RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: Evidenced in vivo by an anti-farnesyl cysteine antibody

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

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Functional Analysis of a Peptide Sequence (Thr323 - Cys337) In ADAMTS13 Disintegrin-Like Domain Revealed Two Discontinuous Sequences Involved In the Interaction with VWF

Blood ◽

10.1182/blood.v116.21.2200.2200 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2200-2200

Author(s):

Atsuko Igari ◽

Takanori Moriki ◽

Terumichi Nakagawa ◽

Yusuke Yamaguchi ◽

Mitsuru Murata

Keyword(s):

Amino Acids ◽

Catalytic Activity ◽

Synthetic Peptides ◽

Inhibitory Effect ◽

Peptide Sequence ◽

Recent Analysis ◽

Inhibitory Effects ◽

Amino Terminal ◽

Terminal Amino ◽

Carboxyl Terminal

Abstract Abstract 2200 ADAMTS13 specifically cleaves multimeric von Willebrand factor (VWF) into smaller molecules to reduce its high reactivity with platelets. The disintegrin-like (D) domain, adjacent to the catalytic domain of ADAMTS13, plays an important role in the process of VWF cleavage. In this study, we aimed to elucidate critical peptide sequences in D-domain involved in the interaction with VWF. A series of partially overlapping peptide sequences, approximately 20 amino acids in length, covering the D-domain, were synthesized and the inhibitory effects on the catalytic activity of plasma ADAMTS13 was examined using FRETS-VWF73 assay. Consequently, some synthetic peptides were selected and the minimal length necessary for the inhibitory effect was determined as TFAREHLDMCQALSC (peptide323-337). Removal of the amino-terminal threonine diminished the inhibitory effect moderately, although deletion of the carboxyl-terminal cysteine abolished it completely. According to the amino acids alignment of ADAMTS family, this peptide sequence is not conserved, highlighting the specific role in the interaction with its substrate. From the recent analysis of crystal structure, amino-terminal half of the peptide323-337, TFAREHL (323-329), was disordered and designated as the variable (V) loop, which creates one of VWF-binding exosites (Akiyama, et al. Proc Natl Acad Sci USA. 2009; 106:19274-9). We hypothesized that the amino-terminal amino acids of the peptide323-337 contribute to VWF binding, whereas the carboxyl-terminal amino acids allow the structural stability of the peptide conformation. To evaluate the effect of carboxyl-terminal cysteine at 337, other synthetic peptides with alanine, serine, glycine or phenylalanine instead of the cysteine (C337A, C337S, C337G, or C337F) were tested about their inhibitory effects on the catalytic activity. Interestingly, C337A, C337S, C337G peptides exhibited slightly weaker inhibitory effects on VWF73 catalysis, although C337F peptide showed stronger inhibition than wild-type sequence, suggesting that the residue 337 regulates the characteristics of the peptide323-337. From the results of peptide screening, the amino- and carboxyl-terminal amino acids of the peptide323-337, TFAREHLDMCQALSC, likely play key roles in the inhibitory effects; therefore, the middle part of the sequence, HLDMC, was replaced by 5 alanines (AAAAA) or reversed sequence CMDLH. Surprisingly, the converted peptides still retained the equivalent level of inhibitory effects, indicating both sides of the amino- and carboxyl-terminal amino acids were especially significant in the interaction with VWF. In conclusion, we characterized the peptide sequence, TFAREHLDMCQALSC (323-337), in D-domain. The peptide clearly inhibited the cleavage of VWF73 and the both sides of amino- and carboxyl-terminal amino acids seemed especially important. The peptide sequence is supposed to bind to VWF for the precise cleavage in the process of proteolysis. By modifying this peptide sequence, such variant ADAMTS13 as gain-of-function recombinants might be developed, leading to an alternative anti-thrombotic drug. Disclosures: No relevant conflicts of interest to declare.

