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Results for “Protein Modification, Translational”

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PubMed1987

Antibodies to a fusion protein identify a cDNA clone encoding msp130, a primary mesenchyme-specific cell surface protein of the sea urchin embryo.

In this report we identify a 130-kDa protein encoded by a sea urchin primary mesenchyme-specific cDNA clone, 18C6. The cDNA clone has been partially sequenced, and an open reading frame has been identified. A portion of this open reading frame has been expressed as a beta-galactosidase fusion protein in Escherichia coli, and antibodies to the fusion protein have been generated. These antibodies recognize a 130-kDa protein localized at the surface of primary mesenchyme cells and designated msp130. This is demonstrated to be the same 130-kDa protein recognized by the primary mesenchyme-specific monoclonal antibody B2C2, which recognizes a post-translational modification of the protein. RNA gel blots show that the transcript encoding msp130 is undetectable in egg RNA or 16-cell RNA but can be first detected in premesenchyme blastula embryos. The transcript accumulates significantly after primary mesenchyme cell ingression. Analysis of the expression of msp130 by indirect immunofluorescence staining of embryos and by immunoblots using fusion protein antibodies shows that the msp130 protein is first detectable soon after primary mesenchyme cell ingression.

Amino Acid Sequence↗

PubMed1995

Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Transgenic mice and sheep secrete only low levels of human factor IX in their milk because of an aberrant splicing of the transgene RNA in the mammary gland. Removal of the cryptic 3' splice site prevents this splicing and leads to the production of relatively high levels of factor IX. The purified protein is fully active showing that the mammary gland is capable of the efficient post-translational modification of this protein and that transgenic animals are a suitable means of its production.

Animals↗

PubMed2003

Bcl-2 proteins: targets and tools for chemosensitisation of tumor cells.

Proteins of the Bcl-2 family share one or several Bcl-2 homology (BH) regions and behave as pro- or anti-apoptotic proteins. Prosurvival members such as Bcl-2 and Bcl-X(L) are supposed to preserve mitochondrial outer membrane integrity, thus preventing the release of soluble apoptogenic molecules. Pro-apoptotic members include BH3-only proteins that act as sensors of cellular damage and initiate the death process and Bax-like proteins that act downstream of BH3-only proteins to permeabilise the mitochondrial outer membrane. Whether BH3-only proteins directly activate Bax-like proteins or prevent prosurvival members of the family from inhibiting Bax-like proteins or both remains a matter of controversy. Expression of these proteins is altered in various human tumours and this abnormal expression may contribute to oncogenesis and tumour cell resistance to anticancer drug-induced cell death. Based on these observations, prosurvival proteins are attractive intracellular targets for inducing tumour cell death or sensitising tumour cells to death induced by chemotherapeutic drugs. The use of 18-mer antisense oligonucleotides (G3139 or Genasense) targeting the first six codons of bcl-2 mRNA is currently developed in clinics with phase I studies demonstrating that thrombocytopenia may be the main dose-limiting side effect. This strategy, that efficiently decreases Bcl-2 protein expression in some tumour cells, is currently tested in phase II and phase III trials. Alternative approaches to achieve the functional knock-out of Bcl-2 include the use of either peptides mimicking the BH3 domain of Bcl-2-related proteins or more stable, non peptidic BH3 mimetics and the pharmacological modulation of the post-translational modifications of the protein.

Animals↗

PubMedDate not supplied

Application of automated sequence analysis to the understanding of protein structure and function.

Application of automated Edman degradation to the covalent structural analysis of proteins has had an important impact upon the generation of structural information. The strategies involved in determining the complete sequences of proteins have been simplified such that studies of this kind can be carried out more rapidly and with less expenditure of material. Moreover, the high efficiency of the procedures in the anlaysis of intact polypeptides has allowed the convenient comparison of extensive regions of sequence at the amino-termini of proteins. Such structural studies have revealed a more detailed understanding of the function and post-translational modification of many proteins.

Amino Acid Sequence↗

PubMed2003

Distribution and function of new bacterial intein-like protein domains.

Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein-splicing of their host-protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N-terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein-like (BIL) domains. Bacterial intein-like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C-terminal flank and could also protein-splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.

Amino Acid Sequence↗

PubMed2006

Proteomics-driven progress in neurodegeneration research.

