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Jason E Derouchey - One of the best experts on this subject based on the ideXlab platform.
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entropy based analysis of vertebrate sperm Protamines sequences evidence of potential dityrosine and cysteine tyrosine cross linking in sperm Protamines
BMC Genomics, 2020Co-Authors: Christian D Powell, Jason E Derouchey, Daniel C Kirchoff, Hunter N B MoseleyAbstract:Spermatogenesis is the process by which germ cells develop into spermatozoa in the testis. Sperm Protamines are small, arginine-rich nuclear proteins which replace somatic histones during spermatogenesis, allowing a hypercondensed DNA state that leads to a smaller nucleus and facilitating sperm head formation. In eutherian mammals, the protamine-DNA complex is achieved through a combination of intra- and intermolecular cysteine cross-linking and possibly histidine-cysteine zinc ion binding. Most metatherian sperm Protamines lack cysteine but perform the same function. This lack of dicysteine cross-linking has made the mechanism behind metatherian Protamines folding unclear. Protamine sequences from UniProt’s databases were pulled down and sorted into homologous groups. Multiple sequence alignments were then generated and a gap weighted relative entropy score calculated for each position. For the eutherian alignments, the cysteine containing positions were the most highly conserved. For the metatherian alignment, the tyrosine containing positions were the most highly conserved and corresponded to the cysteine positions in the eutherian alignment. High conservation indicates likely functionally/structurally important residues at these positions in the metatherian Protamines and the correspondence with cysteine positions within the eutherian alignment implies a similarity in function. One possible explanation is that the metatherian protamine structure relies upon dityrosine cross-linking between these highly conserved tyrosines. Also, the human protamine P1 sequence has a tyrosine substitution in a position expecting eutherian dicysteine cross-linking. Similarly, some members of the metatherian Planigales genus contain cysteine substitutions in positions expecting plausible metatherian dityrosine cross-linking. Rare cysteine-tyrosine cross-linking could explain both observations.
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entropy based analysis of vertebrate sperm protamine sequences evidence of dityrosine and cysteine tyrosine cross linking in sperm Protamines
bioRxiv, 2019Co-Authors: Christian D Powell, Jason E Derouchey, Daniel C Kirchoff, Hunter N B MoseleyAbstract:Background: Spermatogenesis is the process by which germ cells develop into spermatozoa in the testis. Sperm Protamines are small, arginine-rich nuclear proteins which replace somatic histones during spermatogenesis, allowing a hypercondensed DNA state that leads to a smaller nucleus and facilitating sperm head formation. In eutherian mammals, the protamine-DNA complex is achieved through a combination of intra- and intermolecular cysteine cross-linking and possibly histidine-cysteine zinc ion binding. Most metatherian sperm Protamines lack cysteine but perform the same function. This lack of dicysteine cross-linking has made the mechanism behind metatherian Protamines folding unclear. Results: Protamine sequences from UniProt9s databases were pulled down and sorted into homologous groups. Multiple sequence alignments were then generated and a gap weighted relative entropy score calculated for each position. For the eutherian alignments, the cysteine containing positions were the most highly conserved. For the metatherian alignment, the tyrosine containing positions were the most highly conserved and corresponded to the cysteine positions in the eutherian alignment. Conclusions: High conservation indicates likely functionally/structurally important residues at these positions in the metatherian Protamines and the correspondence with cysteine positions within the eutherian alignment implies a similarity in function. One explanation is that the metatherian protamine structure relies upon dityrosine cross-linking between these highly conserved tyrosines. Also, the human protamine P1 sequence has a tyrosine substitution in a position expecting eutherian dicysteine cross-linking. Similarly, some members of the metatherian Planigales genus contain cysteine substitutions in positions expecting plausible metatherian dityrosine cross-linking. Rare cysteine-tyrosine cross-linking could explain both observations.
