Tetratricopeptide Repeat Motifs Research Articles

The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions.

The tetratricopeptide repeat (TPR) is a degenerate 34 amino acid sequence identified in a wide variety of proteins, present in tandem arrays of 3-16 motifs, which form scaffolds to mediate protein-protein interactions and often the assembly of multiprotein complexes. TPR-containing proteins include the anaphase promoting complex (APC) subunits cdc16, cdc23 and cdc27, the NADPH oxidase subunit p67 phox, hsp90-binding immunophilins, transcription factors, the PKR protein kinase inhibitor, and peroxisomal and mitochondrial import proteins. Here, we report the crystal structure of the TPR domain of a protein phosphatase, PP5. Each of the three TPR motifs of this domain consist of a pair of antiparallel alpha-helices of equivalent length. Adjacent TPR motifs are packed together in a parallel arrangement such that a tandem TPR motif structure is composed of a regular series of antiparallel alpha-helices. The uniform angular and spatial arrangement of neighbouring alpha-helices defines a helical structure and creates an amphipathic groove. Multiple-TPR motif proteins would fold into a right-handed super-helical structure with a continuous helical groove suitable for the recognition of target proteins, hence defining a novel mechanism for protein recognition. The spatial arrangement of alpha-helices in the PP5-TPR domain is similar to those within 14-3-3 proteins.

Stage-specific expression of the immunophilin FKBP59 messenger ribonucleic acid and protein during differentiation of male germ cells in rabbits and rats.

FKBP59 is an immunophilin that binds the immunosuppressant drugs FK506 and rapamycin. It is a 90-kDa heat shock protein (hsp90)-binding protein that was originally discovered as a member of steroid receptor complexes. FKBP59 is ubiquitous and well conserved, and it appears to be a multifunctional protein. It has peptidylprolyl cis-trans-isomerase activity and therefore may be involved in protein folding as a molecular chaperon. FKBP59 also includes in its structure a tetratricopeptide repeat (TPR) motif and could have a function in the cell division process. In situ hybridization experiments revealed an overexpression of FKBP59 mRNA in rabbit and rat testes in comparison with other organs. This high level of expression was restricted to germ cells of the seminiferous epithelium. Increasing levels of FKBP59 mRNA became obvious from the midpachytene stage, and the strongest signal was observed in the late pachytene, diplotene, and diakinesis primary spermatocytes. The expression then declined progressively in postmeiotic early spermatids. High expression of FKBP59 mRNA did not occur in earlier and later germ cell stages. During prepubertal development, Northern blot and in situ hybridization of rat testes examined at various postnatal ages revealed that FKBP59 mRNA was not expressed at over a basal level until the pachytene stage. High expression of the FKBP59 protein was demonstrated in the rabbit testis by Western blot and was localized by immunohistochemistry from late pachytene spermatocytes to round spermatids. The cell type-specific and developmental stage-specific expression of FKBP59 at a restricted period of male germ cell differentiation suggests that FKBP59 is involved in a specific function during the cell division process.

Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants.

To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

The prp1+ gene required for pre-mRNA splicing in Schizosaccharomyces pombe encodes a protein that contains TPR motifs and is similar to Prp6p of budding yeast.

The prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature. One of the prp mutants, prp1-4, also has a defect in poly(A)+ RNA transport. The prp1+ gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions. The amino acid sequence of Prp1p shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly. No functional complementation was observed between S. pombe prp1+ and S. cerevisiae PRP6. We examined synthetic lethality of prp1-4 with the other known prp mutations in S. pombe. The results suggest that Prp1p interacts either physically or functionally with Prp4p, Prp6p and Prp13p. Interestingly, the prp1+ gene was found to be identical with the zer1+ gene that functions in cell cycle control. These results suggest that Prp1p/Zer1p is either directly or indirectly involved in cell cycle progression and/or poly(A)+ RNA nuclear export, in addition to pre-mRNA splicing.

Ppt-1, a Neurospora crassa PPT/PP5 subfamily serine/threonine protein phosphatase

We isolated a N. crassa cDNA clone encoding a novel-type serine/threonine phosphatase. The gene (mapped to LGVR), designated ppt- 1, encodes a 479 amino acid putative polypeptide which contains a conserved tetratricopeptide repeat (TPR) motif. ppt- 1 transcript levels are abundant in conidia and decrease during germination, indicating that ppt- 1 is developmentally regulated.

