Biomedical subjects

D Pei

Publications and source records attributed to D Pei.

At least 19 recordsLinked to original sources

PubMed2003

The effect of surgical stress on insulin sensitivity, glucose effectiveness and acute insulin response to glucose load.

Hyperglycemia after stress is a very common clinical phenomenon. It is generally hypothesized that the underlying cause is a neuroendocrine-mediated deterioration in glucose metabolism. However, the detailed roles of insulin sensitivity, glucose effectiveness and acute insulin response to glucose load in response to stress have not been well established. Hernioplasty was used as a minor stress model for studying stress-induced hyperglycemia. Eleven healthy young men were enrolled voluntarily in this study. Their mean age was 22.0 +/- 0.9 yr and BMI 23.3 +/- 0.6 kg/m2. Frequently sampled i.v. glucose tolerance tests were performed one day before and one day after the surgery. Insulin sensitivity (SI), glucose effectiveness (EG) and area under acute insulin response (AIR) were calculated from "minimal model" algorithms. We also measured fasting concentrations of human GH, ACTH and F on the days of the test. Compared to the pre-operation data, levels of ACTH and F did not change significantly after the surgery. Only GH levels were marginally significant. On the other hand, the SI (0.75 +/- 0.1, 0.52 +/- 0.9 x 10(-5) min(-1)/pmol, p = 0.04), EG (0.023 +/- 0.03, 0.016 +/- 0.003 min(-1), p = 0.01) and AIR (6738.5 +/- 1111.6, 5130.0 +/- 1047.2 pmol, p = 0.005) were all significantly decreased after surgery. The percentages of decrease were 16.3 +/- 15.5, 32.1 +/- 10.3 and 17.8 +/- 10.3%, respectively. Finally, only the changes of EG positively correlate with the changes of ACTH before and after surgery. No significant changes were noted among other stress hormones and the changes of SI, EG and AIR. In conclusion, hernioplasty results in reduced SI, EG and AIR. Among them, although not statistically significant, the EG showed the most distinct decrease after the surgery, which has not been found in previous literature.

Adolescent↗

PubMed2003

Sagittal abdominal diameter is associated with insulin sensitivity in Chinese hypertensive patients and their siblings.

The purpose of the study is to observe the relation between anthropometric measurements, focusing on sagittal abdominal diameter (SAD), and insulin sensitivity indices in Chinese hypertensive patients and their siblings. In total, 907 participants, 537 hypertensive and 370 nonhypertensive, from 311 Taiwanese families were drawn from the Stanford Asia and Pacific Program for Hypertension and Insulin Resistance for the study. The participants received anthropometric measurements and 75-g oral glucose tolerance tests after an overnight fast. Fasting insulin, homeostasis model assessment for insulin resistance (HOMA-IR), and the insulin sensitivity index ISI(0,120) were chosen as surrogate measures of insulin sensitivity. In addition to Pearson and partial correlations, we used generalized estimating equations (GEEs) to examine the association between anthropometric measurements and insulin sensitivity indices. A small deviance in the GEEs indicates the goodness of model fit, irrespective of the independence among variables. The hypertensive patients were older in age, wider in waist circumference (WC), larger in body mass index (BMI) and SAD, and more insulin resistant than the nonhypertensive counterparts. The logarithmic transformation of fasting insulin, HOMA-IR, and ISI(0,120) significantly correlated with SAD, WC, and BMI before and after adjustments for age and sex. The deviances of SAD in the GEEs were similar to those of WC in all subjects, while BMI had smaller deviances than SAD and WC in the hypertensive patients. Our results suggest that the performance of SAD in predicting insulin sensitivity is comparable with WC in Chinese hypertensive patients and their siblings. BMI, however, seems to have better association with insulin sensitivity than SAD and WC in the patients with hypertension.

Abdomen↗

PubMed2001

Characterization of an eukaryotic peptide deformylase from Plasmodium falciparum.

Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Until recently, PDF has been thought as an enzyme unique to the bacterial kingdom. Searches of the genomic DNA databases identified several genes that encode proteins of high sequence homology to bacterial PDF from eukaryotic organisms. The cDNA encoding Plasmodium falciparum PDF (PfPDF) has been cloned and overexpressed in Escherichia coli. The recombinant protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is inhibited by specific PDF inhibitors. Western blot analysis indicated expression of mature PfPDF in trophozoite, schizont, and segmenter stages of intraerythrocytic development. These results provide strong evidence that a functional PDF is present in P. falciparum. In addition, PDF inhibitors inhibited the growth of P. falciparum in the intraerythrocytic culture.

Amidohydrolases↗

PubMed2001

Regulation of membrane-type matrix metalloproteinase 1 activity by dynamin-mediated endocytosis.

Membrane-type matrix metalloproteinase 1 (MT1-MMP) plays a critical role in extracellular matrix remodeling under both physiological and pathological conditions. However, the mechanisms controlling its activity on the cell surface remain poorly understood. In this study, we demonstrate that MT1-MMP is regulated by endocytosis. First, we determined that Con A induces proMMP-2 activation in HT1080 cells by shifting endogenous MT1-MMP from intracellular compartments to cell surface. This phenotype was mimicked by the cytoplasmic truncation mutant MT1 Delta C with more robust pro-MMP-2 activation and cell surface expression than wild-type MT1-MMP in transfected cells. MT1 Delta C was subsequently shown to be resistant to Con A treatment whereas MT1-MMP remains competent, suggesting that Con A regulates MT1-MMP activity through cytoplasmic domain-dependent trafficking. Indeed, MT1-MMP was colocalized with clathrin on the plasma membrane and with endosomal antigen 1 in endosomes. Internalization experiments revealed that MT1-MMP is internalized rapidly in clathrin-coated vesicles whereas MT1 Delta C remains on cell surface. Coexpression of a dominant negative mutant of dynamin, K44A, resulted in elevation of MT1-MMP activity by interfering with the endocytic process. Thus, MT1-MMP is regulated by dynamin-dependent endocytosis in clathrin-coated pits through its cytoplasmic domain.

Animals↗

PubMed2001

Genetic variation in aldosterone synthase predicts plasma glucose levels.

The mineralocorticoid hormone, aldosterone, is known to play a role in sodium homeostasis. We serendipitously found, however, highly significant association between single-nucleotide polymorphisms in the aldosterone synthase gene and plasma glucose levels in a large population of Chinese and Japanese origin. Two polymorphisms--one in the putative promoter (T-344C) and another resulting in a lysine/arginine substitution at amino acid 173, which are in complete linkage disequilibrium in this population--were associated with fasting plasma glucose levels (P = 0.000017) and those 60 (P = 0.017) and 120 (P = 0.0019) min after an oral glucose challenge. A C/T variant in intron 1, between these polymorphisms, was not associated with glucose levels. Arg-173 and -344C homozygotes were most likely to be diabetic [odds ratio 2.51; 95% confidence interval (C.I.) 1.39-3.92; P = 0.0015] and have impaired fasting glucose levels (odds ratio 3.53; 95% C.I. 2.02-5.5; P = 0.0000036). These results suggest a new role for aldosterone in glucose homeostasis.

Adult↗

PubMed2001

Genetic variation in the human urea transporter-2 is associated with variation in blood pressure.