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Role of Disulfide Bonds on DNA Packaging Forces in Bull Sperm Chromatin
Biophysical journal, 2017Co-Authors: Jason E Derouchey, Donald C Rau, James M. HutchisonAbstract:Abstract Short arginine-rich proteins called Protamines mediate the near crystalline DNA packaging in most vertebrate sperm cells. Protamines are synthesized during spermiogenesis and condense the paternal genome into a transcriptionally inactive state in late-stage spermatids. Protamines from eutherian mammals, including bulls and humans, also contain multiple cysteine residues that form intra- and interprotamine sulfur-sulfur bonds during the final stages of sperm maturation. Although the cross-linked protamine network is known to stabilize the resulting nucleoprotamine structure, little is known about the role of disulfide bonds on DNA condensation in the mammalian sperm. Using small angle x-ray scattering, we show that isolated bull nuclei achieve slightly lower DNA packing densities compared to salmon nuclei despite salmon protamine lacking cysteine residues. Surprisingly, reduction of the intermolecular sulfur-sulfur bonds of bull protamine results in tighter DNA packing. Complete reduction of the intraprotamine disulfide bonds ultimately leads to decondensation, suggesting that disulfide-mediated secondary structure is also critical for proper protamine function. Lastly, comparison of multiple bull collections showed some to have aberrant x-ray scattering profiles consistent with incorrect disulfide bond formation. Together, these observations shed light on the biological functions of disulfide linkages for in vivo DNA packaging in sperm chromatin.
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role of amino acid insertions on intermolecular forces between arginine peptide condensed dna helices implications for protamine dna packaging in sperm
Biophysical Journal, 2012Co-Authors: Jason E Derouchey, Donald C RauAbstract:In spermatogenesis, chromatin histones are replaced by arginine-rich Protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. We have previously observed that the net attraction between salmon protamine condensed DNA helices was much smaller for DNA condensed by the equivalent homo-arginine peptide. We hypothesized that this is caused by the neutral amino acids present in Protamines. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. The component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance have been determined by the osmotic stress technique coupled with x-ray scattering as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short-range repulsion; while only slightly decreasing the longer-ranged attraction. The decrease in net attraction between salmon protamine condensed helices compared with arginine homopeptides can be well explained by amino acid content alone. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both these observation have biological implications for protamine-DNA packaging in sperm heads.
Pierre Sautière - One of the best experts on this subject based on the ideXlab platform.
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DNA-interacting proteins in the spermiogenesis of the mollusc Murex brandaris.
The Journal of biological chemistry, 1999Co-Authors: Carme Càceres, D. Wouters-tyrou, Arlette Martinage, Pierre Sautière, Enric Ribes, Sylviane Muller, Pepita Gimenez-bonafé, Mostafa Kouach, Jaume Palau, Juan A. SubiranaAbstract:Sperm chromatin of Murex brandaris (a neogastropod mollusc) undergoes a series of structural transitions during spermiogenesis. The DNA-interacting proteins responsible for these changes as well as the mature Protamines present in the ripe sperm nucleus have been characterized. The results reveal that spermiogenic nuclear proteins are protamine precursors that are subjected to a substantial number of small N-terminal deletions that gradually modify their overall charge. The composition of mature Protamines is remarkably simple in turn, promoting an efficient and extremely tight packaging of DNA. The pattern of spermiogenic chromatin condensation in M. brandaris clearly departs from that corresponding to vertebrate chromatin.
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NUCLEAR BASIC PROTEINS IN SPERMIOGENESIS
Biochimie, 1998Co-Authors: D. Wouters-tyrou, Arlette Martinage, Philippe Chevaillier, Pierre SautièreAbstract:Abstract 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 prutamine 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.
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phosphorylation of human sperm Protamines hp1 and hp2 identification of phosphorylation sites
Biochimica et Biophysica Acta, 1993Co-Authors: Frederic Chira, Arlette Martinage, Philippe Chevaillier, Ahmed Arkhis, Michel Jaquinod, Pierre SautièreAbstract: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.
