Carboxy Terminus Of Protein Research Articles

Carboxy-terminal blockade of sortilin binding enhances progranulin gene therapy, a potential treatment for frontotemporal dementia.

The effectiveness of progranulin gene therapy in models of FTD is improved by blocking the protein's carboxy terminus, which prevents sortilin binding.

Organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core.

The difference in membrane (M) protein compositions between the transmissible gastroenteritis coronavirus (TGEV) virion and the core has been studied. The TGEV M protein adopts two topologies in the virus envelope, a Nexo-Cendo topology (with the amino terminus exposed to the virus surface and the carboxy terminus inside the virus particle) and a Nexo-Cexo topology (with both the amino and carboxy termini exposed to the virion surface). The existence of a population of M molecules adopting a Nexo-Cexo topology in the virion envelope was demonstrated by (i) immunopurification of (35)S-labeled TGEV virions using monoclonal antibodies (MAbs) specific for the M protein carboxy terminus (this immunopurification was inhibited only by deletion mutant M proteins that maintained an intact carboxy terminus), (ii) direct binding of M-specific MAbs to the virus surface, and (iii) mass spectrometry analysis of peptides released from trypsin-treated virions. Two-thirds of the total number of M protein molecules found in the virion were associated with the cores, and one-third was lost during core purification. MAbs specific for the M protein carboxy terminus were bound to native virions through the M protein in a Nexo-Cexo conformation, and these molecules were removed when the virus envelope was disrupted with NP-40 during virus core purification. All of the M protein was susceptible to N-glycosidase F treatment of the native virions, which indicates that all the M protein molecules are exposed to the virus surface. Cores purified from glycosidase-treated virions included M protein molecules that completely or partially lost the carbohydrate moiety, which strongly suggests that the M protein found in the cores was also exposed in the virus envelope and was not present exclusively in the virus interior. A TGEV virion structure integrating all the data is proposed. According to this working model, the TGEV virion consists of an internal core, made of the nucleocapsid and the carboxy terminus of the M protein, and the envelope, containing the spike (S) protein, the envelope (E) protein, and the M protein in two conformations. The two-thirds of the molecules that are in a Nexo-Cendo conformation (with their carboxy termini embedded within the virus core) interact with the internal core, and the remaining third of the molecules, whose carboxy termini are in a Nexo-Cexo conformation, are lost during virus core purification.

Exogenous Peptide and Protein Expression Levels Using Retroviral Vectors in Human Cells

Pseudotyped retroviral vectors combine the advantages of broad host range, high expression, stable chromosomal integration, and ease of preparation. These vectors greatly facilitate delivery into mammalian cells of sequences encoding individual peptide inhibitors—including those with therapeutic utility—and inhibitor libraries. However, retroviral vectors vary in behavior, particularly with respect to expression levels in different cell lines. Expression level is especially important in transdominant experiments because the concentration of an inhibitor (for example, an expressed peptide) is one of the key determinants in the degree of complex formation between the inhibitor and its target. Thus, inhibitor concentration should have an impact on the expressivity and/or penetrance of an induced phenotype. Here, we compare several retroviral vectors and human cell lines for relative expression levels using a green fluorescent protein reporter. We show for a subset of these lines that cellular protein concentrations produced by single-copy vectors range up to about 2 μM. We also examine other variables that contribute to expression level, such as the nature of the expressed protein's carboxy terminus. Finally, we test the effect of increased concentration on phenotype with a nine-amino-acid peptide derived from the human papilloma virus protein E7 which overcomes E7-mediated cell growth.

Specific recognition of protein carboxy-terminal sequences by natural IgM antibodies in normal serum.

Our previous study indicated that normal serum contains complement-fixing natural IgM antibodies reacting with a large variety of randomly generated protein carboxy-termini. Here we show that the "carboxy-terminal" IgM (C-IgM) antibodies specifically react with short peptide sequences located immediately at the protein carboxy-terminus. The specificity of C-IgM-peptide interactions is tentatively defined by three to four amino acid residues. All carboxy-terminal peptides in a large peptide library apparently react with C-IgM antibodies. Immobilized synthetic peptides also react with C-IgM antibodies. No interaction of C-IgM antibodies with internal peptide sequences has been observed. C-IgM antibodies are present in germ-free and in athymic adult rats and are absent in newborn rats. The natural ubiquity of protein carboxy-termini in biological structures suggests that C-IgM could play an important role in antigen clearance and presentation to the immune system. From a practical viewpoint, the recognition of carboxy-terminal peptides by complement-fixing C-IgM antibodies has profound implications for the use of peptide- and protein-derivatized delivery vehicles and artificial materials.