The kidney, by regulating the volume of fluid in the body, plays a key role in regulating blood pressure (BP). The kidney uses primarily sodium and, to a lesser extent, urea to maintain the appropriate volume of fluid. Genetic variation in proteins that determine sodium reabsorption and excretion is known to significantly influence BP. However, the influence of genetic variation in urea transporters on BP has not been examined. We determined therefore whether nucleotide variation in the kidney-specific human urea transporter, HUT2, is associated with variation in BP. After determining the genomic structure of the coding sequence, seven single nucleotide polymorphisms (SNPs) were identified. Two of the SNPs result in Val/Ile and Ala/Thr amino acid substitutions at positions 227 and 357 in the HUT2 open reading frame, respectively. Another SNP is silent and four others are in introns or the 3' untranslated region. Over 1000 hypertensive and low-normotensive individuals of Chinese origin were typed for five of these SNPs using a high-throughput genotyping method. The Ile227 and Ala357 alleles were associated with low diastolic BP in men but not women, with odds ratios 2.1 [95% confidence interval (CI) 1.5-2.7, P < 0.001] and 1.5 (95% CI 1.2-1.8, P < 0.001), respectively. There was a similar trend for systolic BP, and odds ratios for the Ile227 and Ala357 alleles were 1.7 (95% CI 1.2-2.3, P = 0.002) and 1.3 (95% CI 1.1-1.6, P = 0.007), respectively, in men.

Blood Pressure↗

PubMed2001

Shedding of membrane type matrix metalloproteinase 5 by a furin-type convertase: a potential mechanism for down-regulation.

The shedding of membrane-associated proteins has been recognized as a regulatory mechanism to either up-regulate or down-regulate cellular functions by releasing membrane-bound growth factors or removing ectodomains of adhesion molecules and receptors. We have reported previously that the ectoenzyme of membrane type matrix metalloproteinase 5 (MT5-MMP) is shed into extracellular milieu (Pei, D. (1999) J. Biol. Chem. 274, 8925-8932). Here we present evidence that MT5-MMP is shed by a furin-type convertase activity in the trans-Golgi network. Among proteinase inhibitors screened, only decanoyl-Arg-Val-Lys-Arg-chloromethylketone, a known inhibitor for furin-type convertases, blocked the shedding of MT5-MMP in a dose-dependent manner. As expected, decanoyl-Arg-Val-Lys-Arg-chloromethylketone also prevented the activation of MT5-MMP, raising the possibility that the observed shedding could be autolytic. However, an active site mutant devoid of any catalytic activity, is also shed efficiently, thus ruling out the autolytic pathway. The shedding cleavage was subsequently mapped to the stem region immediately upstream of the transmembrane domain, where a cryptic furin recognition site, (545)RRKERR, was recognized. Indeed, MT5-MMP and furin are co-localized in the trans-Golgi network and the shed species could be detected inside the cells. Furthermore, deletion mutations removing this cryptic site prevented MT5-MMP from shedding. The resulting mutants express a gain-of-function phenotype by mediating more robust activation of proMMP-2 than the wild type molecule. Thus, shedding provides a potential mechanism to regulate proteolytic activity of membrane-bound MMPs.

Amino Acid Sequence↗

PubMed2001

Subcellular distribution and cytokine- and chemokine-regulated secretion of leukolysin/MT6-MMP/MMP-25 in neutrophils.

Leukolysin, originally isolated from human leukocytes, is the sixth member of the membrane-type matrix metalloproteinase (MT-MMP) subfamily with a potential glycosylphosphatidylinositol (GPI) anchor. To understand its biological functions, we screened subpopulations of leukocytes and localized the expression of leukolysin at the mRNA level to neutrophils. Polyclonal and mono-specific antisera raised against a synthetic peptide from its hinge region recognized a major protein species at 56 kDa and several minor forms between 38 and 45 kDa in neutrophil lysates. In resting neutrophils, leukolysin is distributed among specific granules ( approximately 10%), gelatinase granules ( approximately 40%), secretory vesicles ( approximately 30%), and the plasma membrane ( approximately 20%), a pattern distinct from that of neutrophil MMP-8 and MMP-9. Consistent with its membrane localization and its reported GPI anchor, leukolysin partitions into the detergent phase of Triton X-114 and can be released from intact resting neutrophils by glycosylphosphatidylinositol-specific phospholipase C. Phorbol myristate acetate stimulates neutrophils to discharge 100% of leukolysin from specific and gelatinase granules and approximately 50% from the secretory vesicles and plasma membrane, suggesting that leukolysin can be mobilized by physiological signals to the extracellular milieu as a soluble enzyme. Indeed, interleukin 8, a neutrophil chemoattractant, triggered a release of approximately 85% of cellular leukolysins by a process resistant to a mixture of proteinase inhibitors, including aprotinin, BB-94, pepstatin, and E64. Finally, purified recombinant leukolysin can degrade components of the extracellular matrix. These results not only establish leukolysin as the first neutrophil-specific MT-MMP but also implicate it as a cytokine/chemokine-regulated effector during innate immune responses or tissue injury.

