phosphorylation may result in a conformational change that aids multimer formation. We constructed a model for the Bcl-2 homodimer based on the experimentally determined 3D structure of the Bcl-xL: Bad peptide complex. The model shows that it will require approximately a half turn in helix 2 to expose hydrophobic residues important for the formation of a multimer. Helices 5 and 6 of the monomeric subunit Bcl-2 have been proposed to form an ion-channel by associating with helices 5 and 6 of another monomeric subunit in the higher-order complex. In the multimeric model of Bcl-2, helices 5 and 6 of each subunit were placed distantly apart. From our model, we conclude that a global conformational change may be required to bring helices 5 and 6 together during ion-channel formation.

Amino Acid Sequence↗

PubMed1990

Protein synthesis initiation factor modifications during viral infections: implications for translational control.

Infection of tissue culture cells with certain viruses results in the shutoff of host cell protein synthesis. We have examined virally infected cell lysates using two-dimensional gel electrophoresis and immunoblotting to ascertain whether initiation factor protein modifications are correlated with translational repression. Moderate increases in eukaryotic initiation factor (eIF)-2 alpha phosphorylation are detected in reovirus- and adenovirus-infected cells, as reported previously (Samuel et al., 1984; O'Malley et al., 1989). Neither vesicular stomatitis virus, vaccinia virus, frog virus III, rhinovirus, nor encephalomyocarditis virus caused significantly increased 2 alpha phosphorylation. There were no reproducible, significant changes in eIF-4A, eIF-4B, or eIF-2 beta in cells infected by any of these viruses. The cleavage of eIF-4F subunit p220, such as has been previously demonstrated to occur in poliovirus (Etchison et al., 1982) and rhinovirus (Etchison and Fout, 1985), was not detected in any of the other virus infections analyzed.

Adenoviridae↗

PubMed1998

On-line liquid chromatography/electrospray tandem mass spectrometry to investigate acrylamide adducts with cysteine residues: implications for polyacrylamide gel electrophoresis separations of proteins.

Adduction between acrylamide and cysteine residues is a post-translational modification associated with proteins separated by gel electrophoresis. In the present article, three model peptides containing 2-4 cysteine residues were reduced with dithiothreitol, incubated with acrylamide monomers and examined by on-line liquid chromatography coupled to electrospray tandem mass spectrometry. Each of the solutions examined in this work revealed the presence of four distinct components: the free peptide, two different peptide-acrylamide 1:1 adducts involving two cysteine residues at different positions within the same sequence, and the peptide-acrylamide 1:2 adducts. The use of liquid chromatography allowed the separation of components which differed only by the site of complexation of acrylamide, while the application of tandem mass spectrometry furnished reliable sequencing information permitting the identification of most cysteine residues involved in such complexation.

Acrylamides↗

PubMed2003

The on-off story of protein palmitoylation.

Palmitoylation is one of the most frequent post-translational modifications found on proteins. It contributes to membrane association, protein sorting and many other processes. Through its reversibility, palmitoylation also provides mechanisms to regulate the functional activities of integral and peripheral membrane proteins. Here we discuss evidence that proteins can be palmitoylated at different locations in the cell, how targeting to these locations might be directed, and aspects of the proposed functions of palmitoylation.

Amino Acid Sequence↗

PubMed2004

Modulation of erythrocyte membrane mechanical function by protein 4.1 phosphorylation.

Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of alpha- and beta-spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, beta-spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties.

Actins↗

PubMed2006

The impact of protein characterization in structural proteomics.

Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.

Crystallization↗