Cloning and Characterization of CPVL, a Novel Serine Carboxypeptidase, from Human Macrophages

Carboxypeptidases are proteases that cleave single amino acids from the carboxy termini of proteins or peptides. In addition to degradative functions in the gut, carboxypeptidases activate or inactivate bioactive peptides such as angiotensin, bradykinin, and endothelin I. Using differential display PCR, we cloned a novel carboxypeptidase expressed in human macrophages but not in other leukocytes. The 476-amino-acid gene product has a putative signal sequence but no transmembrane domain and has striking sequence similarity to serine carboxypeptidases, a large family of enzymes in eukaryotes. Only one serine carboxypeptidase, lysosomal protective protein, has previously been reported in mammals. Among known proteins, this gene is most similar (43% amino acid identity) to vitellogenic carboxypeptidase, a serine carboxypeptidase expressed in mosquito ovaries. Therefore, we have named this new gene carboxypeptidase, vitellogenic-like (CPVL). In addition to monocyte/macrophage-rich sources such as spleen, leukocytes, and placenta, CPVL mRNA is abundantly expressed in heart and kidney, suggesting a separate role for CPVL outside the immune system. The CPVL gene contains at least 13 exons spread over more than 150 kb on human chromosome 7p14–p15. An affinity-purified polyclonal antiserum recognized a protein of approximately 57 kDa in macrophage lysates, but not in lysates from lymphocytes, neutrophils, or monocytes. CPVL protein expression was induced during maturation of monocytes into macrophages. Possible functions for CPVL in macrophages include digestion of phagocytosed particles in the lysosome, participation in an inflammatory protease cascade, and trimming of peptides for antigen presentation.

Molecular pathology and mechanism of action of the steroidogenic acute regulatory protein, StARProceedings of Xth International Congress on Hormonal Steroids, Quebec, Canada, 17–21 June 1998.

The first and rate-limiting step in the synthesis of all steroid hormones is the conversion of cholesterol to pregnenolone by the mitochondrial enzyme, P450scc. Tropic hormones such ACTH and gonadotropins induce steroidogenesis via cAMP by elaborating intracellular cAMP which stimulates P450scc activity in two distinct ways. Chronic stimulation (h to days) occurs through the induction of P450scc gene transcription leading to increased P450scc protein and consequent increased steroidogenic capacity. Acute regulation, over minutes, occurs through the phosphorylation of preexisting StAR and the rapid synthesis of new StAR protein. StAR, the steroidogenic acute regulatory protein, increases the flow of cholesterol into mitochondria, thus regulating substrate availability to whatever amount of P450scc is available. In the absence of StAR, up to 14% of maximal StAR-induced level of steroidogenesis persists as StAR-independent steroidogenesis. Congenital lipoid adrenal hyperplasia, an autosomal recessive disorder in which conversion of cholesterol to pregnenolone is severely impaired, results in female genitalia in 46,XY genetic males, variable onset of a severe salt-losing crisis in the first months of life, but normal feminization and cyclical vaginal bleeding in 46,XX females. Lipoid CAH was once thought to be due to P450scc mutations, but in fact homozygous P450scc mutations cannot exist in human beings as they would prohibit placental progesterone production, causing spontaneous abortion of the affected fetus. Lipoid CAH is caused by StAR mutations, which result in tropic hormone-induced intracellular accumulation of cholesterol in the adrenals and gonads. Our two-hit model, which considers the persistence of StAR-independent steroidogenesis and the differences in the fetal and postnatal ages at which the testis, adrenal zona glomerulosa, adrenal zona fasciculata and ovary are stimulated, predicts and explains all of the various clinical manifestations of lipoid CAH. Structure–function studies of StAR show that the critical domains for biological activity reside in the protein's carboxy-terminus. When the N-terminal mitochondrial targeting sequences are deleted and the resulting N-62 StAR remains in the cytoplasm, it retains the ability to stimulate steroidogenesis both in intact cells or when added to isolated mitochondria in vitro. These observations suggest that StAR acts on the outer mitochondrial membrane to promote sterol translocation to P450scc, and that the importation of StAR into mitochondria terminates its action. Data from circular dichroism and Fourier-transform infrared spectroscopy show that the mutant StAR proteins in lipoid CAH are misfolded, suggesting disrupted interaction with another protein. Preliminary data suggest that StAR facilitates cholesterol desorption from membranes, stimulating transfer from the outer mitochondrial (donor) membrane to the inner mitochondrial (acceptor) membrane.

