Isolation of a cDNA clone for transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids

We have isolated a cDNA clone for rat transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids. The clone was identified initially by hybrid selection of TP1 mRNA. The sequence of the 251-nucleotide cDNA includes the entire coding region for the protein, thereby confirming the identity of the clone as well as predicting two changes in the published amino acid sequence.     

A cytochemical study of the transcriptional and translational regulation of nuclear transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids

Immunocytochemical localization and in situ hybridization techniques were used to investigate the presence of spermatid nuclear transition protein 1 (TP1) and its mRNA during the various stages of spermatogenesis in the rat. A specific antiserum to TP1 was raised in a rabbit and used to show that TP1 is immunologically crossreactive among many mammals including humans. During spermatogenesis the protein appears in spermatids as they progress from step 12 to step 13, a period in which nuclear condensation is underway. The protein is lost during step 15. An asymmetric RNA probe generated from a TP1 cDNA clone identified TP1 mRNA in late round spermatids beginning in step 7. The message could no longer be detected in spermatids of step 15 or beyond. Thus, TP1 mRNA first appears well after meiosis in haploid cells but is not translated effectively for the several days required for these cells to progress to the stage of chromatin condensation. Message and then protein disappear as the spermatids enter step 15. In agreement with a companion biochemical study (Heidaran, M.A., and W.S. Kistler. J. Biol. Chem. 1987. 262:13309-13315), these results establish that translational control is involved in synthesis of this major spermatid nuclear protein. In addition, they suggest that TP1 plays a role in the completion but not the initiation of chromatin condensation in elongated spermatids.     

Characterization of the human 36-kDa carboxyl terminal LIM domain protein (hCLIM1)

We characterized a human cDNA clone encoding a 36-kDa carboxyl terminal LIM domain protein with a PDZ domain at the amino terminal. This full-length cDNA clone has a predicted open reading frame (ORF) of 329 amino-acid residues. The ORF of this cDNA encodes the human homolog of rat CLP36, and the putative protein is named human 36-kDa carboxyl terminal LIM domain protein (hCLIM1, nomenclature approved by the HUGO/GDB Nomenclature Committee). The hCLIM1 probe was used to hybridize with poly(A)+ RNA of various human tissues. Strong signals were detected in heart and skeletal muscle; moderate signals were detected in spleen, small intestine, colon, placenta, and lung; weaker levels were detected in liver, thymus, kidney, prostate, and pancreas; and no observable signals were detected in brain, testis, ovary, and peripheral blood leukocytes. The hCLIM1 gene was studied by fluorescence in situ hybridization (FISH), somatic cell hybrid analysis, and radiation hybrid mapping, and it is located at the human chromosome 10q26.     

Transcriptional and translational control of the message for transition protein 1, a major chromosomal protein of mammalian spermatids

The spermatid transition proteins comprise a set of basic chromosomal proteins that appear during the period when spermatids are undergoing nuclear elongation and condensation, about midway between the end of meiosis and the release of spermatozoa from the seminiferous tubule. The transition proteins replace the histones but are themselves subsequently replaced by protamines, and they are not found in sperm nuclei. We have used a cDNA clone for the smallest transition protein (TP1, 54 amino acids) to show that its message first appears postmeiotically in late round spermatids. Thus production of TP1 is an example of haploid gene expression. The message remains translationally inactive for some 3-4 days before translation occurs in early elongating spermatids. While translationally repressed, TP1 message is nonpolysomal and has a discrete size of about 590 bases, including a 140 residue poly(A) tail. In contrast, polysome-associated message is of heterogeneous size due to variability of poly(A) lengths.     

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs)

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)⁺ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.     

