Journal articles on the topic 'Propeptide de la sortiline'
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1
Hivelin, C., J. Mazella, and T. Coppola. "CA-143: Le propeptide de la sortiline module l'entrée de glucose dans les adipocytes et les myocytes." Diabetes & Metabolism 42 (March 2016): A74. http://dx.doi.org/10.1016/s1262-3636(16)30275-0.
2
Hivelin, Céline, Jean Mazella, and Thierry Coppola. "Sortilin derived propeptide regulation during adipocyte differentiation and inflammation." Biochemical and Biophysical Research Communications 482, no. 1 (January 2017): 87–92. http://dx.doi.org/10.1016/j.bbrc.2016.10.139.
3
Petersen, C. M. "Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding." EMBO Journal 18, no. 3 (February 1, 1999): 595–604. http://dx.doi.org/10.1093/emboj/18.3.595.
4
Mazella, J., C. Devader, M. Roulot, M. Borsotto, and C. Heurteaux. "Regulation of serum spadin propeptide: An antidepressant response probe." European Psychiatry 33, S1 (March 2016): S417. http://dx.doi.org/10.1016/j.eurpsy.2016.01.1507.
Abstract:
ObjectivesWe previously discovered that spadin, a short analogue of the propeptide (PE) released from the maturation of sortilin, displays potent antidepressant properties. Since the PE level can be measured in the blood, we aimed to investigate how the PE serum concentration is regulated in mice. We wondered whether the PE serum levels vary between healthy subjects and patients with major depressive disorder (MDD).MethodsWe developped a dosing method based on the AlphaScreen™ technology (Perkin) which allow to selectively detect both PE, spadin and metabolic products from these peptides with a detection range of 1 ng/mL.ResultsWe found that insulin significantly up-regulated serum PE concentration from 26.15 ± 2.63 to 41.43 ± 6.27 nM (P = 0.0318). Analysis during circadian cycle in mice revealed that the amount of PE and its derivatives significantly varied during the cycle being higher during the period of maximal activity (dark period). We also measured serum insulin concentration between 1 and 7 pm and observed a significant rise confirming the relationships between insulin and PE concentration. We showed that the serum level of PE is lower in depressive patients than in healthy non-psychiatric. We observed that the weaker level of PE in depressive patients can recover the level of healthy subjects after a chronic antidepressant treatment.ConclusionsDosing the serum level of PE could be a promising approach for the diagnosis of depression and to determine the remission of the disease.Disclosure of interestThe authors have not supplied their declaration of competing interest.
5
Roulot, Morgane, Alessandra Minelli, Marco Bortolomasi, Elisabetta Maffioletti, Massimo Gennarelli, Marc Borsotto, Catherine Heurteaux, and Jean Mazella. "Increased serum levels of sortilin-derived propeptide after electroconvulsive therapy in treatment-resistant depressed patients." Neuropsychiatric Disease and Treatment Volume 14 (September 2018): 2307–12. http://dx.doi.org/10.2147/ndt.s170165.
6
Devader, Christelle, Morgane Roulot, Sébastien Moréno, Alessandra Minelli, Marco Bortolomasi, Chiara Congiu, Massimo Gennarelli, Marc Borsotto, Catherine Heurteaux, and Jean Mazella. "Serum sortilin-derived propeptides concentrations are decreased in major depressive disorder patients." Journal of Affective Disorders 208 (January 2017): 443–47. http://dx.doi.org/10.1016/j.jad.2016.10.049.
7
Mazella, Jean, and Jean-Pierre Vincent. "La sortiline : une protéine associée à de multiples fonctions." médecine/sciences 20, no. 6-7 (June 2004): 629–31. http://dx.doi.org/10.1051/medsci/2004206-7629.
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Kaufman, Beth D., Nancy Videon, Xuemei Zhang, Matthew A. Harris, Robert E. Shaddy, and Elizabeth Goldmuntz. "Procollagen type III amino-terminal propeptide: a serum biomarker of left ventricular remodelling in paediatric dilated cardiomyopathy." Cardiology in the Young 25, no. 2 (November 6, 2013): 228–36. http://dx.doi.org/10.1017/s1047951113001820.
Abstract:
AbstractBackgroundProcollagen type III amino-terminal propeptide is a collagen III cleavage product released in blood. The serum levels of this propeptide in adults with dilated cardiomyopathy are associated with cardiac remodelling and prognosis. The utility of procollagen type III amino-terminal propeptide as a biomarker in paediatric dilated cardiomyopathy is unknown.MethodsThis was a prospective, longitudinal study of children with dilated cardiomyopathy and changes in procollagen type III amino-terminal propeptide. The serum level of propeptide was measured serially, compared with paediatric normal values, and correlated with clinical status and left ventricular size and function on echocardiograms and cardiac magnetic resonance imaging.ResultsProcollagen type III amino-terminal propeptide was measured serially in 149 samples from 39 patients, age 9.0±6.4 years, followed up for 16.8±16.3 months. Procollagen type III amino-terminal propeptide in dilated cardiomyopathy was higher than in normal children. On multivariate analyses, procollagen type III amino-terminal propeptide had a positive correlation with left ventricular dilation, left ventricular end-diastolic diameter index (p<0.0001), and left ventricular end-diastolic diameter Z-score (p=0.0003), and a negative correlation with shortening fraction changes over time (p=0.001). Patients with myocarditis (n=12) had higher procollagen type III amino-terminal propeptide values than those with idiopathic dilated cardiomyopathy (n=20).ConclusionsProcollagen type III amino-terminal propeptide increases with left ventricular dilation and decreases with improvement in systolic function in paediatric dilated cardiomyopathy, indicating a role as a biomarker of cardiac remodelling in children. The diagnostic utility of procollagen type III amino-terminal propeptide to differentiate myocarditis from idiopathic dilated cardiomyopathy warrants further investigation.
9
Meiring, S. M., B. D. P. Setlai, C. Theron, and R. Bragg. "The Use of Phage Display and Yeast Based Expression System for the Development of a Von Willebrand Factor Propeptide Assay: Development of a Von Willebrand Factor Propeptide Assay." BioMed Research International 2018 (2018): 1–7. http://dx.doi.org/10.1155/2018/6232091.
Abstract:
Background. The diagnosis of von Willebrand disease is complex due to the heterogeneity of the disease. About eighty percent of von Willebrand disease patients are diagnosed with a quantitative defect of von Willebrand factor (VWF) where fifty percent is due to an increased clearance of von Willebrand factor. These patients do not respond well to the treatment of choice, Desmopressin (DDAVP) due to decreased efficacy. The ratio between the VWF propeptide and the mature VWF antigen is used to diagnose these patients. Commercial VWF propeptide assays are too expensive for use in developing countries. In this study, we developed a cost-effective ELISA assay. Methods. We first displayed VWF propeptide on yeast. Antibody fragments were selected against the displayed VWF propeptide by using phage display technology. The antibodies were used to develop a cost-effective VWF propeptide assay and compared to a commercial VWF propeptide assay. Results. Two of these antibody fragments bound specific to the VWF propeptide and not to the yeast used for the expression of the propeptides. These purified antibody fragments were able to detect VWF propeptide in normal plasma. Conclusion. Our assay performed well when compared to a commercial kit. It also showed a higher binding affinity for VWF propeptide in plasma at especially lower plasma concentrations.
10
Emeis, Jef, Henk Bilo, Coen Stehouwer, Claus Thomsen, Ole Rasmussen, Kjeld Hermansen, Claes Wollheim, Jørgen Ingerslev, and Ulrich Vischer. "von Willebrand Factor (vWf) as a Plasma Marker of Endothelial Activation in Diabetes: Improved Reliability with Parallel Determination of the vWf Propeptide (vWf:AgII)." Thrombosis and Haemostasis 80, no. 12 (1998): 1002–7. http://dx.doi.org/10.1055/s-0037-1615401.