Proteomics technologies have been widely used in the investigation of neurodegenerative and psychiatric disorders, and in particular in the detection of differences between healthy individuals and patients suffering from such diseases. Thus, brain and cerebrospinal fluid (CSF) samples from patients with Alzheimer's disease, Down syndrome, Pick's disease, Parkinson's disease, schizophrenia, and other disorders as well as brain and CSF from animals serving as models of neurological disorders have been analyzed by proteomics. 2-DE followed by MALDI-TOF-MS has been mainly applied as this proteomics approach provides the possibility of convenient quantification of protein levels and detection of post-translational modifications. About 330 unique proteins with deranged levels and modifications have been detected by proteomics approaches to be related to neurodegeneration and psychiatric disorders. They are mainly involved in metabolism pathways, cytoskeleton formation, signal transduction, guidance, detoxification, transport, and conformational changes. In this article, we provide a summary of the major contributions of proteomics technologies in the study of neurodegenerative and psychiatric diseases, in particular, in the detection of changes in protein levels and modifications related to these disorders.

Animals↗

PubMed1995

Development of inhibitors of protein farnesylation as potential chemotherapeutic agents.

Protein prenylation, adding either the 15-carbon isoprenoid farnesyl or the 20-carbon isoprenoid geranylgeranyl to cysteine residue(s) at or near the C-termini of proteins, is a recently identified post-translational modification that localizes some proteins to a membrane compartment. One of the most intensely studied prenylated proteins is Ras, a low molecular weight GTP-binding protein that plays an important role in the regulation of cell proliferation. Proteins encoded by ras genes with oncogenic mutations are capable of transforming cells in culture. Such mutated ras genes are frequently found in a wide variety of human tumors. Localization of the Ras oncoprotein to the cytoplasmic face of the plasma membrane via farnesylation is essential for efficient cell transforming ability. Thus, inhibition of the Ras farnesylation reaction is a possible anti-cancer strategy. Several strategies have been employed to inhibit Ras farnesylation, including inhibition of isoprenoid biosynthesis and inhibition of the enzyme which catalyzes the farnesylation reaction, farnesyl-protein transferase (FPTase). Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate limiting enzyme in isoprenoid biosynthesis, inhibit Ras farnesylation and block the growth of ras-transformed cells. However, antiproliferative effects do not result from specific inhibition of Ras farnesylation; they are also observed in cells transformed by raf, which is independent of Ras farnesylation. A more specific approach to inhibiting Ras farnesylation is to inhibit FPTase. Using random screening of natural products and a rational design approach, a variety of compounds that specifically inhibit FPTase have been isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1996

Human placental ATP diphosphohydrolase is a highly N-glycosylated plasma membrane enzyme.

Human placental ATP diphosphohydrolase (ATP-DPH), has been previously characterized as an azide-sensitive, Ca(2+)- or Mg(2+)-dependent triphospho- and diphosphonucleosidase which migrates as an 82 kDa protein band on SDS-PAGE (Christoforidis, S. et al. (1995) Eur. J. Biochem. 234, 66-74). In this paper we have studied the subcellular localization of placental ATP-DPH by differential centrifugation and flotation experiments. Using specific enzymatic markers it was found that ATP-DPH is localized on plasma membrane. ATP-DPH was found to be a highly N-glycosylated protein which is a common post-translational modification of plasma membrane proteins. Extensive incubation of the native pure enzyme with N-glycosidase F resulted in the elimination of the 82 kDa form and the concurrent formation of a deglycosylated product of 57.5 kDa and four other intermediate products, indicating the presence of at least five N-glycosylation sites within the ATP-DPH molecule. The partially deglycosylated sample retained its activity in solution and in native gel electrophoresis and activity staining.

Amidohydrolases↗

PubMed2000

Episomal vectors for gene expression in mammalian cells.

An important reason for preferring mammalian cells for heterologous gene expression is their ability to make authentic proteins containing post-translational modifications similar to those of the native protein. The development of expression systems for mammalian cells has been ongoing for several years, resulting in a wide variety of effective expression vectors. The aim of this review is to highlight episomal expression vectors. Such episomal plasmids are usually based on sequences from DNA viruses, such as BK virus, bovine papilloma virus 1 and Epstein-Barr virus. In this review we will mainly focus on the improvements made towards the usefulness of these systems for gene expression studies and gene therapy.

Animals↗

PubMedDate not supplied

Does PRNP gene control the clinical and pathological phenotype of human spongiform transmissible encephalopathies?