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p2 Protamines from human sperm are zinc finger proteins with one cys2 his2 motif
Biochemical and Biophysical Research Communications, 1992Co-Authors: Fabien Bianchi, Pierre Sautière, Roselyne Rousseauxprevost, Jean RousseauxAbstract:P1 (HP1) and P2 (HP2, HP3, HP4) Protamines were isolated from human sperm nuclei in the reduced form and their interaction with zinc and cobalt was studied. One zinc atom per molecule of P2 Protamines but not of P1 protamine was found. Absorption spectra of P2 Protamines with cobalt were characteristic of a tetrahedral complex involving two histidine and two cysteine residues and with one cobalt per molecule. A tetrahedral complex was found neither in P1 Protamines nor in P2 Protamines alkylated at cysteine or at histidine residues. The zinc finger motif Cys2/His2 of P2 Protamines may play a role in stabilization of human sperm chromatin and in inhibition of transcription.
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the immune response to synthetic peptides of human Protamines hp1 and hp2
Molecular Immunology, 1991Co-Authors: Roselyne Rousseauxprevost, Pierre Sautière, Patrick Hublau, Jean RousseauxAbstract:Peptides representing the amino-terminal sequence of Protamines HP1 (sequence 1-12) and HP2 (sequence 1-11), the two major nuclear proteins of human sperm, have been synthesized. Rabbits were immunized either with peptide conjugated with a carrier or with free peptide. The resulting antisera were examined for their capacities to bind the homologous peptide, other peptides from Protamines HP1, HP2, from ram protamine, a protein resembling HP1, and finally with the whole protamine. Only free peptides were immunogenic. Antisera were found to react with the homologous peptide, but also with some other peptides. More especially, antibodies to peptide HP1 1-12 were found to recognize an epitope shared by the homologous peptide, peptide HP1 37-49 and peptide 1-12 of ram protamine. The common antigenic determinant seems to depend on the conformation of the peptides, rather than strictly related to common sequences. Anti-peptide antibodies react poorly and in a non-specific manner with the parent protein. The failure of reactivity with the Protamines strongly suggest that these small basic proteins are folded and probably globular molecules in contrast with the totally random model postulated by several previous works.
Pekka H Maenpaa - One of the best experts on this subject based on the ideXlab platform.
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In Vitro Phosphorylation Sites of Stallion and Bull P1-Protamines for Cyclic Adenosine 3',5'-Monophosphate-Dependent Protein Kinase and Protein Kinase C'
2016Co-Authors: Arja Pirhonen, Pirjo Valtonen, A. Linnala-kankkunen, Pekka H MaenpaaAbstract:Fish and mammalian Protamines are phosphorylated after their synthesis during sperm cell maturation. Cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), both requiring basic amino acids at their recognition sites, have previously been found to phosphorylate fish Protamines in vitro. In this study, these enzymes were used to phosphorylate stallion and bull sperm Pl-Protamines in vitro. A species-specific difference was found, since PKA was able to phosphorylate both Protamines while PKC phosphorylated only stallion protamine. Thr-41, the only threonine residue in stallion Pl-protamine, and most probably the homologous Thr-43 in bull Pl-protamine are the sites for PKA phosphorylation in addition to an internally located Ser-29 present only in stallion protamine. This Ser residue was phosphorylated in vitro by both kinases. Protamine phosphorylation by PKA was found to be almost independent of cAMP and was inhibited only by a tenfold concentration of PKI when compared to phosphorylation of a model peptide, kemptide. Addition of calcium, phosphatidylserine, and diolein caused a twofold stimulation in phosphorylation of stallion protamine by PKC, indicating that specific cofactors of PKC may have a role in mammalian prot-amine phosphorylation. We suggest that PKA is a good universal candidate for in vivo phosphorylation of Pl-Protamines
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P2 Protamines are phosphorylated in vitro by protein kinase C, whereas P1 Protamines prefer cAMP-dependent protein kinase. A comparative study of five mammalian species.