A Novel Cytoplasmic Protein That Interacts with the Ah Receptor, Contains Tetratricopeptide Repeat Motifs, and Augments the Transcriptional Response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

To identify new proteins involved in dioxin-dependent signal transduction and transcriptional regulation, we used a yeast two-hybrid system to identify proteins that interact with the Ah receptor (AhR). We cloned a mouse cDNA, which encodes a novel approximately 37-kDa protein that binds to AhR; we have designated the protein as Ah receptor-interacting protein (AIP). The amino acid sequence of mouse AIP exhibits homology with members of the FK506-binding protein family. AIP also contains three tetratricopeptide repeat (TPR) motifs; the TPR sequence is present in proteins required for cell cycle control and RNA synthesis and in steroid receptor-binding immunophilins. Coimmunoprecipitation experiments in mouse hepatoma cells reveal that AIP is cytoplasmic and associates with unliganded Ah receptor and with hsp90; 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment disrupts the AhR-AIP-hsp90 interaction. Overexpression of AIP augments the response of the CYP1A1 gene to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Our data suggest that AIP influences ligand receptivity and/or nuclear targeting of AhR.

Dynamic Glycosylation of Nuclear and Cytosolic Proteins: CLONING AND CHARACTERIZATION OF A UNIQUE O-GlcNAc TRANSFERASE WITH MULTIPLE TETRATRICOPEPTIDE REPEATS

O-Linked N-acetylglucosamine (O-GlcNAc) glycosylation is a dynamic modification of eukaryotic nuclear and cytosolic proteins analogous to protein phosphorylation. We have cloned and characterized a novel gene for an O-GlcNAc transferase (OGT) that shares no sequence homology or structural similarities with other glycosyltransferases. The OGT gene is highly conserved (up to 80% identity) in all eukaryotes examined. Unlike previously described glycosyltransferases, OGT is localized to the cytosol and nucleus. The OGT protein contains multiple tandem repeats of the tetratricopeptide repeat motif. The presence of tetratricopeptide repeats, which can mediate protein-protein interactions, suggests that OGT may be regulated by protein interactions that are independent of the enzyme's catalytic site. The OGT is also modified by tyrosine phosphorylation, indicating that tyrosine kinase signal transduction cascades may play a role in modulating OGT activity.

A novel plant peptidyl-prolyl-cis-trans-isomerase (PPIase): cDNA cloning, structural analysis, enzymatic activity and expression.

A novel cDNA encoding for a peptidyl-prolyl-cis-trans-isomerase (PPIase) belonging to the FK506-binding protein (FKBP) family was isolated from wheat. It contains an open reading frame of 559 amino acids and it represents the first plant FKBP-PPIase to be cloned. It possesses a unique sequence which is composed of three FKPB-like domains, in addition to a putative tetratricopeptide repeat (TPR) motif and a calmodulin-binding site. The recombinant FKBP-PPIase expressed in and purified from Escherichia coli exhibits PPIase activity that is efficiently inhibited by the immunosuppressive drugs FK506 and rapamycin. Northern blot analysis showed that wheat FKBP was found mainly in young tissues. Polyclonal antibodies revealed the presence of cross-reacting proteins in embryos, roots and shoots. The unique structural features, the enzymatic activity and the presence of putative isoforms in wheat tissues indicate the possibility of the involvement of wheat PPIase in essential biological functions, similar to other members of the FKBP gene family.

Identification and cloning of a novel cDNA belonging to tetratricopeptide repeat gene family from Down syndrome-critical region 21q22.2.