Abstract:
SummaryElevated plasma von Willebrand factor (vWf) levels are found in diabetes and other vasculopathies, and predict cardiovascular mortality. vWf is stored and released from endothelial cell secretory granules, along with equimolar amounts of its propeptide (vWf:AgII). In the present study, we examined plasma propeptide levels as a marker of endothelial secretion in vivo, using an ELISA based on monoclonal antibodies. vWf but not propeptide levels are influenced by blood groups, explaining in part the smaller variation in plasma propeptide levels among normal individuals. In both controls and insulin-dependent diabetic patients, we found a close correlation between propeptide and immunoreactive vWf levels (r2 = 0.54, p <0.0001). vWf and propeptide were elevated in patient subgroups with microalbuminuria or overt diabetic nephropathy, whereas only the propeptide was significantly elevated in the normoalbuminuric subgroup. This observation suggests that in conjunction with vWf, propeptide measurements may improve the identification of endothelial activation, which occurs frequently even without increased urinary albumin excretion. In 12 NIDDM patients, a 3-week diet enriched in monounsaturated fat (MUFA) resulted in parallel decreases in vWf (-22%, p <0.05) and propeptide (-17%, p <0.05) levels, indicating that the experimental diet affected endothelial secretion rather than vWf catabolism. A carbohydrate-enriched control diet did not significantly influence either marker.Our results suggest that concomittant determinations of plasma vWf and propeptide are useful tools to assess endothelial activation in vivo, and reinforce our previous conclusion that a diet rich in MUFA can improve endothelial function in NIDDM.
11
Bendetowicz, Ana Victoria, Jill A. Morris, Robert J. Wise, Gary E. Gilbert, and Randal J. Kaufman. "Binding of Factor VIII to von Willebrand Factor Is Enabled by Cleavage of the von Willebrand Factor Propeptide and Enhanced by Formation of Disulfide-Linked Multimers." Blood 92, no. 2 (July 15, 1998): 529–38. http://dx.doi.org/10.1182/blood.v92.2.529.
Abstract:
Abstract von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.
12
Bendetowicz, Ana Victoria, Jill A. Morris, Robert J. Wise, Gary E. Gilbert, and Randal J. Kaufman. "Binding of Factor VIII to von Willebrand Factor Is Enabled by Cleavage of the von Willebrand Factor Propeptide and Enhanced by Formation of Disulfide-Linked Multimers." Blood 92, no. 2 (July 15, 1998): 529–38. http://dx.doi.org/10.1182/blood.v92.2.529.414k31_529_538.
Abstract:
von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.
13
ZHANG, Zhen-Zhong, Satoru NIRASAWA, Yoshiaki NAKAJIMA, Michiteru YOSHIDA, and Kiyoshi HAYASHI. "Function of the N-terminal propeptide of an aminopeptidase from Vibrio proteolyticus." Biochemical Journal 350, no. 3 (September 8, 2000): 671–76. http://dx.doi.org/10.1042/bj3500671.
Abstract:
An aminopeptidase from Vibrio proteolyticus was translated as a preproprotein consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. Protein expression and analysis of the activity results demonstrated that the N-terminal propeptide was essential to the formation of the active enzyme in Escherichia coli. Urea dissolution of inclusion bodies and dialysis indicated that the N-terminal propeptide could facilitate the correct folding of the enzyme in vitro. Using l-Leu-p-nitroanilide as the substrate, the kinetic parameters (kcat and Km) of the pro-aminopeptidase and processed aminopeptidases were analysed. The results suggested that the N-terminal propeptide inhibited enzyme activity of the mature region. In contrast, the C-terminal propeptide did not show evidence of forming an active enzyme, of correctly folding in vitro or of inhibiting the active region.
14
Wit, Thalia Romani de, and Jan van Mourik. "Von Willebrand Factor Propeptide in Vascular Disorders." Thrombosis and Haemostasis 86, no. 07 (2001): 164–71. http://dx.doi.org/10.1055/s-0037-1616214.
Abstract:
SummaryVon Willebrand factor (VWF) is a multifunctional plasma protein that plays a prominent role in haemostasis. In endothelial cells, processing of its precursor pro-VWF results in the formation of two large polypeptides, mature VWF and a propeptide. These proteins are co-secreted on an equimolar basis but are cleared from the circulation at different rates. VWF levels are frequently elevated in response to vascular disorders. Similarly, propeptide levels are increased under these conditions, although primarily in fulminant vascular disease, such as thrombotic thrombocytopenic purpura and septicemia. In chronic vascular disease, e.g. diabetes or peripheral vascular disease, propeptide levels are much less elevated. The differential response of VWF and propeptide levels to vascular disease could provide a means to assess the extent and time course of endothelial cell activation. After secretion, the propeptide may play a role in modulating cellular adhesion processes. Thus, enhanced propeptide secretion seems not to be of merely diagnostic significance.
15
Li, Songtao, Janet L. Smith, and Howard Zalkin. "Mutational Analysis of Bacillus subtilisGlutamine Phosphoribosylpyrophosphate Amidotransferase Propeptide Processing." Journal of Bacteriology 181, no. 5 (March 1, 1999): 1403–8. http://dx.doi.org/10.1128/jb.181.5.1403-1408.1999.
Abstract:
ABSTRACT Glutamine phosphoribosylpyrophosphate amidotransferase fromBacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.
16
Zhu, Yong, Anush Oganesian, Douglas R. Keene, and Linda J. Sandell. "Type IIA Procollagen Containing the Cysteine-rich Amino Propeptide Is Deposited in the Extracellular Matrix of Prechondrogenic Tissue and Binds to TGF-β1 and BMP-2." Journal of Cell Biology 144, no. 5 (March 8, 1999): 1069–80. http://dx.doi.org/10.1083/jcb.144.5.1069.
Abstract:
Type II procollagen is expressed as two splice forms. One form, type IIB, is synthesized by chondrocytes and is the major extracellular matrix component of cartilage. The other form, type IIA, contains an additional 69 amino acid cysteine-rich domain in the NH2-propeptide and is synthesized by chondrogenic mesenchyme and perichondrium. We have hypothesized that the additional protein domain of type IIA procollagen plays a role in chondrogenesis. The present study was designed to determine the localization of the type IIA NH2-propeptide and its function during chondrogenesis. Immunofluorescence histochemistry using antibodies to three domains of the type IIA procollagen molecule was used to localize the NH2-propeptide, fibrillar domain, and COOH-propeptides of the type IIA procollagen molecule during chondrogenesis in a developing human long bone (stage XXI). Before chondrogenesis, type IIA procollagen was synthesized by chondroprogenitor cells and deposited in the extracellular matrix. Immunoelectron microscopy revealed type IIA procollagen fibrils labeled with antibodies to NH2-propeptide at ∼70 nm interval suggesting that the NH2-propeptide remains attached to the collagen molecule in the extracellular matrix. As differentiation proceeds, the cells switch synthesis from type IIA to IIB procollagen, and the newly synthesized type IIB collagen displaces the type IIA procollagen into the interterritorial matrix. To initiate studies on the function of type IIA procollagen, binding was tested between recombinant NH2-propeptide and various growth factors known to be involved in chondrogenesis. A solid phase binding assay showed no reaction with bFGF or IGF-1, however, binding was observed with TGF-β1 and BMP-2, both known to induce endochondral bone formation. BMP-2, but not IGF-1, coimmunoprecipitated with type IIA NH2-propeptide. Recombinant type IIA NH2-propeptide and type IIA procollagen from media coimmunoprecipitated with BMP-2 while recombinant type IIB NH2-propeptide and all other forms of type II procollagens and mature collagen did not react with BMP-2. Taken together, these results suggest that the NH2-propeptide of type IIA procollagen could function in the extracellular matrix distribution of bone morphogenetic proteins in chondrogenic tissue.
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Aziz, Mohamed Talaat Abdel, Mohamed Abdel Aziz Wassef, Maha Kamel, Mona El Zein, and Hany El Hassan. "Clinical Evaluation of Serum Aminoterminal Propeptide of Type III Procollagen as Tumor Marker in Gynecologic Malignancies." Tumori Journal 79, no. 3 (June 1993): 219–23. http://dx.doi.org/10.1177/030089169307900313.
Abstract:
Aims The aim of this study was to determine the possible usefulness of the assay of the aminoterminal propeptide of type III procollagen and fibronectin in detecting connective tissue changes associated with gynecologic malignancies. Study Design Serum aminoterminal propeptide of type III procollagen and plasma fibronectin were measured in 36 women with gynecologic malignancies, 20 women with benign gynecologic tumors and 10 healthy women serving as controls. Results A significant serum propeptide was significantly high In the group with gynecologic malignancies and normal in the benign tumor group. The serum propeptide levels were related to of disease stage and presence of ascites in patients with ovarian carcinoma but not in those with cervical or endometrial carcinoma. In the follow-up study, a favorable clinical response was associated with normalizing propeptide levels whereas in rapidly progressive disease the levels fell initially but rose again. In partial response with ultimate progression, the propeptide concentration decreased but remained clearly above the normal range. No difference in plasma fibronectin was found among the malignant tumor, benign tumor and control groups. Conclusions The present study indicates that the aminoterminal propeptide of type III procollagen could serve as an additional, non specific marker to follow the clinical behavior of gynecologic malignancies and consequently of connective tissue metabolism reflecting tumor matrix interaction.