European journal of biochemistry, 1994Co-Authors: Arja Pirhonen, Annikka Linnala-kankkunen, Pekka H MaenpaaAbstract:P1 Protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 Protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). All P1 Protamines were phosphorylated by PKA, whereas P2 Protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 Protamines were phosphorylated by PKC. After phosphoamino acid analysis, the Protamines showing positive signals for phosphoserine (P-Ser) were subjected to P-Ser conversion reaction and protein sequencing. Only stallion (St1) and human (HP1) P1 Protamines contained P-Ser after PKA phosphorylation, located in the middle region of the molecule, i.e., at Ser29 in St1 and Ser28 in HP1. All other phosphorylated P1 Protamines contained only P-Thr, which could not be further localized in the sequence with the present methods. After PKC phosphorylation, the internally located Ser residues in human (ser21) and stallion (Ser29) P1 Protamines were phosphorylated and, in boar P1 protamine, only Thr43 was slightly phosphorylated. The N-terminally located Ser residues in P1 Protamines, which are known to be phosphorylated in vivo, were not phosphorylated by either kinase, indicating that there must still be other types of protamine kinases in sperm cells responsible for their phosphorylation. Within P2 Protamines, HP2 was equally well phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas, in St2, Ser32 was the main target for PKC phosphorylation in vitro. Collectively, PKC is a good candidate for in vivo phosphorylation of P2 Protamines and PKA for phosphorylation of some hydroxyamino acid residues in P1 Protamines.
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identification of phosphoseryl residues in Protamines from mature mammalian spermatozoa
Biology of Reproduction, 1994Co-Authors: Arja Pirhonen, Annikka Linnalakankkunen, Pekka H MaenpaaAbstract:Protamines isolated from ejaculated human, stallion, bull, boar, and ram spermatozoa were subjected to phosphoserine conversion reaction and protein sequencing. Phosphoserines were detected as S-ethylcysteines. Endogenously phosphorylated Protamines have previously been found only in ejaculated human sperm. In this study, we demonstrate that ejaculated sperm from other species also contain Protamines phosphorylated at serine residues. In P1-Protamines, the endogenously phosphorylated serines were located at the N-terminal region in all species studied, whereas in major forms of human and stallion P2-Protamines, the serine residues located in the middle region of the molecule were predominantly phosphorylated. These results support the current DNA binding model in the case of the P1-Protamines. The internal location of the phosphorylated serines in the P2-Protamines indicates, however, that the binding of these proteins to DNA or their interaction with other protamine molecules may differ from that of P1-Protamines. This also suggests that, during sperm maturation, P2-Protamines may have a role different from that of P1-Protamines.
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in vitro phosphorylation sites of stallion and bull p1 Protamines for cyclic adenosine 3 5 monophosphate dependent protein kinase and protein kinase c
Biology of Reproduction, 1993Co-Authors: Arja Pirhonen, Pirjo Valtonen, Annikka Linnalakankkunen, Pekka H MaenpaaAbstract:Fish and mammalian Protamines are phosphorylated after their synthesis during sperm cell maturation. Cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), both requiring basic amino acids at their recognition sites, have previously been found to phosphorylate fish Protamines in vitro. In this study, these enzymes were used to phosphorylate stallion and bull sperm P1-Protamines in vitro. A species-specific difference was found, since PKA was able to phosphorylate both Protamines while PKC phosphorylated only stallion protamine. Thr-41, the only threonine residue in stallion P1-protamine, and most probably the homologous Thr-43 in bull P1-protamine are the sites for PKA phosphorylation in addition to an internally located Ser-29 present only in stallion protamine. This Ser residue was phosphorylated in vitro by both kinases. Protamine phosphorylation by PKA was found to be almost independent of cAMP and was inhibited only by a tenfold concentration of PKI when compared to phosphorylation of a model peptide, kemptide. Addition of calcium, phosphatidylserine, and diolein caused a twofold stimulation in phosphorylation of stallion protamine by PKC, indicating that specific cofactors of PKC may have a role in mammalian protamine phosphorylation. We suggest that PKA is a good universal candidate for in vivo phosphorylation of P1-Protamines.