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Des-serine-proline brain natriuretic peptide 3–32 in cardiorenal regulation

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00569.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. R897-R901 ◽

Cited By ~ 73

Author(s):

Guido Boerrigter ◽

Lisa C. Costello-Boerrigter ◽

Gail J. Harty ◽

Harald Lapp ◽

John C. Burnett

Keyword(s):

Amino Acids ◽

Brain Natriuretic Peptide ◽

Natriuretic Peptide ◽

Urinary Sodium Excretion ◽

Urine Flow ◽

Accelerated Degradation ◽

Equimolar Dose ◽

Terminal Amino ◽

Physiologic Role

Brain natriuretic peptide (BNP 1–32) plays an important physiologic role in cardiorenal homeostasis. Recently, it has been reported that BNP 1–32 is rapidly cleaved by the ubiquitous enzyme dipeptidyl peptidase IV to BNP 3–32, which lacks the two NH2-terminal amino acids of BNP 1–32. The bioactivity of BNP 3–32 in cardiorenal regulation is unknown. We hypothesized that BNP 3–32 has reduced vasodilating and natriuretic bioactivity compared with BNP 1–32 in vivo. Synthetic human BNP 3–32 and BNP 1–32 were administered to eight anesthetized normal canines. After baseline measurements, BNP 1–32 at 30 ng·kg−1·min−1 was administered, followed by a washout, a postinfusion clearance, and a clearance with an equimolar dose of BNP 3–32. In four studies, the sequence of BNP 1–32 and BNP 3–32 infusion was reversed. Peptides were compared by analyzing the changes from the respective preinfusion clearance to the respective infusion clearance. * P < 0.05 between peptides. BNP 3–32, unlike BNP 1–32, did not decrease mean arterial pressure (0 ± 1 vs. −7 ± 2* mmHg, respectively) and did not increase renal blood flow (+12 ± 10 vs. +52 ± 10* ml/min). Effects on heart rate and cardiac output were similar. Urinary sodium excretion increased 128 ± 18 μeq/min with BNP 3–32 and 338 ± 40* μeq/min with BNP 1–32. Urine flow increased 1.1 ± 0.2 ml/min with BNP 3–32 and 2.8 ± 0.4* ml/min with BNP 1–32. Plasma BNP immunoreactivity was lower with BNP 3–32, suggesting accelerated degradation. In this study, BNP 3–32 showed reduced natriuresis and diuresis and a lack of vasodilating actions compared with BNP 1–32.

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The processing pathway of prelamin A

Journal of Cell Science ◽

10.1242/jcs.107.1.61 ◽

1994 ◽

Vol 107 (1) ◽

pp. 61-67 ◽

Cited By ~ 12

Author(s):

M. Sinensky ◽

K. Fantle ◽

M. Trujillo ◽

T. McLain ◽

A. Kupfer ◽

...

Keyword(s):

Amino Acids ◽

Cell Line ◽

Genetic Background ◽

Initial Step ◽

Carboxyl Terminus ◽

Lamin A ◽

Prelamin A ◽

Carboxyl Terminal ◽

Prenylated Proteins

The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known prenylated mammalian proteins. Since the carboxyl terminus of prelamin A is removed during maturation, it is not obvious that this protein would undergo the two reactions subsequent to prenylation observed in other CAAX box proteins--the endoproteolytic removal of the carboxyl-terminal 3 amino acids and the subsequent methylation of the now carboxyl-terminal cysteine. To characterize the maturation of prelamin A further, we have developed a CHO-K1 cell line that possesses a dexamethasone-inducible human prelamin A against a genetic background of high mevalonate uptake. Utilizing this cell line in association with antibodies specific to the transgenic prelamin A, we have been able to demonstrate directly in vivo that prelamin A undergoes farnesylation and carboxymethylation prior to conversion to lamin A, as is the case for other prenylated proteins. We have demonstrated previously that in the absence of isoprenylation, conversion of prelamin A to lamin A is blocked, but that unprocessed prelamin A is transported to the nucleus where it can still undergo maturation. Consistent with the implications of these prior studies, we now demonstrate the presence of both subunits of farnesyl-protein transferase in the nucleus.

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The C Terminus of Substrates Is Critical but Not Sufficient for Their Degradation by the Pseudomonas aeruginosa CtpA Protease

Journal of Bacteriology ◽

10.1128/jb.00174-20 ◽

2020 ◽

Vol 202 (16) ◽

Author(s):

Sammi Chung ◽

Andrew J. Darwin

Keyword(s):