18
Boertjes, Ria, Jan van Mourik, and Perry van Genderen. "Quantitative Analysis of von Willebrand Factor and Its Propeptide in Plasma in Acquired von Willebrand Syndrome." Thrombosis and Haemostasis 80, no. 09 (1998): 495–98. http://dx.doi.org/10.1055/s-0037-1615235.
Abstract:
SummaryMeasurement of the von Willebrand factor (vWF) propeptide, also known as von Willebrand antigen II, has been suggested to be helpful in the discrimination of congenital von Willebrand disease type I from type 2 and in assessing the extent of activation of the endothelium. We performed a quantitative analysis of mature vWF and its propeptide in plasma in 8 patients with acquired von Willebrand syndrome (AvWS) and in 20 normal individuals. Mature vWF levels were significantly lower in AvWS as compared with normal individuals (13.4 ± 3.5 vs 35.6 ± 3.3 nM, p <0.001). In contrast, propeptide levels were significantly higher in AvWS (11.4 ± 1.1 vs 4.7 ± 0.2 nM, p < 0.001), probably reflecting a compensatory increase in vWF synthesis or increased perturbation of the endothelium in AvWS. After treatment with DDAVP, propeptide and mature vWF levels rose 5-fold in AvWS, whereas propeptide levels were not altered by the infusion of a vWF concentrate or treatment with high dose intravenous immunoglobulins, indicating that plasma propeptide levels are a reliable reflection of vWF synthesis. Measurement of propeptide levels may provide additional information in AvWS as to whether decreased levels of mature vWF in the circulation are due to a decrease in synthesis or due to an accelerated removal of vWF from the circulation.
19
Borchiellini, A., K. Fijnvandraat, JW ten Cate, D. Pajkrt, SJ van Deventer, G. Pasterkamp, F. Meijer-Huizinga, L. Zwart-Huinink, J. Voorberg, and JA van Mourik. "Quantitative analysis of von Willebrand factor propeptide release in vivo: effect of experimental endotoxemia and administration of 1- deamino-8-D-arginine vasopressin in humans." Blood 88, no. 8 (October 15, 1996): 2951–58. http://dx.doi.org/10.1182/blood.v88.8.2951.bloodjournal8882951.
Abstract:
The results of studies with cultured endothelial cells have shown that most von Willebrand factor (vWF) synthesized is directly secreted (constitutive pathway) and consists of both mature vWF, its precursor molecule pro-vWF, and the cleaved vWF prosequence. Only fully processed, functionally mature vWF is stored within the cell, together with the propeptide, and leaves the cell only on stimulation (regulated secretion). Both in resting and stimulated cultured endothelial cells, the stoichiometry of the released propeptide to the released mature vWF is essentially equimolar. In the present study, we have measured the molar ratio of propeptide to mature vWF in vivo, both under resting conditions and conditions that reflect activation of the endothelium. To this end, we devised a method that allows the measurement of the propeptide (vW antigen II) on a quantitative, is, molar basis, using purified recombinant propeptide as a standard. Our results show that the molar concentration of the propeptide in normal plasma is about one tenth of the concentration of mature vWF (expressed as half-dimer concentration). This ratio is approximately 1:1 in the medium of cultured endothelial cells. On administration in healthy subjects of either 1-deamino-8-D-arginine vasopressin or endotoxin, both agents being known to elicit an intravascular increase of vWF, the molar ratio of propeptide to mature vWF increased fourfold to fivefold. The propeptide concentration returned to baseline values after about 6 to 7 hours of injection of each stimulus, whereas the increase of mature vWF was much more sustained. Because the respective half-lives of mature vWF and its propeptide clearly differ, measurement of the concentration of these proteins could provide a means to assess the extent of activation of the endothelium under physiological and pathophysiological conditions.
20
van Mourik, Jan A., Ria Boertjes, Inge A. Huisveld, Karin Fijnvandraat, Dasja Pajkrt, Perry J. J. van Genderen, and Rob Fijnheer. "von Willebrand Factor Propeptide in Vascular Disorders: A Tool to Distinguish Between Acute and Chronic Endothelial Cell Perturbation." Blood 94, no. 1 (July 1, 1999): 179–85. http://dx.doi.org/10.1182/blood.v94.1.179.413k18_179_185.
Abstract:
Before de novo synthesized von Willebrand factor (vWF) leaves the endothelial cell, it undergoes endoproteolytic cleavage of its propeptide (vW antigen II). The processed vWF and propeptide are either released constitutively or, following activation of the endothelium, released through the regulated pathway. In a recent study (Borchiellini et al, Blood 88:2951, 1996), we showed that the half-life of mature vWF and of its propeptide differ fourfold to fivefold. We postulated that the molar ratio of the propeptide to mature vWF could serve as a tool to assess the extent of endothelial cell activation under physiologic and clinical conditions. To test this hypothesis, we measured mature vWF and propeptide in patients with documented acute and chronic vascular disease, including patients with thrombotic thrombocytopenic purpura (TTP), acute septicemia, and diabetes mellitus. These data were compared with experimental conditions in healthy subjects in which perturbation of the endothelium was simulated by physical exercise or by administration of 1-deamino-8-D-arginine vasopressin (DDAVP) or endotoxin. In all individuals of the latter study group, both vWF and propeptide levels were elevated during the acute phase of the experimentally induced vascular perturbation; at later time points after stimulation, only vWF levels remained elevated. In patients with sepsis and TTP, both vWF and propeptide were elevated several-fold. Thus, this pattern can readily be explained in terms of acute perturbation of the endothelium. In contrast, in patients with diabetes mellitus propeptide levels were only slightly elevated, whereas vWF levels were elevated twofold to threefold. This pattern is a typical feature of chronic, low-grade activation of the endothelium. These observations support our hypothesis that measurement of both propeptide and vWF levels allows to discriminate between chronic and acute phases of endothelial cell activation in vivo. Measurement of only vWF is less indicative in this respect.
21
Plíhal, O., J. Sklenář, J. Kmoníčková, P. Man, P. Pompach, V. Havlíček, V. Křen, and K. Bezouška. "N-glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase." Biochemical Society Transactions 32, no. 5 (October 26, 2004): 764–65. http://dx.doi.org/10.1042/bst0320764.
Abstract:
β-N-Acetylhexosaminidase from a filamentous fungus Aspergillus oryzae is a secreted enzyme known to be an important component of the binary chitinolytic system. Cloning of the hexA gene and sequencing of the enzyme revealed its unique preproprotein structure. While the enzyme's zincin-like and catalytic domain had significant similarities with members of the glycohydrolase 20 family, the propeptide was unique for the fungal enzyme. Detailed pulse–chase and inhibition studies revealed that propeptide was processed during the biosynthesis of the enzyme. Moreover, the presence of propeptide was necessary for enzyme activation, dimerization and secretion. The catalytic unit was N-glycosylated, and the propeptide was O-glycosylated, both in their C-terminal parts. Deglycosylation experiments revealed that the N-glycosylation increased the stability and solubility of the enzyme. In contrast, O-glycosylated propeptide was necessary to attain the full enzymic activity.
22
Yeung, P. S. Marie, Nicholas Zagorski, and Hélène Marquis. "The Metalloprotease of Listeria monocytogenes Controls Cell Wall Translocation of the Broad-Range Phospholipase C." Journal of Bacteriology 187, no. 8 (April 15, 2005): 2601–8. http://dx.doi.org/10.1128/jb.187.8.2601-2608.2005.
Abstract:
ABSTRACT Listeria monocytogenes is a gram-positive bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from secondary vacuoles. PC-PLC requires cleavage of an N-terminal propeptide for activation, and Mpl, a metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. Previously, we showed that cell wall translocation of PC-PLC is inefficient, resulting in accumulation of PC-PLC at the membrane-cell wall interface. In infected cells, rapid cell wall translocation of PC-PLC is triggered by a decrease in pH and correlates with cleavage of the propeptide in an Mpl-dependent manner. To address the role of the propeptide and of Mpl in cell wall translocation of PC-PLC, we generated a cleavage site mutant and a propeptide deletion mutant. The intracellular behavior of these mutants was assessed in pulse-chase experiments. We observed efficient translocation of the proform of the PC-PLC cleavage site mutant in a manner that was pH sensitive and Mpl dependent. However, the propeptide deletion mutant was efficiently translocated into host cells independent of Mpl and pH. Overall, these results suggest that Mpl regulates PC-PLC translocation across the bacterial cell wall in a manner that is dependent on the presence of the propeptide but independent of propeptide cleavage. In addition, similarly to Mpl-mediated cleavage of PC-PLC propeptide, Mpl-mediated translocation of PC-PLC across the bacterial cell wall is pH sensitive.