We identified and cloned a novel 9,078-bp cDNA, designated TPRDI, from the Down syndrome-critical region by exon trapping. The cDNA encodes a putative protein (TPRDI) of 2,025 amino acid residues. Two isoforms, TPRDII (8,992 bp) and TPRDIII (7,416 bp), were also isolated. TPRDII, which is probably an alternative splicing product from the TPRD gene transcript, encodes two large open reading frames (ORFs) of 200 amino acid residues and 1,792 amino acid residues, respectively. TPRDIII, which is probably generated by transcription from an alternative start site of the TPRD gene, encodes a putative protein of 1,715 amino acid residues (TPRDIII). Northern blot analysis revealed that TPRDI and its isoforms are present in 7-17 day mouse embryo and in all the human adult and fetal tissues examined. TPRDI has three units of a 34-amino-acid repeat similar to the tetratricopeptide repeat (TPR) motif, which may mediate interaction with various proteins. A larger ORF encoded by TPRDII also has three units of TPR motif, but TPRDIII has only two-thirds of this motif unit. Thus, the TPRD gene may belong to the TPR gene family. Near-central and C terminal regions of TPRDs showed some homology to several matrix proteins such as trichohyalin and bullous pemphigoid antigen. It is possible that the TPRD gene is one of the genes whose overexpression causes several morphological anomalies observed in Down syndrome.

Identification and characterization of two novel tetratricopeptide repeat-containing genes.

The tetratricopeptide repeat (TPR) is a degenerate, repeating amino acid motif of 34 residues that has been identified in a variety of proteins; however, no biochemical function has been established for it. In a two-hybrid screen for interaction with the GAP-related domain of neurofibromin, the product of the NF1 gene, we have identified two novel human genes encoding proteins with TPR motifs. The first, represented by cDNA tpr1, is located in chromosome 5q32-33.2. It is ubiquitously expressed as a 1.6-kb transcript that encodes three tandem TPR motifs, but is not related to any other known gene outside this domain. The second, defined by cDNA tpr2, maps to human chromosome 17q11.2-23. It is ubiquitously expressed as a 2.2-kb transcript encoding seven TPR units. Interestingly, a separate region of the tpr2 cDNA has striking similarity to the "J region" of the DnaJ family. The products of the tpr1 and tpr2 cDNAs interact preferentially with a truncated form of the NF1 GAP-related domain via their TPR regions, suggesting that they may be targeted to an abnormality of protein folding.

Identification of two CyP-40-like cyclophilins in Saccharomyces cerevisiae, one of which is required for normal growth.

We report the analysis of two Saccharomyces cerevisiae cyclophilins, Cpr6 and Cpr7, identified by their ability to interact in vivo with the transcriptional regulator Rpd3. Both cyclophilins have an extended carboxy-terminal region containing a three-unit tetratricopeptide repeat (TPR) motif and share significant amino acid identity with the mammalian cyclophilin CyP-40. Neither CPR6 nor CPR7 is essential but deletion of CPR7 results in a significant impairment of the rate of cell division. This is the first demonstration that a member of the cyclophilin family is required for normal cell growth.

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58IPK, a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58IPK- and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58IPK-interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58IPK but was dependent on the presence of the eukaryotic initiation factor 2-alpha homology region, mapping to the 34 aa within the sixth P58IPK TPR motif. Consistent with other TPR proteins, P58IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58IPK , K3L interacted exclusively with the PKR protein kinase catalytic domain. In contrast, however, the K3L-binding region of PKR was localized to within aa 367 to 551, demonstrating that each inhibitor bound PKR in unique, nonoverlapping domains. These data, taken together, suggest that P58IPK and K3L may mediate PKR inhibition by distinct mechanisms. Finally, we will propose a model of PKR inhibition in which P58IPK or a P58IPK complex binds PKR and interferes with nucleotide binding and autoregulation, while formation of a PKR-K3L complex interferes with active-site function and/or substrate association.

Interactions of p60, a mediator of progesterone receptor assembly, with heat shock proteins hsp90 and hsp70.

Previous studies on the assembly of progesterone receptor (PR) complexes in vitro have suggested that PR assembly is a dynamic, ordered process involving at least eight nonreceptor proteins. One of these proteins, p60, appears transiently during assembly and is not a component of functionally mature PR complexes. In the present study we observe that a monoclonal antibody specific for p60 can, on the one hand, inhibit formation of mature PR complexes containing heat shock protein 90 (hsp90), p23, and immunophilins and, on the other, enhance recovery of early PR complexes containing hsp70 and Hip (p48). This observation supports a model in which p60 functions at an intermediate stage of PR assembly to facilitate formation of subsequent PR complexes lacking p60. Since p60 is typically found in a complex with hsp90 and hsp70, we have further characterized its interactions with these proteins. P60 can bind either hsp70 or hsp90 independently and in an ATP-independent manner. Since hsp90 and hsp70 do not readily associate on their own, it appears that p60 is the central organizing component of an hsp90-p60-hsp70 complex. Mutational analysis of p60 indicates that the N terminus is required for hsp70 binding, and a central region containing tetratricopeptide repeat motifs is necessary for binding hsp90 and hsp70. The hsp90-p60-hsp70 multichaperone complex is highly dynamic and does not appear to be affected by the hsp90-binding drug geldanamycin. The interactions of hsp70 and hsp90 in intermediate PR complexes are shown to be distinct from their separate interactions in early PR complexes (hsp70) or in mature PR complexes (hsp90). From these results, it appears that p60 is a key mediator in the chaperoned assembly and functional maturation of PR complexes.

