Biomedical subjects

P Chevaillier

Publications and source records attributed to P Chevaillier.

At least 19 recordsLinked to original sources

PubMed1999

SR protein-specific kinase 1 is highly expressed in testis and phosphorylates protamine 1.

Arginine/serine protein kinases constitute a novel class of enzymes that can modify arginine/serine (RS) dipeptide motifs. SR splicing factors that are essential for pre-mRNA splicing are among the best characterized proteins that contain RS domains. TwoSRprotein-specifickinases, SRPK1 and SRPK2, have been considered as highly specific for the phosphorylation of these proteins, thereby contributing to splicing regulation. However, despite the fact that SR proteins are more or less conserved among metazoa and have a rather ubiquitous tissue distribution we now demonstrate that SRPK1 is predominantly expressed in testis. In situ expression analysis on transverse sections of adult mouse testis shows that SRPK1 mRNA is abundant in all germinal cells but not in mature spermatozoa. RS kinase activity was found primarily in the cytosol and only minimal activity was detected in the nucleus. In a search for testis-specific substrates of SRPK1 we found that the enzyme phosphorylates human protamine 1 as well as a cytoplasmic pool of SR proteins present in the testis. Protamine 1 belongs to a family of small basic arginine-rich proteins that replace histones during the development of mature spermatozoa. The result of this progressive replacement is the formation of a highly compact chromatin structure devoid of any transcriptional activity. These findings indicate that SRPK1 may have a role not only in pre-mRNA splicing, but also in the condensation of sperm chromatin.

Amino Acid Sequence↗

PubMed1999

Basic homopolyamino acids, histones and protamines are potent antagonists of angiogenin binding to ribonuclease inhibitor.

A radio-ribonuclease inhibitor assay based on the interaction of 125I-angiogenin with ribonuclease inhibitor (RI) was used to detect pancreatic-type ribonucleases and potential modulators of their action. We show that highly basic proteins including the homopolypeptides poly-arginine, poly-lysine and poly-ornithine, core histones, spermatid-specific S1 protein and the protamines HP3 and Z3 were strong inhibitors of angiogenin binding to RI. A minimum size of poly-arginine and poly-lysine was required for efficient inhibition. The inhibition likely resulted from direct association of the basic proteins with the acidic inhibitor, as RI bound to poly-lysine and protamines while 125I-angiogenin did not. Antagonists of the angiogenin-RI interaction are potential regulators of either angiogenin-triggered angiogenesis and/or intracellular RI function, depending on their preferential target.

Amino Acids↗

PubMed1998

The amino acid sequence of the ram spermatidal protein 3--a transition protein TP3 or TP4?

As in other mammals, several nuclear basic proteins replace histones during the differentiation of germinal cells into spermatozoa in the ram. These proteins called transition proteins (TP) are later replaced by protamines. The amino acid sequence of the ram spermatidal protein 3 has been established by Edman degradation of the protein and of its fragments generated from digestion with endoproteinase Lys-C and pepsin and from the coding sequence of the gene and of the cDNA. The ram protein 3 is a basic protein of 109 residues (calculated Mr 13,200) with arginine and lysine residues uniformly distributed along the polypeptide chain. Of the 13 serine and threonine residues, 9 are located in structural motifs where they could be phosphorylated and, thus, modulate the binding of the protein to DNA. The tyrosine residues at position 33 and position 93, located in a basic environment, and the tryptophan residue at position 29 could be involved in the interactions of the protein with DNA through the stacking of their aromatic ring between the nucleotide bases. The ram protein 3 differs completely from the two well-defined transition protein families TP1 and TP2, which are also synthesised transiently during mammal spermiogenesis. In contrast with the rat TP3, ram protein 3 does not correspond to a precursor of a protamine. However, it shares structural similarities with both transition proteins TP3 and TP4 of the boar. The ram protein 3 and the boar transition proteins TP3 and TP4 probably belong to the same transition-protein group and would play similar functions in the chromatin remodelling during spermiogenesis. As protamine and transition-protein TP1 and TP2 genes from mammals, the coding sequence of the gene of ram protein 3 is interrupted by one intron but its organisation is different.

Amino Acid Sequence↗

PubMed1998

Nuclear basic proteins in spermiogenesis.