23
Vischer, Ulrich M., Jørgen Ingerslev, Claes B. Wollheim, Jean-Claude Mestries, Dimitrios A. Tsakiris, Walter E. Haefeli, and Egbert K. O. Kruithof. "Acute von Willebrand Factor Secretion from the Endothelium In Vivo: Assessment through Plasma Propeptide (vWf:AgII) Levels." Thrombosis and Haemostasis 77, no. 02 (1997): 387–93. http://dx.doi.org/10.1055/s-0038-1655973.
Abstract:
SummaryElevated plasma concentrations of von Willebrand factor (vWf) are increasingly recognized as a cardiovascular risk factor, and are used as a marker of endothelial activation. However, the factors which determine the rate of vWf release from the endothelium in vivo have not been defined clearly. In addition, vWf plasma levels may also be influenced by adhesion of vWf to the vascular wall or to platelets, and by its rate of degradation. The propeptide of vWf (also called vWf:AgII) is stored and released in equimolar amounts with vWf. In the present study we attempted to determine whether this propeptide could be a more reliable marker of endothelial secretion than vWf itself. To accomplish this we developed an ELISA based on monoclonal antibodies. The propeptide levels in normal plasma were found to be 0.7 µg/ml, more than 10 times lower than vWf itself. Administration of desmopressin (DDAVP) induced a rapid relative increase in propeptide (from 106 to 879%) and in vWf (from 112 to 272%). However, the increases in vWf and propeptide were equivalent when expressed in molar units. A time course study indicated a half-life of the propeptide of 3 h or less. In a baboon model of disseminated intravascular coagulation (DIC) induced by FXa, vWf increased by less than 100%, whereas the propeptide concentrations increased by up to 450%. In view of the massive thrombin generation (as assessed by fibrinogen depletion), the increases in vWf are small, compared to the strong secretory response to thrombin and fibrin previously observed in vitro. Our results suggest that due to its rapid turnover, the propeptide could provide a sensitive plasma marker of acute endothelial secretion.
24
Oda, M. N., S. V. Scott, A. Hefner-Gravink, A. D. Caffarelli, and D. J. Klionsky. "Identification of a cytoplasm to vacuole targeting determinant in aminopeptidase I." Journal of Cell Biology 132, no. 6 (March 15, 1996): 999–1010. http://dx.doi.org/10.1083/jcb.132.6.999.
Abstract:
Aminopeptidase I (API) is a soluble leucine aminopeptidase resident in the yeast vacuole (Frey, J., and K.H. Rohm. 1978. Biochim. Biophys. Acta. 527:31-41). The precursor form of API contains an amino-terminal 45-amino acid propeptide, which is removed by proteinase B (PrB) upon entry into the vacuole. The propeptide of API lacks a consensus signal sequence and it has been demonstrated that vacuolar localization of API is independent of the secretory pathway (Klionsky, D.J., R. Cueva, and D.S. Yaver. 1992. J. Cell Biol. 119:287-299). The predicted secondary structure for the API propeptide is composed of an amphipathic alpha-helix followed by a beta-turn and another alpha-helix, forming a helix-turn-helix structure. With the use of mutational analysis, we determined that the API propeptide is essential for proper transport into the vacuole. Deletion of the entire propeptide from the API molecule resulted in accumulation of a mature-sized protein in the cytosol. A more detailed examination using random mutagenesis and a series of smaller deletions throughout the propeptide revealed that API localization is severely affected by alterations within the predicted first alpha-helix. In vitro studies indicate that mutations in this predicted helix prevent productive binding interactions from taking place. In contrast, vacuolar import is relatively insensitive to alterations in the second predicted helix of the propeptide. Examination of API folding revealed that mutations that affect entry into the vacuole did not affect the structure of API. These data indicate that the API propeptide serves as a vacuolar targeting determinant at a critical step along the cytoplasm to vacuole targeting pathway.
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McLeod, R. S., C. Robbins, A. Burns, Z. Yao, and P. H. Pritchard. "Deletion of the propeptide of apolipoprotein A-I impairs exit of nascent apolipoprotein A-I from the endoplasmic reticulum." Biochemical Journal 302, no. 3 (September 15, 1994): 641–48. http://dx.doi.org/10.1042/bj3020641.
Abstract:
Human apolipoprotein (apo) A-I is secreted as a proprotein of 249 amino acids and is processed extracellularly to the mature form (243 amino acids) by removal of a six-residue propeptide segment. We have examined the role of the apoA-I propeptide in intracellular transport and secretion using transfected baby hamster kidney cells that secreted either proapoA-I (from the wild-type cDNA, A-Iwt) or mature-form apoA-I (from A-I delta pro, a cDNA in which the propeptide sequence was deleted). Deletion of the propeptide from the apoA-I sequence did not affect the rate of apoA-I synthesis, nor did it affect the fidelity of proteolytic removal of the prepeptide. However, the propeptide deletion caused mature-form apoA-I to accumulate within the cells as determined by pulse-chase experiments; the intracellular retention times for the mature-form apoA-I in which the propeptide was prematurely removed was three times longer than that of proapoA-I (t1/2 > 3 h compared with approximately 50 min). There was no detectable degradation of either form of newly synthesized apoA-I. Immunofluorescence microscopy revealed that, whereas the proapoA-I was located predominantly in the Golgi apparatus, large quantities of the mature-form apoA-I were detected in the endoplasmic reticulum and very little was in the Golgi apparatus of A-I delta pro-transfected cells. These findings suggest that the propeptide sequence may be involved in the intracellular transport of apoA-I from the endoplasmic reticulum to the Golgi apparatus. We propose that the function of the propeptide sequence is to facilitate efficient transport of apoA-I through the secretory pathway.
26
Lee, SN, E. Prodhomme, and I. Lindberg. "Prohormone convertase 1 (PC1) processing and sorting: effect of PC1 propeptide and proSAAS." Journal of Endocrinology 182, no. 2 (August 1, 2004): 353–64. http://dx.doi.org/10.1677/joe.0.1820353.
Abstract:
Prohormone convertase 1 (PC1) is a serine proteinase responsible for the proteolytic processing of many precursor proteins within the regulated secretory pathway. The activity of PC1 is potentially regulated by two endogenous inhibitors, the PC1 propeptide and proSAAS. Here we have investigated the effect of proSAAS and propeptide-containing constructs on PC1 carboxy-terminal processing and activity. In AtT-20 cells, proSAAS expression inhibited both C-terminal PC1 processing and proopiomelanocortin (POMC) processing under pulse/chase conditions. SAAS CT peptide-propeptide chimeric constructs had no effect on the cleavage of PC1 and POMC under pulse/chase conditions. However, a construct containing the propeptide alone reduced C-terminal PC1 processing under pulse/chase conditions and also inhibited POMC processing. In contrast, experiments using HEK293 cells transiently expressing PC1 plus the respective constructs demonstrated significant inhibition of zymogen processing and decreased C-terminal processing of PC1 by the SAAS CT peptide portion of the chimera. Our results suggest that the PC1 propeptide expressed in trans is able to act as an endogenous inhibitor of PC1, but that SAAS CT peptide-containing/propeptide constructs cannot function as effective inhibitors of precursor maturation in the regulated pathway.
27
Bristol, JA, JV Ratcliffe, DA Roth, MA Jacobs, BC Furie, and B. Furie. "Biosynthesis of prothrombin: intracellular localization of the vitamin K-dependent carboxylase and the sites of gamma-carboxylation." Blood 88, no. 7 (October 1, 1996): 2585–93. http://dx.doi.org/10.1182/blood.v88.7.2585.bloodjournal8872585.
