Protein Carboxy 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.

Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.

Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.

Proteolytic Post-translational Modification of Proteins: Proteomic Tools and Methodology

Proteolytic processing is a ubiquitous and irreversible post-translational modification involving limited and highly specific hydrolysis of peptide and isopeptide bonds of a protein by a protease. Cleavage generates shorter protein chains displaying neo-N and -C termini, often with new or modified biological activities. Within the past decade, degradomics and terminomics have emerged as significant proteomics subfields dedicated to characterizing proteolysis products as well as natural protein N and C termini. Here we provide an overview of contemporary proteomics-based methods, including specific quantitation, data analysis, and curation considerations, and highlight exciting new and emerging applications within these fields enabling in vivo analysis of proteolytic events.

Protective efficacy of aMycoplasma pneumoniaeP1C DNA vaccine fused with the B subunit ofEscherichia coliheat-labile enterotoxin

In the present study, we investigated the immunomodulatory responses of a DNA vaccine constructed by fusing Mycoplasma pneumoniae P1 protein carboxy terminal region (P1C) with the Escherichia coli heat-labile toxin B subunit (LTB). BALB/c mice were immunized by intranasal inoculation with control DNAs, the P1C DNA vaccine or the LTB-P1C fusion DNA vaccine. Levels of the anti-M. pneumoniae antibodies and levels of interferon-γ and IL-4 in mice were increased significantly upon inoculation of the LTB-P1C fusion DNA vaccine when compared with the inoculation with P1C DNA vaccine. The LTB-P1C fusion DNA vaccine efficiently enhanced the M. pneumoniae-specific IgA and IgG levels. The IgG2a/IgG1 ratio was significantly higher in bronchoalveolar lavages fluid and sera from mice fusion with LTB and P1C than mice receiving P1C alone. When the mice were challenged intranasally with 10(7) CFU M. pneumoniae strain (M129), the LTB-P1C fusion DNA vaccine conferred significantly better protection than P1C DNA vaccine (P < 0.05), as suggested by the results, such as less inflammation, lower histopathological score values, lower detectable number of M. pneumoniae strain, and lower mortality of challenging from 5 × 10(8) CFU M. pneumoniae. These results indicated that the LTB-P1C fusion DNA vaccine efficiently improved protective efficacy against M. pneumoniae infection and effectively attenuated development of M. pneumoniae in mice.

Proteome-wide analysis of protein carboxy termini: C terminomics

As proteome-wide C-terminal sequence analysis has been largely intractable, we developed a polymer-based enrichment approach to profile protein C-terminal peptides by mass spectrometry and identified hundreds of C-terminal peptides in the Escherichia coli proteome. We isotopically labeled GluC protease-digested and undigested samples and identified GluC substrates and their cleavage sites by quantification of neo-C-terminal peptides. Our method thus enables global annotation of protein C-terminal posttranslational modifications, including proteolytic truncations.

BRCA1 E1644X: a deleterious mutation in an African American individual with early onset breast cancer

An African American individual with early onset breast cancer has a unique BRCA1 germline mutation, E1644X, that truncates the protein's carboxy terminal region. DNA sequencing for E1644X mutation and five BRCA1 exon-11 single nucleotide polymorphisms showed tumor LOH. Clinical history suggests paternal transmission of the deleterious allele, and tumor polymorphisms provide some insight into the ancestral origins of the mutation.

Importance of the Penultimate Positive Charge in Mouse Hepatitis Coronavirus A59 Membrane Protein

The coronavirus membrane (M) protein carboxy tail interacts with the nucleocapsid during virus assembly. Previous studies demonstrated that the two terminal residues are important, and the charged residue (R227) in the penultimate position in the mouse hepatitis coronavirus (MHV) A59 M protein was suggested to participate in intermolecular interactions with negative charges in the nucleocapsid (N) protein. To determine the significance of the positive charge at position 227, we substituted the arginine with lysine (K), aspartic acid (D), glutamic acid (E), or alanine (A) and studied these by reverse genetics in the context of a MHV full-length infectious clone. Viruses with wild-type phenotype were readily recovered with the K or A substitutions. In contrast, negative-charge substitutions were not tolerated as well. In all recovered R227D viruses the negative charge was replaced with heterologous residues resulting from apparent template switching during negative-strand synthesis of subgenomic RNA 7. An additional second-site compensatory V202I substitution was present in some viruses. Recovered R227E viruses had second-site changes within the M protein carboxy tail that were partially compensatory. Significantly, most of the second site changes in the R227E mutant viruses were previously shown to compensate for the removal of negative charges in the N protein. Our results strongly indicate that a positive charge is not absolutely required. It is clear that other regions within the tail must also be involved in helping mediate interactions between the M protein and the nucleocapsid.