In animal species, spermiogenesis, the late stage of spermatogenesis, is characterized by a dramatic remodelling of chromatin which involves morphological changes and various modifications in the nature of the nuclear basic proteins. According to the evolution of species, three situations can be observed: a) persistence of somatic histones or appearance of sperm-specific histones; b) direct replacement of histones by generally smaller and more basic proteins called protamines; and c) occurrence of a double nuclear basic protein transition: histones are not directly replaced by protamines but by intermediate basic proteins which are themselves replaced by one or several protamines. However, in some species, two kinds of intermediate basic proteins can be distinguished in spermatid nuclei: transition proteins and protamine precursors. Whereas transition proteins are not structurally related either to histones or to protamines, protamine precursors are further processed at the end of spermiogenesis to give rise to the mature protamine. The molecular characteristics of the protamines as well as number of protamine types present in the spermatozoon vary from species to species. In some cases, protamine-encoding genes, although present, are not expressed to a significant level. The diversity and the precise function of intermediate basic proteins remain open to discussion. Some of them are the precursors of protamines but the mechanism, sequential or not, as well as the enzyme(s) involved in the proteolytic processing, remain to be discovered.

Amino Acid Sequence↗

PubMed1995

Persistence of protamine precursors in mature sperm nuclei of the mouse.

During mouse spermiogenesis, two protamines, mP1 and mP2, are synthesized in replacement of histones. One of them (protamine mP2, 63 residues) appears at first in elongating spermatid nuclei as a protamine of 106 residues (pmP2) with an amino-terminal extension that is progressively excised. The two protamines were previously described as the only proteins associated with DNA in sperm chromatin. This paper shows that the nuclear proteins of mouse spermatozoa are indeed heterogeneous: at least six minor polypeptides in addition to protamines can be identified. The primary structure of four of them has been established. They are intermediate in the maturation of the precursor of protamine mP2 and correspond to polypeptides pmP2/11, pmP2/16, pmP2/20, and pmP2/32, characterized previously in mouse testis. Therefore, these intermediates of proteolysis generated from pmP2 inside spermatid nuclei persist in mature sperm, whereas the largest precursors, pmP2 and pmP2/5, disappear. These findings clearly indicate that limited proteolysis events still occur outside of the testis.

Amino Acid Sequence↗

PubMed1993

Phosphorylation of human sperm protamines HP1 and HP2: identification of phosphorylation sites.

Human sperm is characterized by a high heterogeneity of its basic nuclear protein complement of pro-protamines, protamines and histones. This heterogeneity is increased by the persistence of phosphorylated protamines in mature spermatozoa. Alkaline phosphatase treatment of whole protein indicated that protamines HP1 and HP2 were phosphorylated to various degrees. Presence of non-phosphorylated and phosphorylated protamines HP1 and HP2 was further demonstrated by electrospray mass spectrometry. Phosphorylation sites of mono- and di-phosphorylated protamine HP1 were identified by automatic Edman degradation of the protein after phosphoserine derivatization to S-ethylcysteine. In both phosphorylated forms, Ser-10 was found phosphorylated; in the di-phosphorylated form, Ser-8 was identified as the second site of phosphorylation. In protamine HP2, the unique site of phosphorylation (Ser-14) was located after limited acid hydrolysis of enzymic peptides and thin-layer electrophoresis.

Alkaline Phosphatase↗

PubMed1993

Amino acid sequence of the human intermediate basic protein 2 (HPI2) from sperm nuclei. Structural relationship with protamine P2.

Human intermediate basic protein 2 (HPI2) is a low-molecular-mass basic protein present in small amounts in human sperm nuclei. The amino acid composition of the protein, its N-terminal amino acid sequence and peptide maps obtained after digestion with endoproteinases Lys-C and Glu-C, reveal that HPI2 is structurally related to human protamine species P2 (HP2), which is rich in Arg, His and Cys residues. Compared to HP2, which is one of the two major sperm protamines, HPI2 has an N-terminal extension of 24 residues which includes six acidic residues and does not possess any Arg residues. The amino acid sequence of HPI2 (81 residues) is identical to the sequence of the C-terminal region of another minor sperm nuclear protein, human intermediate basic protein 1 (HPI1, 101 residues), which was sequenced previously [Martinage, A., Arkhis, A., Alimi, E., Sautière, P. & Chevaillier, P. (1990) Eur. J. Biochem. 191, 449-451]. Due to this structural similarity, HPI2 must be considered as an intermediate in the maturation of proprotamine HPI1 limited proteolysis.

Amino Acid Sequence↗

PubMed1993

A corrected primary structure for dog-fish Scylliorhinus caniculus protamine Z3.

We have redetermined the primary structure for dog-fish protamine using automated amino-acid sequencing associated to mass spectrometry techniques and report, on the basis of these findings, that the previously published amino-acid sequence is incorrect. The correct protamine sequence is 37 amino acids long and differs from the original published sequence by the C-terminal hexapeptide Arg32-Gly-Arg-Arg-Ser-Arg37.