Abstract:
Prothrombin is a vitamin K-dependent blood coagulation protein that undergoes posttranslational gamma-carboxylation and propeptide cleavage during biosynthesis. The propeptide contains the gamma-carboxylation recognition site that directs gamma-carboxylation. To identify the intracellular sites of carboxylation and propeptide cleavage, we monitored the synthesis of prothrombin in Chinese hamster ovary cells stably transfected with the prothrombin cDNA by immunofluorescent staining. The vitamin K-dependent carboxylase was located in the endoplasmic reticulum and Golgi complex. Antibodies specific to prothrombin processing intermediates were used for immunocytolocalization. Anti-des-gamma-carboxyprothrombin antibodies stained only the endoplasmic reticulum whereas antiproprothrombin antibodies (specific for the propeptide) and antiprothrombin:Mg(II) antibodies (which bind the carboxylated forms of proprothrombin and prothrombin) stained both the endoplasmic reticulum and the Golgi complex. Antiprothrombin:Ca(II)-specific antibodies (which bind only to the carboxylated form of prothrombin lacking the propeptide) stained only the Golgi complex and secretory vesicles, and colocalized with antimannosidase II and anti-p200 in the juxtanuclear Golgi complex. These results indicate that uncarboxylated proprothrombin undergoes complete gamma-carboxylation in the endoplasmic reticulum and that gamma-carboxylation precedes propeptide cleavage during prothrombin biosynthesis.
28
HOUBEN, Roger J. T. J., Dayun JIN, Darrel W. STAFFORD, Paul PROOST, Rob H. M. EBBERINK, Cees VERMEER, and Berry A. M. SOUTE. "Osteocalcin binds tightly to the γ-glutamylcarboxylase at a site distinct from that of the other known vitamin K-dependent proteins." Biochemical Journal 341, no. 2 (July 8, 1999): 265–69. http://dx.doi.org/10.1042/bj3410265.
Abstract:
Vitamin K-dependent proteins contain a propeptide that is required for recognition by the enzyme γ-glutamylcarboxylase. Substrates used in vitro for carboxylation studies lacking a prosequence are characterized by Km values in the millimolar range, whereas the Kmfor peptides containing a prosequence is three or four orders of magnitude smaller. Here we report that descarboxy-osteocalcin is an exception in this respect. With descarboxy-osteocalcin in purified propeptide-free recombinant carboxylase, the Km was 1.8 μM. Furthermore, osteocalcin was an inhibitor of descarboxy-osteocalcin carboxylation with a Ki of 76 μM. In contrast with the other vitamin K-dependent proteins, free propeptides do not inhibit descarboxy-osteocalcin carboxylation. Moreover, propeptide-containing substrates were inhibited neither by osteocalcin nor by its propeptide. From our studies we conclude that descarboxy-osteocalcin must have an internal recognition sequence that binds to γ-glutamylcarboxylase at a site different from the propeptide-recognition site.
29
Grande, Kerian K., Jean K. Gustin, Efrat Kessler, and Dennis E. Ohman. "Identification of Critical Residues in the Propeptide of LasA Protease of Pseudomonas aeruginosa Involved in the Formation of a Stable Mature Protease." Journal of Bacteriology 189, no. 11 (March 9, 2007): 3960–68. http://dx.doi.org/10.1128/jb.01828-06.
Abstract:
ABSTRACT LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.
30
Pitkänen, Sari, Matti K. Salo, Kim Vettenranta, Kirsti Näntö‐Salonen, and Markku Heikinheimo. "Serum Type III Procollagen in Children with Type I Hereditary Tyrosinemia." Journal of Pediatric Gastroenterology and Nutrition 29, no. 1 (July 1999): 38–41. http://dx.doi.org/10.1002/j.1536-4801.1999.tb02358.x.
Abstract:
ABSTRACTBackground:Type I hereditary tyrosinemia leads to hepatic dysfunction and fibrosis and is associated with a high risk of hepatic malignancy. Serum N‐terminal propeptide of type III procollagen is a sensitive marker of organ fibrosis of diverse origins. The current study was conducted to determine whether analysis of serum levels of type III procollagen in hereditary tyrosinemia would be useful in the follow‐up of the progressive liver disease and eventually in detecting hepatic malignancy.Methods:Serum N‐terminal propeptide of type III procollagen was sequentially studied in 10 children with type I hereditary tyrosinemia.Results:At diagnosis of type I hereditary tyrosinemia, serum N‐terminal propeptide of type III procollagen ranged from 0.6 to 2.9 multiples of age‐related median. During follow‐up, serum N‐terminal propeptide of type III procollagen decreased, yet remained elevated 0.2 to 2.6 years after diagnosis. Children with the acute type of the disease tended to have higher serum N‐terminal propeptide of type III procollagen than did those with the chronic type. Porphyria crises were associated with elevated serum type III procollagen. The one patient receiving 2‐(2‐nitro‐4‐trifluoromethyl‐benzoyl)‐1,3‐cyclohexanedione (NTBC) did not differ from the other ones in serum type III procollagen levels. Serum N‐terminal propeptide of type III procollagen did not increase with developing hepatocellular carcinoma.Conclusions:Serum N‐terminal propeptide of type III procollagen may be useful in monitoring the hepatopathy in type I hereditary tyrosinemia but is not useful in detecting malignant transformation in the liver.
31
Oganesian, Anush, Yong Zhu, and Linda J. Sandell. "Type IIA Procollagen Amino Propeptide Is Localized in Human Embryonic Tissues." Journal of Histochemistry & Cytochemistry 45, no. 11 (November 1997): 1469–80. http://dx.doi.org/10.1177/002215549704501104.
Abstract:
Type II procollagen is synthesized in two forms generated by the alternative splicing of its precursor mRNA. The alternatively spliced domain, exon 2, encodes the 69-amino-acid cysteinerich region of the NH2 propeptide. Studies of mRNA expression have shown that the longer form, designated Type IIA procollagen, is synthesized by chondroprogenitor cells and various noncartilaginous tissues. The shorter form, Type IIB procollagen, is synthesized by differentiated chondrocytes. As the initial step in our investigations of the function of the Type IIA procollagen, the protein domain corresponding to exon 2 was created as a recombinant fusion protein and used to raise antibodies in rabbits. The resulting antiserum was specific for Type IIA procollagen NH2 propeptide as shown by ELISA, Western blotting, and immunofluorescent co-localization with the triple-helical domain of Type II collagen. Type IIA procollagen was identified in tissue culture medium of 54-day human fetal ribs. Confocal microscopy was used to localize the Type IIA NH2 propeptide in Day 50 and 53 human embryos. In the digital rays of the developing hand, where only Type IIA procollagen mRNA was detected, Type IIA procollagen NH2 propeptide was observed in the extracellular matrix. The presence of Type IIA procollagen NH2 propeptide was observed in the cartilage of the developing long bones of the lower arm and vertebral bodies even though these tissues synthesize Type IIB mRNA at this developmental stage. Type IIA procollagen NH2 propeptide was localized in the developing trachea, a cartilage that does not undergo endochondral bone formation. Type IIA NH2 propeptide was also localized in noncartilaginous tissues known to synthesize Type IIA mRNA, such as the intervertebral area, perichondrium, notochordal sheath, and neuroepithelium of the otic vesicle. In most tissues, co-localization with antiserum against the triple-helical domain of Type II collagen was observed. Positive immunoreactivity with the Type IIA NH2 propeptide antiserum indicates, for the first time, that this propeptide is present in the tissue. Co-localization of NH2 propeptide antibodies with the triple-helical domain of the collagen molecule suggests that Type IIA procollagen is intact in the extracellular matrix of these tissues. Taken together, these results strongly suggest that around cells that synthesize Type IIA procollagen mRNA, Type IIA procollagen NH2 propeptide is secreted and deposited into the extracellular matrix. In light of these results, we predict that Type IIA procollagen plays a role in differentiation of tissues that augments its purely architectural function. (J Histochem Cytochem 45:1469–1480, 1997)
32
Pulido, Marian, Kenji Saito, Shun-Ichi Tanaka, Yuichi Koga, Masaaki Morikawa, Kazufumi Takano, and Shigenori Kanaya. "Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding." Applied and Environmental Microbiology 72, no. 6 (June 2006): 4154–62. http://dx.doi.org/10.1128/aem.02696-05.
Abstract:
ABSTRACT Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80�C. This maturation process was completed within 30 min at 80�C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80�C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80�C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ∼5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a Ki of 25 � 3.0 nM at 20�C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.
33
Kalafatis, Michael, Daniela Tormene, Sonia Luni, Patrizia Zerbinati, Luisa Barzon, Giorgio Palù, Antonio Girolami, and Paolo Simioni. "Abnormal Propeptide Processing Resulting in the Presence of Two Abnormal Species of Protein C in Plasma." Thrombosis and Haemostasis 86, no. 10 (2001): 1017–22. http://dx.doi.org/10.1055/s-0037-1616527.
