A novel brain-expressed protein related to carnitine palmitoyltransferase I

Malonyl-CoenzymeA acts as a fuel sensor, being both an intermediate of fatty acid synthesis and an inhibitor of the two known isoforms of carnitine palmitoyltransferase I (CPT I), which control mitochondrial fatty acid oxidation. We describe here a novel CPT1 family member whose mRNA is present predominantly in brain and testis. Chromosomal locations and genome organization are reported for the mouse and human genes. The protein sequence contains all the residues known to be important for both carnitine acyltransferase activity and malonyl-CoA binding in other family members. Yeast expressed protein has no detectable catalytic activity with several different acyl-CoA esters that are good substrates for other carnitine acyltransferases, including the liver isoform of CPT I, which is also expressed in brain; however, it displays high-affinity malonyl-CoA binding. Thus this new CPT I related protein may be specialized for the metabolism of a distinct class of fatty acids involved in brain function.     

Mimicking phosphorylation of the small heat-shock protein alphaB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles.

The mammalian small heat shock protein alphaB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild-type, nonphosphorylatable and all possible pseudophosphorylation mutants of alphaB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/S59D) sites induced the partial translocation of alphaB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The alphaB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated alphaB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against alphaB-crystallin endogenously phosphorylated at Ser45, our findings suggest that alphaB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.     

Structure and expression of the gene encoding mouse F-box protein, Fwd2

A novel class of ubiquitin ligases, termed the SCF complex, consists of invariable components, Skp1 and Cullin, and variable components called F-box proteins, which have a primary role in determining substrate specificity. We have isolated a cDNA encoding the mouse F-box protein Fwd2 (also known as MD6) as a possible constituent of an SCF-type ubiquitin ligase. Fwd2 cDNA contains 1890 bp with a 1362-bp open reading frame and encodes an approximately 51.5-kDa protein. Fwd2 is expressed predominantly in liver and, to a lesser extent, in the testis, lung, heart, and skeletal muscle. Immunofluorescence staining for Fwd2 protein shows a pattern with the cytoplasm. A coimmunoprecipitation assay has revealed the in vivo interaction between Skp1 and Fwd2 through the F-box domain. Fwd2 also interacts with Cul1 through Skp1, suggesting that Skp1, Cul1, and the F-box protein Fwd2 form an SCF complex (SCF(Fwd2)). We have also isolated and determined the nucleotide sequence and genomic organization of the gene that encodes mouse Fwd2. This gene spans approximately 17 kb and consists of six exons and five introns. Our results suggest that Fwd2 is an F-box protein that constitutes an SCF ubiquitin ligase complex and that it plays a critical role in the ubiquitin-dependent degradation of proteins expressed in the liver.     

Characterization of G25K, a GTP-binding protein containing a novel putative nucleotide binding domain

Amino acid sequences were obtained for four peptides (p1, -2, -3 and 4) generated by chemical or proteolytic cleavage of a 25 kDa GTP-binding protein purified from human placental and platelet membranes. The peptides shared sequence similarities with those contained in several of the ras-related GTP-binding proteins. Peptide p2, a 12-mer, was homologous with a region of the GTP-binding proteins that contains a structural motif proposed to contribute to the nucleotide binding site. However, whereas nearly all GTP-binding proteins exhibit the residues NKXD as this motif, p2 contains TQID. Antisera (Ap1 and Ap3) raised against synthetic peptides corresponding to p1 and p3 specifically reacted on Western blots with the 25 kDa GTP-binding protein purified from human placenta, human platelet and bovine brain as well as with a 25 kDa polypeptide in various cell lines. These results demonstrate the widespread existence of an abundant 25 kDa GTP-binding protein which contains a putative nucleotide binding domain that is chemically distinct from that described for all GTP-binding proteins of known primary structure.     

Characterization of a cDNA encoding a novel heat-shock protein that binds to calmodulin.