Amino Acid Sequence↗

PubMed2001

Melanoma chondroitin sulfate proteoglycan regulates matrix metalloproteinase-dependent human melanoma invasion into type I collagen.

Tumor cell adhesion and proteolysis of the extracellular matrix proteins surrounding the cells are tightly linked processes in tumor invasion. In this study, we sought to identify components of the cell surface of a vertical growth phase melanoma cell line, WM1341D, that mediate invasive cellular behavior. We determined by antisense inhibition that melanoma chondroitin sulfate proteoglycan (MCSP) and membrane-type 3 matrix metalloproteinase (MT3-MMP) expressed on WM1341D are required for invasion of type I collagen and degradation of type I gelatin. MT3-MMP co-immunoprecipitated with MCSP in WM1341D melanoma cells cultured on type I collagen or laminin. The association between MT3-MMP and MCSP was largely disrupted by removing chondroitin sulfate glycosaminoglycan (CS) from the cell surface, suggesting CS could mediate the association between the two cell surface core proteins. Recombinant MT3-MMP and MT3-MMP from whole cell lysates of WM1341D cells were specifically eluted from CS- conjugated affinity columns. The results indicate that MT3-MMP possesses the potential to promote melanoma invasion and proteolysis and that the formation of a complex between MT3-MMP and MCSP may be a crucial step in activating these processes.

Animals↗

PubMed2001

Overexpression of matrix metalloproteinases leads to lethality in transgenic Xenopus laevis: implications for tissue-dependent functions of matrix metalloproteinases during late embryonic development.

The extracellular matrix (ECM) functions as the structural support of cells and as a medium for cell-cell interactions. It is understood to play critical roles in development. ECM remodeling is mediated largely through the action of matrix metalloproteinases (MMPs), a family of Zn2+-dependent proteases capable of degrading various proteinaceous components of the ECM. MMPs are expressed in many developmental and pathologic processes. However, few studies have been carried out to investigate the function of MMPs during embryogenesis and postembryonic organogenesis. By using Xenopus development as a model system, we have previously shown that several MMP genes are expressed from neurulation to the completion of embryogenesis in distinct tissues/organs, suggesting that ECM remodeling during mid- to late embryogenesis occurs in an organ-specific manner. By using the recently developed transgenic technology for Xenopus laevis, we overexpressed Xenopus MMPs stromelysin-3 (ST3) and collagenase-4 (Col4) under the control of a ubiquitous promoter and observed that embryos with overexpressed ST3 or Col4, but not the control green fluorescent protein (GFP), died in a dose-dependent manner during late embryogenesis. The specificity of this embryonic lethal phenotype was confirmed by the failure of a catalytically inactive mutant of ST3 to affect development. Finally, overexpression of a mammalian membrane type-MMP also led to late embryonic lethality in Xenopus embryos, suggesting that membrane type-MMPs have functions in vivo for ECM remodeling, in addition to being activators of other pro-MMPs. These data together with the developmental expression of several MMPs during Xenopus development, suggest that MMPs play important roles during mid- to late embryogenesis and that proper regulation of MMP genes is critical for tissue morphogenesis and organogenesis.

Animals↗

PubMed2001

Screening combinatorial libraries for optimal enzyme substrates by mass spectrometry.