Abstract:
SummaryA heterozygous GT transversion at position 1388 of the protein C (PC) gene which predicted the substitution of Arg-1 to a Leu (PCR-1L) was identified in a thrombophilic patient. The PCR-1L was purified from the patient’s plasma by immunoaffinity chromatography using Ca++-independent and Ca++-dependent monoclonal antibodies. NH2-terminal sequencing of the light chain of PCR-1L revealed two amino acid sequences: one was identical to the complete propeptide sequence of PC, while the other matched the normal PC light chain sequence elongated by one amino acid (Leucine at position 1). Activated PCR-1L/propeptide exhibited normal amidolytic and impaired anticoagulant activity. Thus, the substitution of a Leu for an Arg at position -1 of PC shifts the propeptidase cleavage site by one amino acid. In addition, in PCR-1L/propeptide the propeptide cleavage at Lys-2 is less efficient since approximately 60% of PC variant molecules present in patient’s plasma retained the entire propeptide. Our findings suggest that depending on the specific amino acid substitution at position-1, PC can be secreted in plasma containing the entire propeptide attached to the light chain. Impaired interaction of elongated APC molecules with a membrane-surface and/or factor Va which is the physiological substrate for APC, is manifested in vivo by thrombophilia.
34
Falzon, Liliana, Smita Patel, Yu-Jen Chen, and Masayori Inouye. "Autotomic Behavior of the Propeptide in Propeptide-mediated Folding of Prosubtilisin E." Journal of Molecular Biology 366, no. 2 (February 2007): 494–503. http://dx.doi.org/10.1016/j.jmb.2006.11.019.
35
Steenstrup, Thomas, Claus Kristensen, and Gert Bolt. "All post-translational modifications except propeptide cleavage are required for optimal secretion of coagulation factor VII." Thrombosis and Haemostasis 98, no. 11 (2007): 988–97. http://dx.doi.org/10.1160/th07-05-0332.
Abstract:
SummaryHuman coagulation factor VII (FVII) has two N-glycosylation sites (N145 and N322) and two O-glycosylation sites (S52 and S60). In transiently transfected COS-7 cells, all combinations of N- and O-glycosylation knock-out mutations reduced the release of FVII to the medium. Pulse-chase analysis of CHO-K1 cell lines expressing recombinant FVII demonstrated that virtually all wild-type FVII synthesized was secreted from the cells, whereas both N- and O- glycosylation knock-out mutations induced partial intracellular degradation of the synthesized FVII. Likewise, two thirds of the FVII synthesized in vitamin K-depleted and warfarin-treated CHO cells was degraded intracellularly, demonstrating the importance of gamma-carboxylation for the secretion of FVII. The furin inhibitor decanoyl-R-V-K-R-chloromethylketone inhibited propeptide cleavage, but FVII with propeptide appeared to be secreted equally well as FVII without propeptide. Propeptide cleavage was not inhibited by vitamin K depletion and warfarin treatment, suggesting that for FVII, correct gamma-carboxylation is not required for optimal processing of the propeptide. In conclusion, all post-translational modifications of FVII except propeptide cleavage were important for complete secretion of the synthesized FVII and to avoid intracellular degradation. Thus, the extensive post-translational modification of FVII seems critical for the intracellular stability of the protein and is required for keeping the protein in the secretory pathway.
36
Jensen, Lars T., Jens O. L. Jørgensen, Juha Risteli, Jens S. Christiansen, and Ib Lorenzen. "Type I and III procollagen propeptides in growth hormone-deficient patients: effects of increasing doses of GH." Acta Endocrinologica 124, no. 3 (March 1991): 278–82. http://dx.doi.org/10.1530/acta.0.1240278.
Abstract:
Abstract. The effect of increasing doses of growth hormone on collagen synthesis in GH-treated GH-deficient patients was determined in a short-term study. The synthesis of type I and III collagen was estimated by measurements of the carboxyterminal propeptide of type I procollagen and the aminoterminal propeptide of type III procollagen. Type I collagen is mainly found in bone and type III collagen in loose connective tissue. We observed a GH dose dependency of both procollagen propeptides. Serum type I procollagen propeptide was significantly higher following GH doses of 4 and 6 IU/day for 14 days compared with 2 IU/day (normal replacement dose) (p=0.04). Withdrawal of GH therapy for 14 days resulted in wider variation, but not significantly different from the levels at 2, 4 and 6 IU/day. A dose dependency was found regarding type III procollagen propeptide, showing significantly higher serum concentrations at a GH dose of 4 IU/day compared with 2 IU/day (p=0.001), and of 6 IU/day compared with 4 IU/day (p=0.001). Withdrawal of GH therapy resulted in significantly lower type III procollagen propeptide concentrations compared with those at a GH dose of 4 and 6 IU/day (p=0.03). Serum type III procollagen propeptide increased twice as much as type I procollagen propeptide, by 47 vs 25%, at a GH dose of 6 IU/day compared with 2 IU/day. The differences between the effects on type I and type III collagen may reflect differences in secretion or turn-over rate of collagen in bone and loose connective tissue. Serum type I and type III procollagen propeptides may prove useful as monitors of GH therapy, especially regarding the GH dose levels in the individual patients.
37
Le Loir, Y., S. Nouaille, J. Commissaire, L. Brétigny, A. Gruss, and P. Langella. "Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis." Applied and Environmental Microbiology 67, no. 9 (September 1, 2001): 4119–27. http://dx.doi.org/10.1128/aem.67.9.4119-4127.2001.
Abstract:
ABSTRACT Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SPNuc) by that of L. lactis protein Usp45 (SPUsp) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SPUsp than when directed by SPNuc. This SPUsp effect on Nuc secretion is not due to a better antifolding activity, since SPUsp:Nuc precursor proteins display enzymatic activity in vitro, while SPNuc:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895–1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SPUsp and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.
38
Gao, Wenwen, Yaqi Xu, Hongli Liu, Meng Gao, Qing Cao, Yiyi Wang, Longteng Cui, et al. "Characterization of missense mutations in the signal peptide and propeptide of FIX in hemophilia B by a cell-based assay." Blood Advances 4, no. 15 (August 7, 2020): 3659–67. http://dx.doi.org/10.1182/bloodadvances.2020002520.
Abstract:
Abstract Many mutations in the signal peptide and propeptide of factor IX (FIX) cause hemophilia B. A FIX variants database reports 28 unique missense mutations in these regions that lead to FIX deficiency, but the underlying mechanism is known only for the mutations on R43 that interfere with propeptide cleavage. It remains unclear how other mutations result in FIX deficiency and why patients carrying the same mutation have different bleeding tendencies. Here, we modify a cell-based reporter assay to characterize the missense mutations in the signal peptide and propeptide of FIX. The results show that the level of secreted conformation-specific reporter (SCSR), which has a functional γ-carboxyglutamate (Gla) domain of FIX, decreases significantly in most mutations. The decreased SCSR level is consistent with FIX deficiency in hemophilia B patients. Moreover, we find that the decrease in the SCSR level is caused by several distinct mechanisms, including interfering with cotranslational translocation into the endoplasmic reticulum, protein secretion, γ-carboxylation of the Gla domain, and cleavage of the signal peptide or propeptide. Importantly, our results also show that the SCSR levels of most signal peptide and propeptide mutations increase with vitamin K concentration, suggesting that the heterogeneity of bleeding tendencies may be related to vitamin K levels in the body. Thus, oral administration of vitamin K may alleviate the severity of bleeding tendencies in patients with missense mutations in the FIX signal peptide and propeptide regions.
39
Niemelä, O., L. Risteli, J. Parkkinen, and J. Risteli. "Purification and characterization of the N-terminal propeptide of human type III procollagen." Biochemical Journal 232, no. 1 (November 15, 1985): 145–50. http://dx.doi.org/10.1042/bj2320145.
Abstract:
The N-terminal propeptide of type III procollagen was purified from human ascitic fluid by using (NH4)2SO4 precipitation, DEAE-Sephacel chromatography at pH 8.6, Sephacryl S-300 chromatography and another DEAE-Sephacel chromatography at pH 4.5. The Mr of the human peptide was about 42 000, which corresponds in size to the propeptide released by the specific N-proteinase during the extracellular processing of collagen. Bacterial-collagenase digestion of the human peptide produced three fragments, which could be separated on a Bio-Gel P-10 column. The human propeptide and its collagenase-derived fragments, an N-terminal non-collagenous domain Col 1, a C-terminal non-helical domain Col 2 and a collagenous domain Col 3, resembled those derived from the N-terminal segment of bovine type III procollagen in their amino acid composition. The human peptide was found to contain sulphate, which may explain its extremely low isoelectric point (3.1). Antibodies against the human N-terminal propeptide reacted similarly with both the purified human peptide and a corresponding segment of bovine type III procollagen. The human propeptide could be used in developing radioimmunoassays for monitoring fibrotic processes.