Amino Acids ◽

Pseudomonas Aeruginosa ◽

Cell Wall ◽

Outer Membrane ◽

Content Type ◽

C Terminus ◽

Outer Membrane Lipoprotein ◽

Membrane Lipoprotein ◽

Carboxyl Terminal

ABSTRACT Bacterial carboxyl-terminal processing proteases (CTPs) are widely conserved and have been linked to important processes, including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc suggested that it cleaves proteins with nonpolar and/or structurally unconstrained C termini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a divergent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. In this study, we investigated if the C termini of two nonhomologous CtpA substrates are important for their degradation. We determined that these substrates have extended C termini compared to those of their closest E. coli homologs. Removing 7 amino acids from these extensions was sufficient to reduce their degradation by CtpA both in vivo and in vitro. Degradation of one truncated substrate was restored by adding the C terminus from the other but not by adding an unrelated sequence. However, modification of the C termini of nonsubstrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C termini of CtpA substrates are required but not sufficient for their efficient degradation. Although C-terminal truncated substrates were protected from degradation, they still associated with the LbcA-CtpA complex in vivo. Therefore, degradation of a protein by CtpA requires a C terminus-independent interaction with the LbcA-CtpA complex, followed by C terminus-dependent degradation, perhaps because CtpA normally initiates cleavage at a C-terminal site. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We report an important advance by revealing that efficient degradation by CtpA requires at least two separable phenomena and that one of them depends on information encoded in the substrate C terminus. A C terminus-independent association with the LbcA-CtpA complex is followed by C terminus-dependent cleavage by CtpA. Increased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.

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The six amino-terminal amino acids of p60src are sufficient to cause myristylation of p21v-ras

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3960-3963.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3960-3963

Author(s):

J E Buss ◽

C J Der ◽

P A Solski

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Myristic Acid ◽

Amino Terminus ◽

Directed Mutagenesis ◽

Ras Protein ◽

Amino Terminal ◽

Terminal Amino ◽

Crucial Part

We have used oligonucleotide-directed mutagenesis to replace the N-terminal amino acids of p21v-ras with residues which mimic the amino terminus of p60v-src. p21v-ras protein possessing only the first five amino acids of p60src was not myristylated, while substitution of residue 6 (serine) produced a protein p21(GSSKS) which incorporated [3H]myristic acid that was stable to hydroxylamine, sensitive to inhibitors of protein synthesis, and found in both the normally nonacylated precursor and mature forms of p21(GSSKS). This defines the minimum framework of the p60v-src myristylation signal (glycine 2 and serine 6) and identifies serine 6 as a crucial part of that signal for myristylation of a protein in vivo.

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Features of the pp60v-src carboxyl terminus that are required for transformation

Molecular and Cellular Biology ◽

10.1128/mcb.6.8.2807-2819.1986 ◽

1986 ◽

Vol 6 (8) ◽

pp. 2807-2819

Author(s):

P Yaciuk ◽

D Shalloway

Keyword(s):

Amino Acids ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Cellular Proteins ◽

Carboxyl Terminus ◽

Deletion Mutants ◽

Carboxyl Terminal ◽

Protein Instability ◽

Wide Family

Analysis of the biological and biochemical activities of pp60recombinant-src proteins encoded by 12 carboxyl-terminal mutants showed that a wide family of alternate src carboxyl termini permit complete transforming and kinase activities. src proteins having carboxyl termini which are up to 10 amino acids longer than that of pp60c-src (17 amino acids longer than that of pp60v-src) still permit transformation. Transformation-positive mutations preserve leucine-516, a residue which is highly conserved in protein-tyrosine kinase sequences; removal causes in vivo protein instability. Successive deletion mutants show that this residue is at the boundary of a region required for kinase activity. pp60src which is truncated just outside this point still transforms cells and binds both pp50 and pp90 cellular proteins.

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Features of the pp60v-src carboxyl terminus that are required for transformation.

Molecular and Cellular Biology ◽

10.1128/mcb.6.8.2807 ◽

1986 ◽

Vol 6 (8) ◽

pp. 2807-2819 ◽

Cited By ~ 21

Author(s):

P Yaciuk ◽

D Shalloway

Keyword(s):

Amino Acids ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Cellular Proteins ◽

Carboxyl Terminus ◽

Deletion Mutants ◽

Carboxyl Terminal ◽

Protein Instability ◽

Wide Family

Analysis of the biological and biochemical activities of pp60recombinant-src proteins encoded by 12 carboxyl-terminal mutants showed that a wide family of alternate src carboxyl termini permit complete transforming and kinase activities. src proteins having carboxyl termini which are up to 10 amino acids longer than that of pp60c-src (17 amino acids longer than that of pp60v-src) still permit transformation. Transformation-positive mutations preserve leucine-516, a residue which is highly conserved in protein-tyrosine kinase sequences; removal causes in vivo protein instability. Successive deletion mutants show that this residue is at the boundary of a region required for kinase activity. pp60src which is truncated just outside this point still transforms cells and binds both pp50 and pp90 cellular proteins.

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