Nucleotide Sequence of a Complementary DNA Encoding Pea Cytosolic Copper/Zinc Superoxide Dismutase

The authors now report the nucleotide sequence of the cytosolic Cu/Zn SOD cloned from a {lambda}gt11 cDNA library constructed from mRNA extracted from leaves of 7- to 10-d pea seedlings (Pisum sativum L.). The clone was isolated using a 22-base synthetic oligonucleotide complementary to the amino acid sequence CGIIGLQG. This sequence, found at the protein's carboxy terminus, is highly conserved among plant cytosolic Cu/Zn SODs but not chloroplastic Cu/Zn SODs. The 738-base pair sequence contains an open reading frame specifying 152 codons and a predicted M{sub r} of 18,024 D. The deduced amino acid sequence is highly homologous (79-82% identity) with the sequences of other known plant cytosolic Cu/Zn SODs but less highly conserved (63-65%) when compared with several chloroplastic Cu/Zn SODs including pea (10).

N-Ac-KDEL induces rapid alterations in endoplasmic reticulum structure

KDEL (i.e., Lysine-Aspartate-Glutamate-Leucine) is a tetrapeptide motif that serves as a retention sequence at the carboxy-terminus of proteins which reside within the ER lumen. A specific receptor located in or near the cis-Golgi recognizes proteins bearing carboxyterminal KDEL and mediates their return to the ER. We reasoned that it may be possible to saturate the KDEL receptor with a hapten, thereby competitively inhibiting its function in living cells. We hypothesized that if we blinded the receptor with an excess of hapten then the intermediate compartment should be unable to differentiate between polypeptides containing carboxyterminal KDEL from those that do not. If the receptor could be blinded, then KDEL-polypeptides such as BiP should be secreted.

Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2′-deoxyuridylate and 10-propargyl-5,8-dideazafolate

The crystal structure of an Escherichia coli thymidylate synthase (TS) ternary complex containing 5-fluoro-2′-deoxyuridylate (FdUMP) and 10-propargyl-5,8-dideazafolate (PDDF) has been determined and refined at 2.3 Å resolution. Each of the two chemically identical subunits folds into a three-layer domain anchored by a large six-stranded mixed β-sheet. The backside of one sheet is juxtaposed against the corresponding face of the equivalent sheet in the second protomer creating a β-sandwich. In contrast to other proteins of known structure in which aligned β-sheets stack face to face with a counterclockwise rotation, sheets in the TS dimer are related by a clockwise twist. The substrate-binding pocket is a large funnel-shaped cleft extending some 25 Å into the interior of each subunit and is surrounded by 30 amino acids, 28 from one subunit and two from the other. FdUMP binds at the bottom of this pocket covalently linked through C-6 to the sulfur of Cys146. Up-pointing faces of the pyrimidine and ribose rings are exposed to provide a complementary docking surface for the quinazoline ring of PDDF. The quinazoline inhibitor binds in a partially folded conformation with its p-aminobenzoyl glutamate tail exposed at the entrance to the active site cleft. Ternary complex formation is associated with a large conformational change involving four residues at the protein's carboxy terminus that close down on the distal side of the inhibitor's quinazoline ring, capping the active site and sequestering the bound ligands from bulk solvent.

Crystal structure of Escherichia coli thymidylate synthase with FdUMP and 10-propargyl-5,8-dideazafolate

The crystal structure of an E. coli TS ternary complex containing FdUMP and PDDF has been determined and refined at 2.3Å resolution. Each of the two chemically identical subunits folds into a three-layer domain anchored by a large six-stranded mixed beta sheet. The backside of one sheet is juxtaposed against the corresponding face of the equivalent sheet in the second protomer creating a beta sandwich. In contrast to other proteins of known structure in which aligned beta sheets stack face to face with a counterclockwise rotation, sheets in the TS dimer are related by a clockwise twist. The substrate binding pocket is a large funnel-shaped cleft extending some 25Å into the interior of each subunit and surrounded by 28 amino acids, 26 from one subunit and 2 from the other. FdUMP binds at the bottom of this pocket covalently linked through C6 to the sulfur of Cys-146. Up-pointing faces of the pyrimidine and ribose rings are exposed to provide a complementary docking surface for the quinazoline ring of PDDF. The quinazoline inhibitor binds in a partially folded conformation with its p-aminobenzoylglutamate tail exposed at the extrance to the active site cleft. Ternary complex formation is associated with a large conformational change involving 4 residues at the protein's carboxy-terminus that close down on the distal side of the inhibitor's quinazoline ring, capping the active site and sequestering the bound ligands from bulk solvent.