40
NIRASAWA, Satoru, Yoshiaki NAKAJIMA, Zhen-Zhong ZHANG, Michiteru YOSHIDA, and Kiyoshi HAYASHI. "Intramolecular chaperone and inhibitor activities of a propeptide from a bacterial zinc aminopeptidase." Biochemical Journal 341, no. 1 (June 24, 1999): 25–31. http://dx.doi.org/10.1042/bj3410025.
Abstract:
An aminopeptidase from Aeromonas caviae T-64 was translated as a preproprotein consisting of three domains; a signal peptide (19 amino acid residues), an N-terminal propeptide (101 residues) and a mature region (273 residues). We demonstrated that a proteinase, which was isolated from the culture filtrate of A. caviae T-64, activated the recombinant pro-aminopeptidase by removal of the majority of the propeptide. Using L-Leu-p-nitroanilide as a substrate, the processed aminopeptidase showed a large increase in kcat when compared with the unprocessed enzyme, whereas the Km value remained relatively unchanged. The similar Km values for the pro-aminopeptidase and the mature aminopeptidase indicated that the N-terminal propeptide of the pro-aminopeptidase did not influence the formation of the enzyme-substrate complex, suggesting the absence of marked conformational changes in the active domain. In contrast, the marked difference in kcat suggests a significant decrease in the energy of one or more of the transition states of the enzyme-substrate reaction coordinate. Moreover, we showed that the activity of the urea-denatured pro-aminopeptidase could be recovered by dialysis, whereas the activity of the urea-denatured mature aminopeptidase, which lacked the propeptide, could not. Further to this, the propeptide-deleted aminopeptidase formed an inclusion body in the cytoplasmic space in Escherichia coli and was not secreted at all. These results suggested that the propeptide of the pro-aminopeptidase acted as an intramolecular chaperone that was involved with the correct folding of the enzyme in vitro and was required for extracellular secretion in E. coli.
41
Dai, Zheng, Heting Fu, Yufeng Zhang, Jing Zeng, Bing Tang, and Xiao-Feng Tang. "Insights into the Maturation of Hyperthermophilic Pyrolysin and the Roles of Its N-Terminal Propeptide and Long C-Terminal Extension." Applied and Environmental Microbiology 78, no. 12 (April 13, 2012): 4233–41. http://dx.doi.org/10.1128/aem.00548-12.
Abstract:
ABSTRACTPyrolysin-like proteases from hyperthermophiles are characterized by large insertions and long C-terminal extensions (CTEs). However, little is known about the roles of these extra structural elements or the maturation of these enzymes. Here, the recombinant proform ofPyrococcus furiosuspyrolysin (Pls) and several N- and C-terminal deletion mutants were successfully expressed inEscherichia coli. Pls was converted to mature enzyme (mPls) at high temperatures via autoprocessing of both the N-terminal propeptide and the C-terminal portion of the long CTE, indicating that the long CTE actually consists of the C-terminal propeptide and the C-terminal extension (CTEm), which remains attached to the catalytic domain in the mature enzyme. Although the N-terminal propeptide deletion mutant PlsΔN displayed weak activity, this mutant was highly susceptible to autoproteolysis and/or thermogenic hydrolysis. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of pyrolysin into its thermostable conformation. In contrast, the C-terminal propeptide deletion mutant PlsΔC199 was converted to a mature form (mPlsΔC199), which is the same size as but less stable than mPls, suggesting that the C-terminal propeptide is not essential for folding but is important for pyrolysin hyperthermostability. Characterization of the full-length (mPls) and CTEm deletion (mPlsΔC740) mature forms demonstrated that CTEm not only confers additional stability to the enzyme but also improves its catalytic efficiency for both proteineous and small synthetic peptide substrates. Our results may provide important clues about the roles of propeptides and CTEs in the adaptation of hyperthermophilic proteases to hyperthermal environments.
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O'Neil, Heather S., Brian M. Forster, Kari L. Roberts, Andrew J. Chambers, Alan Pavinski Bitar, and Hélène Marquis. "The Propeptide of the Metalloprotease of Listeria monocytogenes Controls Compartmentalization of the Zymogen during Intracellular Infection." Journal of Bacteriology 191, no. 11 (April 3, 2009): 3594–603. http://dx.doi.org/10.1128/jb.01168-08.
Abstract:
ABSTRACT Integral to the virulence of the intracellular bacterial pathogen Listeria monocytogenes is its metalloprotease (Mpl). Mpl regulates the activity and compartmentalization of the bacterial broad-range phospholipase C (PC-PLC). Mpl is secreted as a proprotein that undergoes intramolecular autocatalysis to release its catalytic domain. In related proteases, the propeptide serves as a folding catalyst and can act either in cis or in trans. Propeptides can also influence protein compartmentalization and intracellular trafficking or decrease folding kinetics. In this study, we aimed to determine the role of the Mpl propeptide by monitoring the behavior of Mpl synthesized in the absence of its propeptide (MplΔpro) and of two Mpl single-site mutants with unstable propeptides: Mpl(H75V) and Mpl(H95L). We observed that all three Mpl mutants mediate PC-PLC activation when bacteria are grown on semisolid medium, but to a lesser extent than wild-type Mpl, indicating that, although not essential, the propeptide enhances the production of active Mpl. However, the mutant proteins were not functional in infected cells, as determined by monitoring PC-PLC maturation and compartmentalization. This defect could not be rescued by providing the propeptide in trans to the mplΔpro mutant. We tested the compartmentalization of Mpl during intracellular infection and observed that the mutant Mpl species were aberrantly secreted in the cytosol of infected cells. These data indicated that the propeptide of Mpl serves to maintain bacterium-associated Mpl and that this localization is essential to the function of Mpl during intracellular infection.
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Gram, Jeppe, Jens Bollerslev, Henning K. Nielsen, and Peter Junker. "Increased serum concentrations of type I procollagen C-terminal propeptide and osteocalcin during a short course of calcitriol administration to adult male volunteers." Acta Endocrinologica 125, no. 6 (December 1991): 609–13. http://dx.doi.org/10.1530/acta.0.1250609.
Abstract:
Abstract. To investigate bone collagen metabolism during vitamin D treatment, 15 healthy males (aged 28-45 years, median 34) were treated orally with calcitriol, 2 μg daily for 7 days and followed for a total of 2 weeks. The serum concentration of calcitriol rose markedly (median difference and 95% confidence limits: 49% (5-82), p<0.005) during treatment, whereas serum levels of calcidiol, and calcium remained unchanged. The serum level of procollagen type I C-terminal propeptide rose 15% (7-33, p<0.003), whereas no alterations were observed concerning serum procollagen type III N-terminal propeptide, and serum hyaluronan. The serum concentration of osteocalcin rose concomitantly (26% (12-45), p<0.003). All values returned to baseline levels within seven days after the treatment week. The serum levels of osteocalcin and procollagen type I C-terminal propeptide were positively correlated (rs=0.71, p<0.004) during the study. Serum procollagen type I C-terminal propeptide and serum osteocalcin did not correlate with serum procollagen type III N-terminal propeptide or serum hyaluronan either at baseline or after treatment. It is concluded that a short course of calcitriol administration to healthy males stimulates the biosynthesis of bone-related matrix proteins. By contrast, connective tissue components of predominantly extraosseous origin are not affected.
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Hinek, A., A. Reiner, and A. R. Poole. "The calcification of cartilage matrix in chondrocyte culture: studies of the C-propeptide of type II collagen (chondrocalcin)." Journal of Cell Biology 104, no. 5 (May 1, 1987): 1435–41. http://dx.doi.org/10.1083/jcb.104.5.1435.
Abstract:
We have shown that when chondrocytes are isolated by collagenase digestion of hyaline cartilage from growth plate, nasal, and epiphyseal cartilages of bovine fetuses they rapidly elaborate an extracellular matrix in culture. Only growth plate chondrocytes can calcify this matrix as ascertained by incorporation of 45Ca2+, detection of mineral with von Kossa's stain and electron microscopy. There is an extremely close direct correlation between 45Ca2+ incorporation in the first 24 h of culture and the content of the C-propeptide of type II collagen, measured by radioimmunoassay, at the time of isolation and during culture. Moreover, growth plate cells have an increased intracellular content of the C-propeptide per deoxyribonucleic acid and, during culture, per hydroxyproline (as a measure of helical collagen) compared with nasal and epiphyseal chondrocytes. In growth plate chondrocytes 24,25-dihydroxycholecalciferol (24,25-[OH]2D3), but not 1,25-dihydroxycholecalciferol alone, stimulates the net synthesis of the C-propeptide and calcification; proteoglycan net synthesis is unaffected. Together, these metabolites of vitamin D further stimulate C-propeptide net synthesis but do not further increase calcification stimulated by 24,25-(OH)2D3. These observations further demonstrate the close correlation between the C-propeptide of type II collagen and the calcification of cartilage matrix.