Phosphorylation of the carboxyl terminal region of dystrophin by mitogen-activated protein (MAP) kinase

Dystrophin is the 427-kDa protein product of the Duchenne muscular dystrophy gene (DMD). The function of this protein remains to be elucidated. We have recently reported that dystrophin is phosphorylated,in vivo, in rat skeletal muscle primary cell culture (RE Milner, JL Busaan, CFB Holmes, JH Wang, M Michalak (1993) J Biol Chem 268: 21901–21905). This observation suggests that protein phosphorylation may have some role in modulating the function of dystrophin or its interaction with membrane associate dystroglycan. We report here that the carboxyl-terminal of dystrophin is phosphorylated by the MAP kinase p44^mpk (mitogen-activated protein kinase), from the sea star oocytes and by soluble extracts of rabbit skeletal muscle. Importantly we showed that native dystrophin in isolated sarcolemmal vesicles is phosphorylated by sea star p44^mpk. Partial purification and immunological analysis show that a mammalian kinase related to p44^mpk is present in the skeletal muscle extracts and that it contributes to phosphorylation of the carboxyl-terminal of dystrophin. This kinase phosphorylates dystrophin on a threonine residue(s). We conclude that phosphorylation of dystrophin may play an important role in the function of this cytoskeletal protein.Abbreviations MAP kinase mitogen-activated protein kinase - DMD Duchenne muscular dystrophy - GST Glutathione S-transferase - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - MOPS 4-morpholinepropanesulfonic acid     

Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase.

Focal adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called focal adhesions. pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals. Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk. In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin. The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified focal adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK. The interaction of paxillin and pp125FAK is independent of the adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin.     

The carboxyl terminus of v-Abl protein can augment SH2 domain function

Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway.     

The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins

SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike–ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.

A study using a combination of surrogate entry assays and live virus suggests that SARS-CoV-2 may have a broad host-range, revealing that the virus''s spike protein can use a broad range of host ACE2 receptors to enter cells and that the sequence of this protein might have changed during the zoonotic jump into humans.     

Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain

SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1. The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.     

Partial characterization of a new basic nuclear protein from rat testis elongated spermatids.

A basic protein designated TP2 has been isolated from rat testis elongated spermatids. This new protein contains basic and acidic amino acids in relative amounts similar to those in histone F2al but is unusually rich in serine and proline. Techniques which were developed for preparing relatively homogeneous populations of spermatid nuclei were used to demonstrate that TP2 is most abundant and most actively synthesized in spermatids representing steps 12 through 15 of spermiogenesis.     

Nuclear basic protein transition during sperm differentiation. Primary structure of the spermatid-specific protein S2 from the dog-fish Scylliorhinus caniculus

The remodeling of nucleoproteins during dog-fish spermiogenesis involves two successive nuclear protein transitions: the first from somatic-type histones to transition proteins during the nuclear elongation of spermatids and the second leading to protamine-DNA association in mature spermatozoa. The chromatin of elongating spermatids contains two transition proteins called S1 and S2. The amino acid sequence of protein S1, a polypeptide of 87 residues was determined previously [Chauvière, M., Martinage, A., Briand, G., Sautière, P. & Chevaillier, Ph. (1987) Eur. J. Biochem. 169, 105-111]. In the present paper, we report the elucidation of the primary structure of the minor transition protein S2 established by automated Edman degradation of the protein and of its fragments generated by cleavage at methionine and aspartate residues. S2 contains 80 residues and has a molecular mass of 9726 Da. S2 is mainly characterized by a high content of basic amino acids mostly represented by lysine, a relatively high level of hydrophobic residues, the presence of six phosphorylatable residues and the lack of cysteine. Its amino acid sequence shows that the N-terminal half is highly basic, while the acidic residues are located in the C-terminal part of the protein where more diversity in amino acids is noticed. The two transition proteins S1 and S2 share striking structural similarities. Few but significative similarities have been detected with the mammalian transition protein TP1 [Kistler, W. S., Noyes, C., Hsu, R. & Heinrikson, R. L. (1975) J. Biol. Chem. 250, 1847-1853], suggesting similar functions for all these proteins in chromatin remodeling during sperm differentiation. By contrast, the two dog-fish spermatid-specific proteins are structurally unrelated to sperm protamines and cannot be considered as their precursors.     

Nuclear basic protein transition during sperm differentiation. Amino acid sequence of a spermatid-specific protein from the dog-fish Scylliorhinus caniculus

During dog-fish spermatogenesis, chromatin undergoes a continuous processing which involves two basic protein transitions: the first from somatic-type histones to spermatid-specific proteins and the second leading to protamines. Two spermatid-specific proteins S1 and S2 were isolated from nuclei of spermatid-enriched testis zone and the amino acid sequence of S1 has been determined. S1 contains 87 amino acids and has a molecular mass of 11179 Da. It is mainly characterized by a high content of basic residues (45%) and the presence of one residue of cysteine. Its primary structure shows that the N-terminal half is highly basic while the hydrophobic residues are preferentially localized in the C-terminal region. Three forms of S1 are present in testis which correspond to di-, mono- and nonphosphorylated molecules. This spermatid-specific protein shares no common structural feature with either histones and dog-fish protamines or rat spermatid-specific protein which has been previously described.     