The Mas20p and Mas70p subunits of the protein import receptor of yeast mitochondria interact via the tetratricopeptide repeat motif in Mas20p: evidence for a single hetero-oligomeric receptor.

Protein import into yeast mitochondria is mediated by four integral outer membrane proteins which function as import receptors. These proteins (termed Mas20p, Mas22p, Mas37p and Mas70p) appear to exist as two subcomplexes: a Mas37p-Mas70p heterodimer and a less well characterized Mas20p-Mas22p complex. The subcomplexes interact functionally during protein import, but it has remained uncertain whether they are in direct contact with each other in vivo. Here we show that Mas20p and Mas70p can be cross-linked in intact mitochondria, or co-immunoprecipitated from digitonin-solubilized mitochondria. Furthermore, the cytosolic domains of these two proteins interact in the 'two-hybrid' system. Association of Mas20p and Mas70p is virtually abolished by a mutation in the single tetratricopeptide motif in Mas20p. This mutation specifically inhibits import of precursors that are first recognized by Mas37p-Mas70p and only then transferred to Mas20p-Mas22p. We conclude that the two receptor subcomplexes of the mitochondrial protein import receptor interact in vivo via their Mas20p and Mas70p subunits and that this interaction is functionally important.

Identification of a novel human gene containing the tetratricopeptide repeat domain from the Down syndrome region of chromosome 21.

The Down syndrome (DS) region on chromosome 21, which is responsible for the DS main features, has been defined by analysis of DS patients with partial trisomy 21. Within the DS region, we constructed a 1.6-Mb P1 contig map previously. To isolate gene fragments from the 1.6-Mb region, we performed direct cDNA library screening and exon trapping using the P1 clones and a human fetal brain cDNA library, and obtained 67 cDNA fragments and 52 possible exons. Among them, 23 cDNA fragments and 4 exons were interpreted to be derived from a single gene by localization on P1 clones and by Northern analysis. To obtain the full-length cDNA sequence, longer cDNA clones were further screened from another human cDNA library which was enriched with longer cDNA species. These clones were sequenced and assembled to a sequence of 9045 bp. This transcribed sequence encodes a novel 2025 amino-acid protein containing tetratricopeptide repeat (TPR) motifs and therefore the gene was designated as TPRD (a gene containing the TPR motifs on the Down syndrome region). The TPR domain has been found in a certain protein phosphatase and in other proteins involved in the regulation of RNA synthesis or mitosis. The TPRD gene, the novel gene which was proved to be in the 1.6-Mb region and to have the interesting features described above, is a candidate for genes responsible for the DS phenotypes.

Novel Protein Phosphatases That May Participate in Cell Signaling

Publisher Summary The major family of protein phosphatases that dephosphorylate serine and threonine residues includes PP1, PP2A, and the calciudcalmodulin-regulated protein phosphatase (PP2B, calcineurin-CNA, CMP), which were identified as enzyme activities in the cell cytosol. On further analysis, these novel phosphatases have been shown to carry out distinct cellular functions. A phylogenetic tree of the fully sequenced phosphatase catalytic subunits belonging to this family that is present in the human species and in the yeast Succbaromyces cereuisiue. Three novel protein phosphatases, termed human PP4 (originally designated PPX), S. cerevzszae SIT4, and PPG, show about 60% identity to PP2A and perform different cellular roles to PP2A. Human PP5 and yeast PPT comprise a catalytic domain preceded by a long N-terminal domain that contains four 34-amino-acid or tetratricopeptide repeat (TPR) motifs. Protein Phosphatase Y (PPY) was identified in Drosophila melanogaster and because no homologs in mammals or yeast have as yet been identified, it is necessary to study Drosophila to investigate the function of this protein phosphatase. The functions of several novel protein phosphatases in the major family of protein serinelthreonine phosphatases are beginning to be delineated. Only a few of the novel protein phosphatases have been tested for inhibition by okadaic acid and microcystin—the tumor promoters. Although a number of novel protein phosphatases, such as PP4 and PPS, are found in all tissues, some are tissue specific.