Arja Pirhonen - One of the best experts on this subject based on the ideXlab platform.
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In Vitro Phosphorylation Sites of Stallion and Bull P1-Protamines for Cyclic Adenosine 3',5'-Monophosphate-Dependent Protein Kinase and Protein Kinase C'
2016Co-Authors: Arja Pirhonen, Pirjo Valtonen, A. Linnala-kankkunen, Pekka H MaenpaaAbstract:Fish and mammalian Protamines are phosphorylated after their synthesis during sperm cell maturation. Cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), both requiring basic amino acids at their recognition sites, have previously been found to phosphorylate fish Protamines in vitro. In this study, these enzymes were used to phosphorylate stallion and bull sperm Pl-Protamines in vitro. A species-specific difference was found, since PKA was able to phosphorylate both Protamines while PKC phosphorylated only stallion protamine. Thr-41, the only threonine residue in stallion Pl-protamine, and most probably the homologous Thr-43 in bull Pl-protamine are the sites for PKA phosphorylation in addition to an internally located Ser-29 present only in stallion protamine. This Ser residue was phosphorylated in vitro by both kinases. Protamine phosphorylation by PKA was found to be almost independent of cAMP and was inhibited only by a tenfold concentration of PKI when compared to phosphorylation of a model peptide, kemptide. Addition of calcium, phosphatidylserine, and diolein caused a twofold stimulation in phosphorylation of stallion protamine by PKC, indicating that specific cofactors of PKC may have a role in mammalian prot-amine phosphorylation. We suggest that PKA is a good universal candidate for in vivo phosphorylation of Pl-Protamines
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P2 Protamines are phosphorylated in vitro by protein kinase C, whereas P1 Protamines prefer cAMP-dependent protein kinase. A comparative study of five mammalian species.
European journal of biochemistry, 1994Co-Authors: Arja Pirhonen, Annikka Linnala-kankkunen, Pekka H MaenpaaAbstract:P1 Protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 Protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). All P1 Protamines were phosphorylated by PKA, whereas P2 Protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 Protamines were phosphorylated by PKC. After phosphoamino acid analysis, the Protamines showing positive signals for phosphoserine (P-Ser) were subjected to P-Ser conversion reaction and protein sequencing. Only stallion (St1) and human (HP1) P1 Protamines contained P-Ser after PKA phosphorylation, located in the middle region of the molecule, i.e., at Ser29 in St1 and Ser28 in HP1. All other phosphorylated P1 Protamines contained only P-Thr, which could not be further localized in the sequence with the present methods. After PKC phosphorylation, the internally located Ser residues in human (ser21) and stallion (Ser29) P1 Protamines were phosphorylated and, in boar P1 protamine, only Thr43 was slightly phosphorylated. The N-terminally located Ser residues in P1 Protamines, which are known to be phosphorylated in vivo, were not phosphorylated by either kinase, indicating that there must still be other types of protamine kinases in sperm cells responsible for their phosphorylation. Within P2 Protamines, HP2 was equally well phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas, in St2, Ser32 was the main target for PKC phosphorylation in vitro. Collectively, PKC is a good candidate for in vivo phosphorylation of P2 Protamines and PKA for phosphorylation of some hydroxyamino acid residues in P1 Protamines.