Nuclear transition protein 1 from ram elongating spermatids. Mass spectrometric characterization, primary structure and phosphorylation sites of two variants

The ram transition protein 1 (TP1) is present in spermatid cell nuclei in the nonphosphorylated, monophosphorylated and diphosphorylated forms. Its primary structure was determined by automated Edman degradation of S-carboxamidomethylated protein and of peptides generated by cleavage with thermolysin and endoproteinase Lys-C. The ram TP1 is a small basic protein of 54 residues and structurally very close to other mammalian TP1. The mass spectrometric data obtained from the protein and its fragments reveal that ram TP1 is indeed a mixture (approximately 5:1) of two structural variants (Mr 6346 and 6300). These variants differ only by the nature of the residue at position 27 (Cys in the major variant and Gly in the minor variant). The study of phosphorylation sites has shown that four different serine residues could be phosphorylated in the monophosphorylated TP1, at positions 8, 35, 36 or 39. From previous physical studies, it has been postulated that the Tyr32 surrounded by two highly conserved basic clusters was responsible for the destabilization of chromatin by intercalation of its phenol ring between the bases of double-stranded DNA. The presence of three phosphorylatable serine residues in the very conserved sequence 29-42 is another argument for the involvement of this region in the interaction with DNA.     

The N-terminal domain of human centrin 2 has a closed structure, binds calcium with a very low affinity, and plays a role in the protein self-assembly

Centrins are well-conserved calcium binding proteins from the EF-hand superfamily implicated in various cellular functions, such as centrosome duplication, DNA repair, and nuclear mRNA export. The intrinsic molecular flexibility and the self-association tendency make difficult the structural characterization of the integral protein. In this paper we report the solution structure, the Ca2+ binding properties, and the intermolecular interactions of the N-terminal domain of two human centrin isoforms, HsCen1 and HsCen2. In the absence of Ca2+, the N-terminal construct of HsCen2 revealed a compact core conformation including four almost antiparallel alpha-helices and a short antiparallel beta-sheet, very similar to the apo state structure of other calcium regulatory EF-hand domains. The first 25 residues show a highly irregular and dynamic structure. The three-dimensional model for the N-terminal domain of HsCen1, based on the high sequence conservation and NMR spectroscopic data, shows very close structural properties. Ca2+ titration of the apo-N-terminal domain of HsCen1 and HsCen2, monitored by NMR spectroscopy, revealed a very weak affinity (10(2)-10(3) M(-1)), suggesting that the cellular role of this domain is not calcium dependent. Isothermal calorimetric titrations showed that an 18-residue peptide, derived from the N-terminal unstructured fragment, has a significant affinity (approximately 10(5) M(-1)) for the isolated C-terminal domain, suggesting an active role in the self-assembly of centrin molecules.     

The ORFa protein, the putative transposase of maize transposable element Ac, has a basic DNA binding domain.

Ac encodes the 807 amino acid ORFa protein which binds specifically to multiple AAACGG motifs that are subterminally located in both ends of Ac. The wild-type ORFa protein and a number of deletion and amino acid exchange mutants were expressed in Escherichia coli, renatured and used for mobility shift assays. At least 136 amino acids from the N-terminus and 537 C-terminal amino acids may be removed from the ORFa protein without destroying the DNA binding domain, whereas a protein starting at amino acid 189 is DNA binding deficient. Certain basic amino acids between positions 190 and 200 are essential for DNA binding, as their substitution with uncharged amino acids leads to the loss of this function. The DNA binding domain of ORFa protein has an overall basic character, but no obvious sequence homology to any other known DNA binding protein. The homologies to the major open reading frames of transposable elements Tam3 from Antirrhinum majus and Hobo from Drosophila are found between the C-terminal two thirds of the three proteins. The ORFa protein forms discrete complexes with target DNA that appear, depending on the protein concentration, as a 'ladder' of bands on gels, indicating the occupation of target DNA by multiple ORFa protein molecules.