A method has been developed for the rapid identification of optimal enzyme substrates from combinatorial libraries. This methodology was validated by screening a 361-member N-terminally formylated tripeptide library, f-XXR (X = 19 different amino acids), for optimal substrates of Escherichia coli peptide deformylase (PDF). The library was synthesized on a solid phase via the split-pool synthesis method. The N-terminal formyl group was added by treating the resin with a 1:1 (mol/mol) mixture of HCO(2)H and DCO(2)D in the presence of dicyclohexylcarbodiimide. In a mass spectrum, each member of the library produced a doublet peak (separated by 1.0063 Da). Limited treatment of this library with E. coli PDF resulted in the deformylation of those peptides that are the most efficient substrates of the enzyme. The deformylated products, due to loss of the mass-degenerate formyl group, each generated a singlet peak in the mass spectrum. Thus, the PDF product peaks were readily identified and sequenced via tandem mass spectrometry. The results showed that PDF strongly prefers a norleucine and, to a lesser extent, a phenylalanine as the N-terminal residue, whereas it has little selectivity at the penultimate position. This result is in excellent agreement with the literature data and therefore demonstrates the methodology as an effective approach to the identification of optimal enzyme substrates. This method should be generally applicable to other enzymes as well as synthetic catalysts.

Amidohydrolases↗

PubMed2001

Sibling-based association study of the PPARgamma2 Pro12Ala polymorphism and metabolic variables in Chinese and Japanese hypertension families: a SAPPHIRe study. Stanford Asian-Pacific Program in Hypertension and Insulin Resistance.

The peroxisome proliferator activated receptor (PPAR) gamma2 is a transcription factor that has been shown to be involved in adipocyte differentiation, adipogenesis, and insulin sensitivity. To address the role of PPARgamma2 in glucose homeostasis and insulin sensitivity, among many other objectives, we conducted a sibling-controlled association study in a multicenter program - the Stanford Asian-Pacific Program in Hypertension and Insulin Resistance (SAPPHIRe). Approximately 2525 subjects in 734 Chinese and Japanese families have been recruited from six field centers for SAPPHIRe. In total, 1702 subjects including parents and siblings from 449 families have been genotyped for PPARgamma2, of which 328 families were Chinese and 121 Japanese. Only 88 subjects of the 1525 siblings screened for the P12A polymorphism were found to be carriers of the A variant, the most common variant of the PPARgamma2 gene. A variant frequencies of the siblings were 4.27% in Chinese and 2.72% in Japanese. A sibling-controlled association study was performed through genetically discordant sibships (i.e., P/P genotype vs. P/A + A/A genotypes). Specifically, we examined whether there were differences in metabolic variables between the discordant siblings within families. In total, 88 subjects carrying either 1 or 2 A alleles had at least one sibling who was discordant for the P12A polymorphism, yielding a total of 180 individuals from 47 families for analyses, among which 92 siblings were homozygous for wild-type P allele. Siblings with the A variant tended to have lower levels of fasting plasma glucose (OG-10), and lower glucose levels at 60 min following oral glucose loading after adjusting for age, gender, and body mass index. Using a mixed model treating family as a random effect, we found that P12A polymorphism of the PPARgamma2 gene contributes significantly to the variance in fasting plasma glucose, glucose level at 60 min, and insulin-resistance homeostasis model assessment. Our results suggest that within families siblings with the A variant in the PPARgamma2 gene may be more likely to have better glucose tolerance and insulin sensitivity independent of obesity in Chinese and Japanese populations.

Adult↗

PubMed2001

Simvastatin treatment on postprandial hypertriglyceridemia in type 2 diabetes mellitus patients with combined hyperlipidemia.