BACKGROUND: Human spongiform transmissible encephalopathies (TSE) are a group of neurodegenerative diseases caused by a transmissible not yet recognized agent; their distinctive neuropathological features are astrocytosis, spongiform lesions of the neuropil, neuronal loss and occasionally amyloid plaques in the cortical and subcortical gray matter. TSE are biochemically characterized by the deposition in the nervous system of an amyloid-type protein, PrPres derived from the post-translational modification of a normal protein, PrPsen. The expression of this protein is controlled by the PRNP gene mapped on chromosome 20 in man. A number of point mutations of the PRNP gene have been described in the familial forms of these TSE. Some of these mutations have been associated with differences in the phenotypic expression of the disease. MATERIAL AND METHODS: This study was designed to verify whether it was possible to identify a selective phenotype depending upon a given PRNP modified genotye; for this purpose, a group of familial TSE cases (CJD 210ILE, CJD 201LYS, FFI 178ASN) were selected and their neuropathological profiles have been compared with those of a large series of sporadic CJD cases. RESULTS: No significant differences were found between the topography and severity of lesions in the cerebral cortex, cerebellum, hippocampus, basal ganglia and thalamus between the two groups. Two differences were found: the clinical duration of the disease which appeared significantly (p = 0.02) shorter in the 210ILE-mutated cases compared to that of non-mutated sporadic cases. The highly selective vulnerability of thalamus in FFI showing a severe pathology especially in its dorso-medial part in comparison with that of the sporadic CJD cases. CONCLUSION: The results of this study confirm that the different polymorphism at codon 129 of the PRNP gene, which could be involved in the structural "domains" of human PrP, might modulate the pathological phenotype of TSE.

Aged↗

PubMedDate not supplied

Endogenous relatives of ADP-ribosylating bacterial toxins in mice and men: potential regulators of immune cell function.

ADP-ribosylation of proteins, like phosphorylation, is a post-translational modification that can modulate protein function. Bacterial mono (ADP-ribosyl)transferases have been well studied, since potent and clinically important pathogenic exoenzymes such as diphtheria, cholera and pertussis toxins belong to this group. Some of these enzymes interfere with signal transduction mechanisms of host cells, and have become widely used as research tools in cell biology because of their high potency and selectivity. Recently, relatives of these toxins have been cloned from vertebrates. Seven members of a novel multigene family have been identified to date. Surprisingly, all are predicted to be extracellular proteins. Preferred tissues of expression are skeletal and cardiac muscle, testis and hematopoietic cells. ADP-ribosylation of target proteins on the cell surface of T cells and leukocytes have been found to modulate the transmission of extracellular signals to the cell interior.

ADP Ribose Transferases↗

PubMed2000

Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system.

We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.

Animals↗

PubMed2005

A role of p73 in mitotic exit.

The p53-related p73 proteins regulate developmental processes, cell growth, and DNA damage response. p73 function is regulated by post-translational modifications and protein-protein interactions. At the G2/M transition, p73 is phosphorylated at Thr-86 by the p34cdc2/cyclin B complex; this is associated with its exclusion from condensed chromosomes and loss of DNA binding and transcriptional activation ability. Here we showed that p73 hypo-phosphorylated species reappear during mitotic exit, concomitant with p73 relocalization to telophase nuclei and recovered ability to activate transcription. Functional knock-out of p73 gene expression by small interfering RNAs (siRNAs) alters mitotic progression, yielding an increase of ana-telophase cells, the accumulation of aberrant late mitotic figures, and the appearance of abnormalities in the subsequent interphase. This p73 activity at the M-to-G1 transition is mediated by its transactivating function because expression of the transcription dominant negative mutant p73DD induces the same mitotic exit phenotype. We also found that the cyclin-dependent kinase inhibitor Kip2/p57 gene is a specific target of p73 regulation during mitotic exit and re-entry into G1. Both knock-out of p73 gene expression by siRNAs and abrogation of p73-dependent transcription by the p73DD mutant abrogate Kip2/p57 increase at the M-to-G1 transition. Moreover, similar abnormalities (e.g. delay in late mitotic stages with the accumulation of aberrant ana-telophase figures, and abnormalities in the following interphase) are observed in cultures in which the expression of Kip2/p57 is abrogated by siRNAs. These results identify a novel p73-Kip2/p57 pathway that coordinates mitotic exit and transition to G1.

Anaphase↗

PubMed2001

Poly(ADP-ribose) polymerase: a guardian angel protecting the genome and suppressing tumorigenesis.

Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage generated either exogenously or endogenously. This post-translational modification is catalyzed by poly(ADP-ribose) polymerase (PARP, PARP-1, EC 2.4.2.30). It is proposed that this protein plays a multifunctional role in many cellular processes, including DNA repair, recombination, cell proliferation and death, as well as genomic stability. Chemical inhibitors of the enzyme, dominant negative or null mutations of PARP-1 cause a high degree of genomic instability in cells. Inhibition of PARP activity by chemical inhibitors renders mice or rats susceptible to carcinogenic agents in various tumor models, indicating a role for PARP-1 in suppressing tumorigenesis. Despite the above observations, PARP-1 knockout mice are generally not prone to the development of tumors. An enhanced tumor development was observed, however, when the PARP-1 null mutation was introduced into severely compromised immune-deficient mice (a mutation in DNA-dependent protein kinase) or mice lacking other DNA repair or chromosomal guardian molecules, such as p53 or Ku80. These studies indicate that PARP-1 functions as a cofactor to suppress tumorigenesis via its role in stabilization of the genome, and/or by interacting with other DNA strand break-sensing molecules. Studies using PARP-1 mutants and chemical inhibitors have started to shed light on the role of this protein and of the specific protein post-translational modification in the control of genomic stability and hence its involvement in cancer.

Animals↗

PubMed2004

Detection and proteomic identification of S-nitrosylated proteins in endothelial cells.

Nitric oxide (NO) is a key signalling molecule in the vascular endothelium. It can induce different post-translational modifications in mammalian proteins that can alter their functionality, among which incorporation of a NO group in cysteine thiols, called S-nitrosation or S-nitrosylation, is one of the best characterized. Identification of the proteins that are susceptible to this modification would help us to determine the relevance of this modification in physiological and pathophysiological conditions. For this purpose, we have used a proteomic approach to identify S-nitrosylated proteins in endothelial cells, which includes replacing S-nitrosylation by a specific biotinylation and subsequent purification (called the "biotin switch" method). We have applied this methodology to identify proteins that are S-nitrosylated in endothelial cells acutely exposed to S-nitroso-l-cysteine, a physiological S-nitrosothiol. We describe the identified proteins and discuss the characterization of their S-nitrosylation, confirming the validity of the methodology for approaching the description of the "nitrosylome."

Animals↗

PubMed2004

Evidence for a post-translational modification, aspartyl aldehyde, in a photosynthetic membrane protein.

In oxygenic photosynthesis, photosystem II (PSII) carries out the oxidation of water and reduction of plastoquinone. Three PSII subunits contain reactive groups that covalently bind amines and phenylhydrazine. It has been proposed that these reactive groups are carbonyl-containing, co- or post-translationally modified amino acids. To identify modified amino acid residues in one of the PSII subunits (CP47), tandem mass spectrometry was performed. Modified residues were affinity-tagged with either biotin-LC-hydrazide or biocytin hydrazide, which are known to label carbonyl groups. The affinity-tagged subunit was isolated by denaturing gel electrophoresis, and tryptic peptides were then subjected to affinity purification and tandem mass spectrometry. This procedure identified a hydrazide-labeled peptide, which has the sequence XKEGR. This result is supported by quantitative results acquired from peptide mapping and methylamine labeling. The gene sequence and these tandem data predict that the first amino acid, X, which is labeled with the hydrazide reagent, is a modified form of aspartic acid. On the basis of these data, we propose that D348 of the CP47 subunit is post- or co-translationally modified to give a novel amino acid side chain, aspartyl aldehyde.

Amino Acids↗

PubMed2002

Chemical strategies for functional proteomics.

With complete genome sequences now available for several prokaryotic and eukaryotic organisms, biological researchers are charged with the task of assigning molecular and cellular functions to thousands of predicted gene products. To address this problem, the field of proteomics seeks to develop and apply methods for the global analysis of protein expression and protein function. Here we review a promising new class of proteomic strategies that utilizes synthetic chemistry to create tools and assays for the characterization of protein samples of high complexity. These approaches include the development of chemical affinity tags to measure the relative expression level and post-translational modification state of proteins in cell and tissue proteomes. Additionally, we discuss the emerging field of activity-based protein profiling, which aims to synthesize and apply small molecule probes that monitor dynamics in protein function in complex proteomes.

Affinity Labels↗

PubMed1994

High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector.

Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially available vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marker, and the dihydrofolate reductase-encoding gene (DHFR) to enable amplification of transfected DNA followed by stable expression in mammalian cell lines. Levels of 5 micrograms/ml of the coagulation proteins, factor VII (FVII) and factor XI (FXI), have been achieved in serum-free media. N-terminal sequencing of the purified proteins, and of their separated chains after proteolytic activation, demonstrated correct processing of the recombinant products. In addition, the ratios of clotting activity to antigen for each are close to unity, and the recombinant and plasma-derived proteins had identical mobilities upon electrophoresis in the presence of SDS. The vector described will be of use for the synthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translational modification of the protein makes it essential to use mammalian cells.

Animals↗