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Higgins, G. C., J. L. Foster, and A. E. Postlethwaite. "Interleukin 1 beta propeptide is detected intracellularly and extracellularly when human monocytes are stimulated with LPS in vitro." Journal of Experimental Medicine 180, no. 2 (August 1, 1994): 607–14. http://dx.doi.org/10.1084/jem.180.2.607.
Abstract:
Human interleukin 1 beta (IL-1 beta) is synthesized as an inactive precursor that is cleaved by IL-1 converting enzyme (ICE) between Asp116 and Ala117 to form COOH-terminal mature IL-1 beta and NH2-terminal IL-1 beta propeptide. Little is known about the fate of the NH2-terminal cleavage product. In this study, human recombinant (hr)IL-1 beta propeptide (amino acids 2-116) was produced and used to prepare specific antibodies which do not recognize mature human IL-1 beta. These anti-propeptide antibodies were used for immunoprecipitation of biosynthetically labeled proteins from lipopolysaccharide-stimulated human monocytes. Analysis of immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed that these antibodies recognize precursor IL-1 beta and two unique proteins: one migrating at 17.5 kD and one at 14 kD. The larger of these two proteins has a migration nearly identical to that of the recombinant IL-1 beta propeptide, and most likely represents naturally derived propeptide. The protein migrating at 14 kD may result from a second cleavage by ICE, between Asp27 and Gly28. These proteins accumulate intracellularly and extracellularly during pulse-chase experiments, and therefore represent stable products of precursor IL-1 beta cleavage.
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Pendefunda, Arina Alice Ciocan, Cosmin Gabriel Popa, Daniela Anistoroaei, Ionut Luchian, Gabriela Iftene, Silvia Martu, and Norina Consuela Forna. "Quantification of N-Terminal Procollagen I Propeptide in Patients With Occlusal Trauma Induced by Prosthetic Factors." Revista de Chimie 69, no. 12 (January 15, 2019): 3556–658. http://dx.doi.org/10.37358/rc.18.12.6813.
Abstract:
Occlusal trauma can be generated by a number of local factors, including incorrect dental prosthetic systems. Proper therapy can lead to improved tissue homeostasis, with an increase in bone neoformation, whose markers include the N-terminal procollagen I propeptide (PINP). The purpose of the study was to determine serum N-terminal procollagen I propeptide levels in patients with occlusal trauma caused by incorrect prosthetic systems, performed before and after the establishment of perio-prosthetic complex therapy. The study was performed on a group of 56 patients with periodontal disease and prosthetic systems generating nonphysiological, traumatic occlusal forces. These patients underwent the clinical examination, appropriate multidisciplinary therapy, and N-terminal procollagen I propeptide levels were evaluated in the serum at baseline, 6 months, and 12 months after the therapy completion. The results demonstrated significant improvements for periodontal parameters in both post-treatment assessments and an increase in N-terminal procollagen I propeptide, more significant at one year after treatment.
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Osokina, A. V., V. N. Karetnikova, O. M. Polikutina, IU S. Slepynina, O. V. Gruzdeva, T. P. Artemova, S. N. Ryzhenkova, T. B. Pecherina, and O. L. Barbarash. "Procollagen propeptides and visceral obesity index in patients with myocardial infarction with preserved emission faction and various metabolic phenotype." Translational Medicine 7, no. 1 (March 11, 2020): 22–32. http://dx.doi.org/10.18705/2311-4495-2020-7-1-22-32.
Abstract:
Purpose. To identify the peculiarities of the dynamics of fibrosis markers of C-terminal procollagen propeptide type I (PICP) and N-terminal collagen propeptide type III (PIIINP) in patients with ST segment elevation myocardial infarction (STEMI) and preserved myocardial contractility.Material and methods. 86 patients with STEMI and preserved left ventricular ejection fraction were examined. In addition to the standard laboratory and instrumental examinations, all the patients underwent the estimation of the concentrations of C-terminal procollagen propeptide type I (PICP) and N-terminal collagen propeptide type III (PIIINP) by enzyme-linked immunoassay using BCM Diagnostics laboratory kits (USA) on the 1st and the 12th days of disease and 1 year later.Results. The concentration of PICP was significantly reduced in all groups during the entire observation period. Significant correlations were found between PICP and visceral obesity index, age and left ventricular ejection fraction. A relationship was found between PIIINP and an increased visceral obesity index.
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Maître, Blandine, Catherine Angénieux, Virginie Wurtz, Emilie Layre, Martine Gilleron, Anthony Collmann, Sabrina Mariotti, et al. "The assembly of CD1e is controlled by an N-terminal propeptide which is processed in endosomal compartments." Biochemical Journal 419, no. 3 (April 14, 2009): 661–68. http://dx.doi.org/10.1042/bj20082204.
Abstract:
CD1e displays unique features in comparison with other CD1 proteins. CD1e accumulates in Golgi compartments of immature dendritic cells and is transported directly to lysosomes, where it is cleaved into a soluble form. In these latter compartments, CD1e participates in the processing of glycolipid antigens. In the present study, we show that the N-terminal end of the membrane-associated molecule begins at amino acid 20, whereas the soluble molecule consists of amino acids 32–333. Thus immature CD1e includes an N-terminal propeptide which is cleaved in acidic compartments and so is absent from its mature endosomal form. Mutagenesis experiments demonstrated that the propeptide controls the assembly of the CD1e α-chain with β2-microglobulin, whereas propeptide-deleted CD1e molecules are immunologically active. Comparison of CD1e cDNAs from different mammalian species indicates that the CD1e propeptide is conserved during evolution, suggesting that it may also optimize the generation of CD1e molecules in other species.
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Slepkov, Emily R., Alan Pavinski Bitar, and Hélène Marquis. "Differentiation of propeptide residues regulating the compartmentalization, maturation and activity of the broad-range phospholipase C of Listeria monocytogenes." Biochemical Journal 432, no. 3 (November 25, 2010): 557–66. http://dx.doi.org/10.1042/bj20100557.
Abstract:
The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28–Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane–cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1′ (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.
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WOJCIK, Emiel G. C., Marieke VAN DEN BERG, Swibertus R. POORT, and Rogier M. BERTINA. "Modification of the N-terminus of human factor IX by defective propeptide cleavage or acetylation results in a destabilized calcium-induced conformation: effects on phospholipid binding and activation by factor XIa." Biochemical Journal 323, no. 3 (May 1, 1997): 629–36. http://dx.doi.org/10.1042/bj3230629.
Abstract:
The propeptide of human coagulation factor IX (FIX) directs the γ-carboxylation of the first 12 glutamic acid residues of the mature protein into γ-carboxyglutamic acid (Gla) residues. The propeptide is normally removed before secretion of FIX into the blood. However, mutation of Arg-4 in the propeptide abolishes propeptide cleavage and results in circulating profactor IX in the blood. We studied three such genetic variants, factor IX Boxtel (Arg-4 → Trp), factor IX Bendorf (Arg-4 → Leu) and factor IX Seattle C (Arg-4 → Gln). These variant profactor IX molecules bind normally to anti-FIX:Mg(II) antibodies, which indicates that the mutations do not seriously affect γ-carboxylation. Metal ion titration of the binding of variant profactor IX to conformation-specific antibodies demonstrates that the calcium-induced conformation is destabilized in the variant molecules. Also the binding of FIX Boxtel to phospholipids and its activation by factor XIa requires a high (> 5 mM) calcium concentration. The three-dimensional structure of the Gla domain of FIX in the presence of calcium indicates that the acylation of the amino-terminus, rather than the presence of the propeptide, was responsible for the destabilization of the calcium-induced conformation. In order to confirm this, the α-amino group of Tyr1 of FIX was acetylated. This chemically modified FIX showed a similar destabilization of the calcium-induced conformation to variant profactor IX. Our data imply that the amino-terminus of FIX plays an important role in stabilizing the calcium-induced conformation of the Gla domain of FIX. This conformation is important for the binding to phospholipids as well as for the activation by factor XIa. Our results indicate that mutations in FIX that interfere with propeptide cleavage affect the function of the protein mainly by destabilizing the calcium-induced conformation.