Recent studies have shown that statins are effective in reducing fasting low-density lipoprotein-cholesterol (LDL-C) and triglyceride levels. However, it remains unknown if treatment with statins also lowers daily postprandial triglyceride concentrations, which may promote atherogenesis in type 2 diabetes subjects. Forty-one subjects with type 2 diabetes and combined hyperlipidemia who had stable glycemic control were randomly assigned to take simvastatin 20 mg (n = 27) or a placebo (n = 14) once daily for 12 weeks. The medication dosage was doubled after 4 weeks if a subject's LDL-C was not less than 130 mg/dL. Among these participants, 24 subjects (15 on simvastatin and 9 on placebo) agreed to take a meal tolerance test with isocaloric mixed meals (carbohydrate, 52%; fat, 33%, and protein, 15% of the daily caloric intake) and daytime hourly blood sampling from 8 AM to 4 PM. Simvastatin treatment reduced the fasting total cholesterol level from 237 +/- 5 to 178 +/- 6 mg/dL (-25%), the LDL cholesterol level from 150 +/- 6 to 87 +/- 5 mg/dL (-40%), and raised high-density lipoprotein-cholesterol (HDL-C) level from 36 +/- 2 to 40 +/- 2 mg/dL (+11%) (all P <.001). Fasting and daily ambient triglyceride concentrations from 8 AM to 4 PM decreased significantly in response to simvastatin administration (P <.001), but not to the placebo (P =.305). Simvastatin treatment not only decreased total cholesterol and LDL-C levels and increased HDL-C levels effectively, it also decreased fasting, as well as daily postprandial triglyceride concentrations, but had no effect on glycemic control in type 2 diabetes subjects with combined hyperlipidemia.

Cholesterol↗

PubMed2001

High-throughput genotyping with single nucleotide polymorphisms.

To make large-scale association studies a reality, automated high-throughput methods for genotyping with single-nucleotide polymorphisms (SNPs) are needed. We describe PCR conditions that permit the use of the TaqMan or 5' nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically. To demonstrate the utility of these methods, we typed >1600 individuals for a G-to-T transversion that results in a glutamate-to-aspartate substitution at position 298 in the endothelial nitric oxide synthase gene, and a G/C polymorphism (newly identified in our laboratory) in intron 8 of the 11-beta hydroxylase gene. The genotyping method is accurate-we estimate an error rate of fewer than 1 in 2000 genotypes, rapid-with five 96-well PCR machines, one fluorescent reader, and no automated pipetting, over one thousand genotypes can be generated by one person in one day, and flexible-a new SNP can be tested for association in less than one week. Indeed, large-scale genotyping has been accomplished for 23 other SNPs in 13 different genes using this method. In addition, we identified three "pseudo-SNPs" (WIAF1161, WIAF2566, and WIAF335) that are probably a result of duplication.

Alleles↗

PubMed2001

Alstrom syndrome in two siblings.

Alstrom syndrome is a very rare autosomal recessive inherited disorder. Only 50 cases have been reported since the syndrome was first described in 1959. This syndrome is characterized by obesity, impaired glucose tolerance with insulin resistance, retinal degeneration, neurosensory deafness, acanthosis nigricans, hepatic dysfunction, and some endocrine disorders. The index case of this report was a 12-year-old girl who became blind at the age of 6 years as the result of progressively impaired vision. At the age of 12, diabetes mellitus was diagnosed and acanthosis nigricans presented in the neck, axilla, and groin regions. Her 10-year-old brother had similar symptoms. Electroretinography and audiometry disclosed generalized pigmentary epithelial change, decreased to absent cone and rod responses, and moderate sensorineural hearing loss in both siblings. Biochemistry and oral glucose tolerance tests showed diabetes mellitus, dyslipidemia, and hepatic dysfunction in the index case. Elevations of insulin, C-peptide, and leptin concentrations were found in both siblings. Insulin resistance was also demonstrated in both siblings using the modified insulin suppression test with constant infusion of somatostatin and exogenous insulin.

Acanthosis Nigricans↗

PubMed2000

Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9.