Identification of Functionally Important Negatively Charged Residues in the Carboxy End of Mouse Hepatitis Coronavirus A59 Nucleocapsid Protein

The coronavirus nucleocapsid (N) protein is a multifunctional viral gene product that encapsidates the RNA genome and also plays some as yet not fully defined role in viral RNA replication and/or transcription. A number of conserved negatively charged amino acids are located within domain III in the carboxy end of all coronavirus N proteins. Previous studies suggested that the negatively charged residues are involved in virus assembly by mediating interaction between the membrane (M) protein carboxy tail and nucleocapsids. To determine the importance of these negatively charged residues, a series of alanine and other charged-residue substitutions were introduced in place of those in the N gene within a mouse hepatitis coronavirus A59 infectious clone. Aspartic acid residues 440 and 441 were identified as functionally important. Viruses could not be isolated when both residues were replaced by positively charged amino acids. When either amino acid was replaced by a positively charged residue or both were changed to alanine, viruses were recovered that contained second-site changes within N, but not in the M or envelope protein. The compensatory role of the new changes was confirmed by the construction of new viruses. A few viruses were recovered that retained the D441-to-arginine change and no compensatory changes. These viruses exhibited a small-plaque phenotype and produced significantly less virus. Overall, results from our analysis of a large panel of plaque-purified recovered viruses indicate that the negatively charged residues at positions 440 and 441 are key residues that appear to be involved in virus assembly.

Apolipoprotein E isoforms and apolipoprotein AI protect from amyloid precursor protein carboxy terminal fragment‐associated cytotoxicity

Inheritance of the apolipoprotein (APO) E gene epsilon4 or epsilon2 allele alters the risk of developing Alzheimer disease (AD), while increased alpha-tocopherol (AT) intake appears to lower the risk of AD. As APOE is a major apolipoprotein in the CNS and AT in vivo is transported in lipoproteins, we tested the hypothesis that CNS lipoproteins, as modeled by relevant concentrations of high density lipoprotein (HDL), and AT would interact to suppress neurotoxicity in a cell culture model of amyloid beta (Abeta)- related toxicity. These cells conditionally express C99-derived peptides, proposed to be a key step in AD pathogenesis; this expression is closely associated with subsequent cell death. We found that physiologic concentrations of lipoproteins present in the CNS protected from C99-associated toxicity and provided evidence for two mechanisms of protection. The first was AT-independent, APOE isoform-dependent, and most potent for the APOE2 isoform. The second was a synergistic protection afforded by a combination of APOAI, or less so APOE, and AT. These data provide a novel explanation for the apparent AD-protective effect of inheriting an epsilon2 APOE allele, and suggest that optimizing AT enrichment of CNS lipoproteins or devising APOAI mimetics may augment AT efficacy in treating AD.

Progressive decrease of amyloid precursor protein carboxy terminal fragments (APP-CTFs), associated with tau pathology stages, in Alzheimer's disease.

Amyloid precursor protein (APP) dysfunction is a key aetiologic agent in Alzheimer's disease (AD). The processing of this transmembrane protein generates carboxy terminal fragments (CTFs) upstream of beta-amyloid peptide (Abeta) production. The physiologic significance of APP-CTFs is still poorly understood, as well as the relationship that could link APP dysfunction and tau pathology in familial and non-familial AD (non-FAD). In the present study, we have investigated the quantitative and qualitative changes of APP-CTFs in different brain areas of non-demented and demented patients from a prospective and multidisciplinary study. A significant decrease of the five APP-CTFs was observed, which correlated well with the progression of tau pathology, in most cases with infraclinical AD and AD, either familial or non-FAD. Furthermore, solubility properties and the ratio between the five bands were also modified, both in the Triton-soluble and/or -insoluble fractions. Together, we show here for the first time a modification directly observed on APP-CTFs upstream of Abeta products and its relationship with tau pathology, which could reflect the basic aetiological mechanisms of AD.

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.

Plasmodium falciparum: binding studies of peptide derived from the sporozoite surface protein 2 to Hep G2 cells.