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identification of phosphoseryl residues in Protamines from mature mammalian spermatozoa
Biology of Reproduction, 1994Co-Authors: Arja Pirhonen, Annikka Linnalakankkunen, Pekka H MaenpaaAbstract:Protamines isolated from ejaculated human, stallion, bull, boar, and ram spermatozoa were subjected to phosphoserine conversion reaction and protein sequencing. Phosphoserines were detected as S-ethylcysteines. Endogenously phosphorylated Protamines have previously been found only in ejaculated human sperm. In this study, we demonstrate that ejaculated sperm from other species also contain Protamines phosphorylated at serine residues. In P1-Protamines, the endogenously phosphorylated serines were located at the N-terminal region in all species studied, whereas in major forms of human and stallion P2-Protamines, the serine residues located in the middle region of the molecule were predominantly phosphorylated. These results support the current DNA binding model in the case of the P1-Protamines. The internal location of the phosphorylated serines in the P2-Protamines indicates, however, that the binding of these proteins to DNA or their interaction with other protamine molecules may differ from that of P1-Protamines. This also suggests that, during sperm maturation, P2-Protamines may have a role different from that of P1-Protamines.
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in vitro phosphorylation sites of stallion and bull p1 Protamines for cyclic adenosine 3 5 monophosphate dependent protein kinase and protein kinase c
Biology of Reproduction, 1993Co-Authors: Arja Pirhonen, Pirjo Valtonen, Annikka Linnalakankkunen, Pekka H MaenpaaAbstract:Fish and mammalian Protamines are phosphorylated after their synthesis during sperm cell maturation. Cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), both requiring basic amino acids at their recognition sites, have previously been found to phosphorylate fish Protamines in vitro. In this study, these enzymes were used to phosphorylate stallion and bull sperm P1-Protamines in vitro. A species-specific difference was found, since PKA was able to phosphorylate both Protamines while PKC phosphorylated only stallion protamine. Thr-41, the only threonine residue in stallion P1-protamine, and most probably the homologous Thr-43 in bull P1-protamine are the sites for PKA phosphorylation in addition to an internally located Ser-29 present only in stallion protamine. This Ser residue was phosphorylated in vitro by both kinases. Protamine phosphorylation by PKA was found to be almost independent of cAMP and was inhibited only by a tenfold concentration of PKI when compared to phosphorylation of a model peptide, kemptide. Addition of calcium, phosphatidylserine, and diolein caused a twofold stimulation in phosphorylation of stallion protamine by PKC, indicating that specific cofactors of PKC may have a role in mammalian protamine phosphorylation. We suggest that PKA is a good universal candidate for in vivo phosphorylation of P1-Protamines.
Rod Balhorn - One of the best experts on this subject based on the ideXlab platform.
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sperm nuclear Protamines a checkpoint to control sperm chromatin quality
Anatomia Histologia Embryologia, 2018Co-Authors: Klaus Steger, Rod BalhornAbstract:Protamines are nuclear proteins which are specifically expressed in haploid male germ cells. Their replacement of histones and binding to DNA is followed by chromatin hypercondensation that protects DNA from negative influences by environmental factors. Mammalian sperm contain two types of Protamines: PRM1 and PRM2. While the proportion of the two Protamines is highly variable between different species, abnormal ratios within a species are known to be associated with male subfertility. Therefore, it is more than likely that correct protamine expression represents a kind of chromatin checkpoint during sperm development rendering Protamines as suitable biomarkers for the estimation of sperm quality. This review presents an overview of our current knowledge on Protamines comparing gene and protein structures between different mammalian species with particular consideration given to man, mouse and stallion. At last, recent insights into the possible role of inherited sperm histones for early embryo development are provided.