Amino Acid Sequence↗

PubMed1993

Production, characterization, and immunocytochemical applications of monoclonal antibodies to human sperm protamines.

Three monoclonal antibodies against human protamines were obtained by immunization with total human basic nuclear proteins or purified protamine HP3. The specificity of antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. They recognized three distinct epitopes: One was specific for the protamine P1 family, another was specific for the protamine P2 family and the third was common to both families. All were specific for the human species. Antibodies were used to detect protamines in germ cells by indirect immunofluorescence and by immunoelectron microscopy. Protamines appeared in spermtid nuclei at steps 4-5 of spermiogenesis, i.e., during the chromatin condensation process, and were not accumulated in the cytoplasm before entering the nucleus.

Animals↗

PubMed1993

Pest sequences in nuclear proteins.

1. Most of proteins which are rapidly degraded inside eukaryotic cells have been found to contain amino acid sequences (PEST sequences) enriched in proline, acidic residues (glutamic acid and/or aspartic acid) and hydrophilic residues (serine and threonine) (Rogers et al. (1986) Science 234, 364-368). 2. This correlation was tested on nuclear proteins and a close relationship was found between nuclear protein stability and the presence of PEST regions. 3. Nuclear proteins with structural functions which can be considered as stable components of cell nuclei generally lack PEST sequences. 4. In contrast, regulatory nuclear factors which have specific and transient functions generally possess at least one PEST sequence.

Amino Acid Sequence↗

PubMed1993

Electroblotting proteins onto carboxymethylcellulose membranes for sequencing.

Proteins and peptides separated by polyacrylamide gel electrophoresis either in acetic acid-urea or in sodium dodecyl sulfate (SDS)-containing gels were electroblotted onto carboxymethylcellulose membrane and eluted from the membrane by dilute acid. Polypeptides thus recovered with a high yield can be used to get structural or biological data.

Amino Acid Sequence↗

PubMed1992

Molecular characterization of six intermediate proteins in the processing of mouse protamine P2 precursor.

In mouse spermatozoa, DNA is compacted by two protamines mP1 and mP2. Protamine mP2 (63 residues) is synthesized in spermatid nuclei as a precursor pmP2 (106 residues) which is subsequently processed at the end of spermiogenesis [Yelick, P.C., Balhorn, R., Johnson, P.A., Corzett, M., Mazrimas, J.A., Kleene, K.C. & Hecht, N.B. (1987) Mol. Cell. Biol. 7, 2173-2179]. Six proteins, three of which were described earlier [Chauvière, M., Martinage, A., Debarle, M., Alimi, E., Sautière, P. & Chevaillier, Ph. (1991) C.R. Acad. Sci. 313, 107-112], have molecular and electrophoretic properties similar to those of pmP2. They were isolated from purified testis nuclei and characterized by amino acid composition, N-terminal sequence and peptide mapping. From the amino acid compositions, it appears that all six proteins are rich in arginine, cysteine and histidine and are closely related to pmP2 and mP2. The N-terminal sequence of each protein overlaps a distinct region of the N-terminal part of pmP2. The C-terminal part of protamine mP2 starting at arginine 15 is common to all proteins as assessed by amino acid compositions and peptide maps. All these structural data demonstrate that the six isolated proteins are products of pmP2 precursor processing. The six intermediate proteins pmP2/5, pmP2/11, pmP2/16, pmP2/20, pmP2/26 and pmP2/32 which contain 102, 96, 91, 87, 81 and 75 residues, respectively, are generated from the pmP2 precursor after N-terminal excision of 4, 10, 15, 19, 25 and 31 residues, respectively. The C-terminal sequence of protamine mP2 is strictly identical to that of its precursor; therefore, no maturation occurs in this part of the molecule. At the present time, the proteolytic pathway involved in the amino-terminal processing leading to the mature form of the protamine mP2 (63 residues) has not been elucidated. However, the different representation of six intermediates in the testis suggests that some stages of processing are faster than others or that some cleavage sites are preferred. The proteins described in this paper could result either from stepwise excision of N-terminal residues or from non-sequential cleavages.

Amino Acid Sequence↗

PubMed1991

Molecular structure of human protamine P4 (HP4), a minor basic protein of human sperm nuclei.