Plasmodium falciparum sporozoite surface protein 2 (Pf SSP2), also called thrombospondin related anonymous protein (TRAP), is involved in the process of sporozoite invasion of hepatocytes. Pf SSP2/TRAP possesses two different adhesion domains sharing sequences and structural homology with von Willebrand factor A-domains and human repeat I thrombospondin (TSP). Pf SSP2/TRAP has also been implicated in sporozoite mobility and in mosquito salivary gland invasion processes. We tested 15-mer long synthetic peptides having five overlapping residues covering the complete protein Pf SSP2 sequence in binding assays to Hep G2 cells. In these 57 peptides, 21 high-activity binding peptides (HABPs) were identified; five were in the adhesion domains already described and 16 were in two regions toward the protein's carboxy and middle terminal part. Six HABPs showed conserved amino acid sequences: 3243 (21FLVNGRDVQNNIVDE35), 3279 (201FLVGCHPSDGKCNLY215), 3287 (241TASCGVWDEWSPCSV255), 3289 (251SPCSVTCGKGTRSRK265), 3327 (441ERKQSDPQSQDNNGNY455) and 3329 (451DNNGNRHVPNSEDREY465). The HABPs show saturable binding and dissociation constants between 140 and 900 nm with 40 000-855 000 binding sites per cell. The 3279 (201FLVGCHPSDGKCNLY215), 3323 (421NDKSDRYIPYSPLSP435) and 3331 (461SEDRETRPHGRNNENY475) HABPs have B epitopes in their sequences; these have previously been recognized by antibodies partially inhibiting hepatocyte invasion and development of the hepatic state. The 3287 (241TASCGVWDEWSPCSV255) and 3289 (251SPCSVTCGKGTRSRK265) HABPs share common sequences with the Pf SSP2/TRAP region II plus, which is present in a great number of adhesion proteins. Based on this information, six new peptides covering the high binding regions identified previously were synthesized and, using a competition assay, the amino acid involved in the binding were determined.

The membrane M protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability.

The architecture of transmissible gastroenteritis coronavirus includes three different structural levels, the envelope, an internal core, and the nucleocapsid that is released when the core is disrupted. Starting from purified virions, core structures have been reproducibly isolated as independent entities. The cores were stabilized at basic pH and by the presence of divalent cations, with Mg(2+) ions more effectively contributing to core stability. Core structures showed high resistance to different concentrations of detergents, reducing agents, and urea and low concentrations of monovalent ions (<200 mM). Cores were composed of the nucleoprotein, RNA, and the C domain of the membrane (M) protein. At high salt concentrations (200 to 300 mM), the M protein was no longer associated with the nucleocapsid, which resulted in destruction of the core structure. A specific ionic interaction between the M protein carboxy terminus and the nucleocapsid was demonstrated using three complementary approaches: (i) a binding assay performed between a collection of M protein amino acid substitution or deletion mutants and purified nucleocapsids that led to the identification of a 16-amino-acid (aa) domain (aa 237 to 252) as being responsible for binding the M protein to the nucleocapsid; (ii) the specific inhibition of this binding by monoclonal antibodies (MAbs) binding to a carboxy-terminal M protein domain close to the indicated peptide but not by MAbs specific for the M protein amino terminus; and (iii) a 26-residue peptide, including the predicted sequence (aa 237 to 252), which specifically inhibited the binding. Direct binding of the M protein to the nucleoprotein was predicted, since degradation of the exposed RNA by RNase treatment did not affect the binding. It is proposed that the M protein is embedded within the virus membrane and that the C region, exposed to the interior face of the virion in a population of these molecules, interacts with the nucleocapsid to which it is anchored, forming the core. Only the C region of the M protein is part of the core.

The Exon-Intron Organization of the Genes (GAD1 and GAD2) Encoding Two Human Glutamate Decarboxylases (GAD 67 and GAD 65) Suggests That They Derive from a Common Ancestral GAD

We have cloned and characterized human genes (GAD1 and GAD2) encoding the two human glutamate decarboxylases, GAD 67 and GAD 65. The coding region of the GAD 65 gene consists of 16 exons, spanning more than 79 kb of genomic DNA. Exon 1 contains the 5′ untranslated region of GAD 65 mRNA, and exon 16 specifies the protein's carboxy terminal and at least part of the mRNA's 3′ untranslated sequence. Similarly, the coding region of the GAD 67 gene consists of 16 exons, spread over more than 45 kb of genomic DNA. The GAD 67 gene contains an additional exon (exon 0) that, together with part of exon 1, specifies the 5′ untranslated region of GAD 67 mRNA. Exon 16 specifies the entire 3′ untranslated region of GAD 67 mRNA. Exons 1-3 encode the most divergent region of GAD 65 and GAD 67. The remaining exon-intron boundaries occur at identical positions in the two cDNAs, suggesting that they derive from a common ancestral GAD gene.