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New monoclonal antibodies specific for mammalian Protamines P1 and P2
2018Co-Authors: Rod Balhorn, Klaus Steger, Martin Bergmann, Hans-christian Schuppe, Stefanie Neuhauser, Monique C. BalhornAbstract:The expression of Protamines and the binding of these small arginine-rich proteins to DNA complete the process of spermatid chromatin reorganization and the global inactivation of the male’s haploid genome that occurs during the final stages of sperm development in mammals. While a number of anti-protamine antibodies have been created during the last 40 years, only a few have proven useful for detecting the presence of the Protamines, determining the timing of their expression and deposition in chromatin, and investigating their structure and function in both maturing spermatids and sperm. The aim of this effort was to develop an additional set of monoclonal antibodies (MAbs) that not only recognize new P1 and P2 protamine epitopes but also work well as IHC reagents for detecting and identifying mammalian Protamines in testicular tissue and ejaculated sperm. Using a combination of native and synthetic human Protamines as antigens, 38 hybridoma clones recognizing human protamine P1 or P2 were generated. Antibodies produced by the 12 best clones were screened for selectivity by enzyme-linked immunosorbent assay, and two were found to recognize only human protamine P1 or P2, while a number of the others bound to both the human and mouse proteins. One MAb recognized every protamine tested. All the antibodies, including one recognizing stallion P1 and another recognizing stallion P2, bound to the native Protamines in the chromatin of spermatids or sperm. While the majority labeled only elongating spermatids or sperm, several of the antibodies were found to also bind to the cytoplasm or nuclei of cells that lack protamine, which indicates these MAbs must recognize epitopes present in the Protamines that are also found in other proteins. Thirteen overlapping human protamine P1 peptides were synthesized and subsequently used to identify the epitopes recognized by the six best antibodies. Abbreviations: BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; HCl: hydrochloric acid; IHC: immunohistochemistry; i.p: intraperitoneal; LIS: lithium diiodosalicylate; MAb: monoclonal antibody; PBS: phosphate buffered saline
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analysis of hamster Protamines primary sequence and species distribution
Molecular Reproduction and Development, 1999Co-Authors: Michele Corzett, Cheryl Kramer, Russell Blacher, Joe Mazrimas, Rod BalhornAbstract:Basic nuclear proteins were isolated from the sperm of the Syrian hamster Mesocricetus auratus and characterized by gel electrophoresis, amino acid analysis, and sequencing. Analyses of the proteins by gel electrophoresis show that sperm of this species contain both Protamines 1 and 2. The two proteins were purified by HPLC and the complete primary sequence of hamster protamine 1 was determined by automated amino acid sequence analysis. The protein sequence was subsequently confirmed by sequencing the PCR-amplified protamine 1 gene. The first forty-two residues of the hamster protamine 2 sequence were obtained by amino acid sequence analysis of the isolated protein, and this sequence was also confirmed and extended by sequencing the gene. Total basic nuclear protein was also isolated from sperm of six other species of hamsters, the Protamines were identified by HPLC and amino acid analysis, and the proportion of Protamines 1 and 2 in each species was determined. Marked differences in the protamine 2 content of sperm were observed among the different species of hamster. This variation and the high level of sequence similarity between mouse and hamster Protamines provide insight into how the two Protamines may be organized in sperm chromatin. Mol. Reprod. Dev. 54:273-282, 1999. Published 1999 Wiley-Liss, Inc.
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protamine induced condensation and decondensation of the same dna molecule
Science, 1999Co-Authors: Laurence R Brewer, Michele Corzett, Rod BalhornAbstract:The DNA in sperm and certain viruses is condensed by arginine-rich proteins into toroidal subunits, a form of packaging that inactivates their entire genome. Individual DNA molecules were manipulated with an optical trap to examine the kinetics of torus formation induced by the binding of protamine and a subset of its DNA binding domain, Arg6. Condensation and decondensation experiments with λ-phage DNA show that toroid formation and stability are influenced by the number of arginine-rich anchoring domains in protamine. The results explain why Protamines contain so much arginine and suggest that these proteins must be actively removed from sperm chromatin after fertilization.
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synthesis and processing of mammalian Protamines and transition proteins
Molecular Reproduction and Development, 1994Co-Authors: G R Green, Rod Balhorn, Dominic Poccia, Norman B HechtAbstract:Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian Protamines and transition proteins. The tubule fragments were incubated with [3H]-arginine, [3H]-histidine, [35S]-cysteine, or [32P]-PO4, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized Protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that Protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized Protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the Protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms.