Cloning and sequencing of a gene encoding a new member of the tetratricopeptide protein family from magnetosomes of Magnetospirillum magnetotacticum

A gene encoding the 22-kDa protein (MAM22) which was localized in the magnetosomes isolated from the magnetotactic bacterium, Magnetospirillum magnetotacticum, was cloned and sequenced. MAM22 was composed of 220 amino acids (aa) with a molecular weight of 24 186 Da. The deduced aa sequence exhibited significant homology with a number of proteins that belong to the tetratricopeptide repeat (TPR) protein family, including mitochondrial protein import receptors and peroxisomal protein import receptors. The presence of three repeats of a degenerate 34-aa consensus sequence, suggest that MAM22 localized in magnetosome membranes may interact with the cytoplasmic proteins containing similar TPR motifs.

Characterization of the human homologue of the mouse Tg737 candidate polycystic kidney disease gene.

We previously identified a gene from the mutant locus in a new mouse mutation that causes recessive polycystic kidney disease. Here we describe the cloning, characterization and mapping of the homologous human gene. The human and mouse genes are 95% identical at the predicted amino acid sequence level, and both genes encode a putative protein that contains a tetratricopeptide repeat motif. The human gene, called hTg737, is expressed with a broad tissue distribution that includes the the kidney and liver, and gives rise to a 2.9 kb mRNA. The gene contains 26 exons and spans a genomic region greater than 100 kb. Chromosome mapping experiments revealed that the hTg737 gene maps near the centromere on the long arm of human chromosome 13, at position 13q12.1. While this gene does not map to the primary locus that has been identified for ARPKD in humans, it may represent a candidate gene for other recessive renal disorders that have yet to be mapped.

Isolation of a cDNA encoding a novel human FK506-binding protein homolog containing leucine zipper and tetratricopeptide repeat motifs

Reduced-stringency PCR was used to isolate a cDNA encoding a novel human FK506-binding protein (FKBP) homolog. The encoded 38-kDa protein (FKBPr38) contains at its N-terminus a domain that is 33% identical to FKBP12. FKBPr38 is a member of a subclass of immunophilins, whose other members include FKBP52 and CyP40 (cyclophilin 40), that contain a three-unit tetratricopeptide repeat (TPR). In addition, FKBPr38 contains a consensus leucine-zipper repeat. The presence of the TPR domain and leucine zipper suggest that FKBPr38 may form homomultimers or interact with other, as yet unidentified, proteins.

Molecular cloning of a protein serine/threonine phosphatase containing a putative regulatory tetratricopeptide repeat domain.

Two novel protein serine/threonine phosphatases were cloned from a rat fat cell library with probes generated by a polymerase chain reaction-based cloning approach. One of these cDNAs encoded a protein presumably representing the rat homologue of PPV from Drosophila (75% identity of amino acids). The other novel cDNA encoded a protein phosphatase of 499 amino acids and was designated PPT. Its catalytic domain contains motifs typical for protein phosphatases but is only distantly related with PP1, PP2A, and PP2B (38-42% identical amino acids). When expressed in Escherichia coli, the catalytic domain of PPT exhibited protein phosphatase activity (dephosphorylation of phosphorylase a) that was inhibitable by okadaic acid. As a unique feature among other members of this gene family, PPT has an amino-terminal extension of 200 amino acids harboring three tandemly arranged tetratricopeptide repeat (TPR) motifs. This domain has previously been found in other proteins involved in the regulation of RNA synthesis or mitosis. mRNA of PPT was predominantly found in brain and, in lower levels, in testis, but was nearly undetectable in spleen, lung, skeletal muscle, kidney, and liver. It is suggested that the TPR domain of PPT may be involved in the regulation of the function of this novel protein phosphatase.