Forkhead-associated (FHA) domains have been shown to recognize both pThr and pTyr-peptides. The solution structures of the FHA2 domain of Rad53 from Saccharomyces cerevisiae, and its complex with a pTyr peptide, have been reported recently. We now report the solution structure of the other FHA domain of Rad53, FHA1 (residues 14-164), and identification of binding sites of FHA1 and its target protein Rad9. The FHA1 structure consists of 11 beta-strands, which form two large twisted anti-parallel beta-sheets folding into a beta-sandwich. Three short alpha-helices were also identified. The beta-strands are linked by several loops and turns. These structural features of free FHA1 are similar to those of free FHA2, but there are significant differences in the loops. Screening of a peptide library [XXX(pT)XXX] against FHA1 revealed an absolute requirement for Asp at the +3 position and a preference for Ala at the +2 position. These two criteria are met by a pThr motif (192)TEAD(195) in Rad9. Surface plasmon resonance analysis showed that a pThr peptide containing this motif, (188)SLEV(pT)EADATFVQ(200) from Rad9, binds to FHA1 with a K(d) value of 0.36 microM. Other peptides containing pTXXD sequences also bound to FHA1, but less tightly (K(d)=4-70 microM). These results suggest that Thr192 of Rad9 is the likely phosphorylation site recognized by the FHA1 domain of Rad53. The tight-binding peptide was then used to identify residues of FHA1 involved in the interaction with the pThr peptide. The results are compared with the interactions between the FHA2 domain and a pTyr peptide derived from Rad9 reported previously.

Amino Acid Motifs↗

PubMed2000

Determination of the binding specificity of the SH2 domains of protein tyrosine phosphatase SHP-1 through the screening of a combinatorial phosphotyrosyl peptide library.

A method for the rapid identification of high-affinity ligands to Src homology-2 (SH2) domains is reported. A phosphotyrosyl (pY) peptide library containing completely randomized residues at positions -2 to +3 relative to the pY was synthesized on TentaGel resin, with a unique peptide sequence on each resin bead (total 2.5 x 10(6) different sequences). The library was screened against the biotinylated N- and C-terminal SH2 domains of protein tyrosine phosphatase SHP-1, and the beads that carry high-affinity ligands of the SH2 domains were identified using an enzyme-linked assay involving a streptavidin-alkaline phosphatase conjugate. Peptide ladder sequencing of the selected beads using matrix-assisted laser desorption ionization mass spectrometry revealed consensus sequences for both SH2 domains. The N-terminal SH2 domain strongly selects for peptides with a leucine at the -2 position; at the C-terminal side of the pY residue, it can recognize two distinct classes of peptides with consensus sequences of LXpY(M/F)X(F/M) and LXpYAXL (X = any amino acid), respectively. The C-terminal SH2 domain exhibits almost exclusive selectivity for peptides of the consensus sequence, (V/I/L)XpYAX(L/V). Several representative sequences selected from the library were individually synthesized and tested for binding to the SH2 domains by surface plasmon resonance and for their ability to stimulate the catalytic activity of SHP-1. Both experiments have demonstrated that the selected peptides are capable of binding to the SH2 domains with dissociation constants (K(D)) in the low micromolar range.

Amino Acid Sequence↗

PubMed2000

Cysteine array matrix metalloproteinase (CA-MMP)/MMP-23 is a type II transmembrane matrix metalloproteinase regulated by a single cleavage for both secretion and activation.

Matrix metalloproteinases characterized so far are either secreted or membrane anchored via a type I transmembrane domain or a glycosylphosphatidylinositol linkage. Lacking either membrane-anchoring mechanism, the newly discovered CA-MMP/MMP-23 was reported to be expressed as a cell-associated protein. In this report, we present evidence that CA-MMP is expressed as an integral membrane zymogen with an N-terminal signal anchor, and secreted as a fully processed mature enzyme. We further demonstrate that L(20)GAALSGLCLLSALALL(36) is required for this unique membrane localization as a signal anchor and its secretion is regulated by a proprotein convertase motif RRRR(79) sandwiched between its pro- and catalytic domains. Thus, CA-MMP is a type II transmembrane MMP that can be regulated by a single proteolytic cleavage for both activation and secretion, establishing a novel paradigm for protein trafficking and processing within the secretory pathway.

Amino Acid Motifs↗