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).

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.

Sites of allosteric shift in the structure of the cyclic AMP receptor protein

We have characterized crp mutations in E. coli that allow CRP to function without cAMP. crp* mutants carrying a deletion of the gene encoding adenylate cyclase (cya) show significant lac expression. Cyclic GMP, normally an ineffective activator of CRP+, can stimulate these mutant CRP*s to permit greater lac expression in vivo. Cyclic AMP binding to the amino-terminal domain of CRP+ induces an allosteric transition that changes the DNA-binding property of the carboxy domain. The CRP* phenotype is caused by substitution of amino acids with bulkier side chains in the D alpha-helix of the protein's carboxy domain, near the hinge connecting the two domains. These results are consistent with a model in which the mutant CRP*s assume, in part, a conformation normally evoked only by cAMP binding: one in which the relative orientation of the C, D, and F alpha-helices is altered. We define precisely the amino acids of these alpha-helices that interact to cause the allosteric shift.

Fibronectin-induced protein carboxy- O-methylation in aortic endothelial cells

In a previous report (Bowersox, J.C. and Sorgente, N. (1982) Cancer Res. 42, 2547–2551) we demonstrated that the glycoprotein fibronectin is a chemoattractant for vascular endothelial cells. In probing the mechanisms by which fibronectin induces endothelial cell chemotaxis, we have discovered that the carboxy- O-methylation of cellular proteins is stimulated by fibronectin. By measuring the incorporation of l-[ methyl- 3H]methionine into alkali-labile, [ 3H]methyl ester linkages, we determined that fibronectin stimulated protein carboxy- O-methylation in aortic endothelial cells in a time- and concentration-dependent manner; the greatest stimulation occurred at 100 μg/ml fibronectin (approx. 35% above controls). When inhibitors of carboxymethylation were added, fibronectin-induced stimulation of protein methylation did not occur. Furthermore, inhibitors of methylation prevented the chemotaxis of endothelial cells in response to fibronectin. These data support our hypothesis that fibronectin mediates endothelial cell chemotaxis, such as that occurring during neovascularization. As carboxy- O-methylation of cell proteins is also effected by fibronectin, transmethylation reactions may be an important component of endothelial cell chemotaxis.

Phospholipid methylation in macrophages is inhibited by chemotactic factors

Chemotaxis by human monocytes has been shown to require methylation mediated by S-adenosyl-L-methionine(AdoMet), but the specific transmethylation reaction necessary for this function was not elucidated. In an attempt to define the methylation requirement for chemotaxis, we examined the effect of chemotactic agonists and antagonists on protein carboxy-O-methylation of protein and methylation of phospholipid in guinea pig macrophages. Chemotactic agents tested over a wide dose and time range produced no alteration in carboxy-O-methylation. However, these agents did produce an effect on the methylation of phosphatidylethanolamine by macrophages. AdoMet-mediated phospholipid methylation was inhibited by as much as 73% by chemotactic factors, and there was excellent correlation (r = 0.99) between their concentrations for producing half-maximal chemotactic responses and for inhibiting phospholipid methylation. The inhibition of methylation by chemotactic factors was observed at all incubation times and could not be explained by an increased turnover of membrane phospholipid. Neither the chemotaxis antagonist fPhe-Met nor the nonchemotactic tripeptide Met-Met-Met significantly depressed phospholipid methylation. Immune phagocytosis by macrophages similarly did not alter phospholipid methylation. The chemotactic factors produced no alteration in total macrophage phospholipid synthesis or in the phospholipid methylation in a nonchemotactic cell type. The formation of newly methylated derivatives of phosphatidylethanolamine in macrophages was decreased by a biologically active dose of chemotactic factor. These findings indicate that chemotactic factors are capable of altering the methylation of phosphatidylethanolamine in chemotactically responsive cells. The inhibition of phospholipid methylation by chemotactic factors may be necessary for the translation of a chemotactic signal on the surface of the cell into directional cell movement.