Protamine HP4 is a minor protein which was purified from human sperm nuclei. It was characterized by its amino acid composition, peptide mapping after digestion with highly specific endoproteinases and finally by its amino acid sequence. Protamine HP4 contains high amounts of arginine, cysteine and histidine. The primary structure of the protein was established by sequence analysis of intact protamine and of its fragments. HP4 is a P2-type protamine of 58 residues (Mr 7783) structurally related to human protamines HP2 and HP3 from which it only differs by an amino-terminal extension of one and four residues, respectively. These three protamines exhibit a close structural relationship with mouse protamine mP2. The heterogeneity of protamines in human sperm nuclei is discussed.

Amino Acid Sequence↗

PubMed1991

[Purification and characterization of precursors of mouse protamine mP2].

Mouse protamine 2 (62 residues) is synthesized as a precursor pmP2 (106 residues) which is subsequently processed during the end of spermiogenesis. Three proteins with molecular and electrophoretic properties similar to those of pmP2 were isolated from testis purified nuclei. The comparison of their amino acid composition and of their N-terminal sequence with those of pmP2 shows that the three isolated proteins are products of pmP2 precursor processing. The three intermediate proteins pmP2/5, pmP2/11 and pmP2/20 which contain 102, 96 and 87 residues respectively, are generated from pmP2 precursors after N-terminal excision of 4, 10 and 19 residues, respectively.

Amino Acid Sequence↗

PubMed1990

Molecular characterization of nuclear basic protein HPI1, a putative precursor of human sperm protamines HP2 and HP3.

The largest intermediate basic protein HPI1 (101 residues) from human sperm chromatin was isolated and characterized. The amino acid composition and sequence analysis of the protein and of tryptic peptides together with peptide mapping of endoproteinases Lys-C and Glu-C hydrolysates showed that the C-terminal region (residues 45-101) of HPI1 is identical to protamine HP2. These structural data strongly suggest that protein HPI1 is a precursor of human sperm protamines HP2 and HP3 (57 and 54 residues, respectively) as well as of two other intermediate basic proteins HPS1 and HPS2 (69 and 66 residues, respectively) sequenced previously.

Amino Acid Sequence↗

PubMed1990

Chromosomal localization of the human protamine genes, PRM1 and PRM2, to 16p13.3 by in situ hybridization.

Protamines are sperm-specific proteins that replace histones in the nuclear chromatin of mature spermatozoa. A chromosomal localization of the genes coding for human protamines has been achieved by in situ hybridization. Two cDNA probes of 423 bp and 397 bp containing the entire coding sequence for human protamine 1 (HP1) and human protamine 2 (HP2), respectively, have been used. The genes, called PRM1 and PRM2, have been found, tightly linked, on band 16p13.3. Arguments are given for the existence of these two genes as single copies, PRM1 coding for the unique HP1 protamine and PRM2 coding for a precursor of several proteins belonging to the HP2 family.

Chromosome Banding↗

PubMed1990

[Protamine precursors in human spermatozoa].

Basic proteins isolated from human sperm nuclei are highly heterogeneous. Three groups of nuclear basic proteins have been characterized: somatic-type as well as testis-specific histones, protamines and basic proteins with an electrophoretic mobility which is intermediate between that of histones and that of protamines. Human protamines can be separated into 2 protein families with different amino acid composition and amino-acid sequence. Protamines HP1 differ in their degree of phosphorylation. Protamines HP2, 3 and 4 differ by their amino-terminal sequence. Intermediate basic proteins (HPI1, HPI2, HPS1, HPS2) share a common C-terminal sequence of 54 residues identical to the amino-acid sequence of protamine HP3; only their N-terminal regions are different. Taking into account these structural homologies, the intermediate basic protein HPI1 appears as a precursor of protamines HP2 and HP3.

Amino Acid Sequence↗

PubMed1990

[Basic nuclear proteins of transition during spermiogenesis in Scylliorhinus caniculus].

During dog-fish spermiogenesis, 2 basic nuclear protein transitions occur: the first from histones to spermatid-specific proteins S1 and S2, the second leading to protamines. S1, the most abundant transition protein, is a polypeptide containing 87 residues (Mr = 11,179 Da) whereas S2, the minor transition protein, contains 80 residues (Mr = 9,726 Da). The 2 proteins are mainly characterized by an asymmetry of the molecule, a very high content of basic residues, a relatively high level of hydrophobic residues and a cluster of acidic residues in the carboxy-terminal quarter of the molecule. The 2 proteins are phosphorylated on serine residues and the degree of phosphorylation is relatively important in protein S1. The 2 transition proteins are structurally unrelated to testis histones or sperm protamines and cannot be considered either as their proteolytic degradation products or as their precursors.

Amino Acid Sequence↗