A cDNA clone (pTCB48) encoding a calmodulin-binding protein was isolated by screening a lambda ZAPII cDNA expression library constructed from cell cultures of heat-shocked tobacco (Nicotiana tabacum L. cv Wisconsin-38) with metabolically labeled [35S]calmodulin. Calmodulin gel overlay analysis indicated that pTCB48 generated major peptides of 53, 36, and 22 kD and two minor peptides of 37 and 16 kD that bound calmodulin in a Ca(2+)-dependent manner. Deletion analysis of pTCB48 indicated that these and the minor calmodulin-binding proteins resulted from the insert. A probe made from the cDNA insert recognized two bands with sizes of 2.1 and 1.8 kb on northern blot analysis. Both species of RNAs were undetectable in the control and were induced after 15 min of heat-shock treatment at 38 degrees C. The intensity of the two bands reached maximum after 1.5 h of heat-shock treatment. The cDNA clone was not full length; however, the complete sequence was determined by 5' rapid amplification of cDNA ends using nested antisense primers. The full-length cDNA contains 1648 bp and a single open reading frame of 1347 bp and is expected to encode a protein of approximately 50 kD. No significant homology with other reported genes and proteins was found. Structural predictions, deletion analysis, and gel overlay analysis suggested that the calmodulin-binding domain was a basic amphiphilic alpha-helix near the C terminus of the protein. The strong induction of the mRNA for this protein suggests a role for Ca2+/calmodulin-mediated process in the heat-shock response.     

The F-box protein FBX4 targets PIN2/TRF1 for ubiquitin-mediated degradation and regulates telomere maintenance

Pin2/TRF1 was identified previously as both a protein (TRF1) that binds to telomeric DNA repeats and as a protein (Pin2) that associates with the kinase NIMA and suppresses its mitosis inducing activity. Pin2/TRF1 negatively regulates telomere length and also plays a critical role in cell cycle checkpoint control. Pin2/TRF1 is down-regulated in many human cancers and may be degraded by the ubiquitin-proteasome pathway, but components of the pathway involved in Pin2/TRF1 turnover have not been elucidated. By using the two-hybrid system, we recently identified Pin2/TRF1-interacting proteins, PinX1-4, and we demonstrated that PinX1 is a conserved telomerase inhibitor and a putative tumor suppressor. Here we report the characterization of PinX3. PinX3 was later found to be identical to Fbx4, a member of the F-box family of proteins, which function as substrate-specific adaptors of Cul1-based ubiquitin ligases. Fbx4 interacts with both Pin2 and TRF1 isoforms and promotes their ubiquitination in vitro and in vivo. Moreover, overexpression of Fbx4 reduces endogenous Pin2/TRF1 protein levels and causes progressive telomere elongation in human cells. In contrast, inhibition of Fbx4 by RNA interference stabilizes Pin2/TRF1 and promotes telomere shortening, thereby impairing cell growth. These results demonstrate that Fbx4 is a central regulator of Pin2/TRF1 protein abundance and that alterations in the stability of Pin2/TRF1 can have a dramatic impact on telomere length. Thus, Fbx4 may play a critical role in telomere maintenance.     

Cloning and characterization of a novel human gene encoding a zinc finger protein with 25 fingers

This study reports cloning and characterization of a human cDNA encoding a novel human zinc finger protein, ZFD25. ZFD25 cDNA is 6118 bp long and has an open reading frame of 2352 bp that encodes a 783 amino acid protein with 25 C2H2-type zinc fingers. The ZFD25 cDNA also contains a region with high sequence similarity to the Krüppel-associated box A and B domain in the 5'-untranslated region, suggesting that ZFD25 belongs to the Krüppel-associated box zinc finger protein family. The ZFD25 gene was localized to chromosome 7q11.2. Northern blot analysis showed that ZFD25 was expressed in a wide range of human organs. In cultured endothelial cells, the mRNA level was decreased upon serum starvation.     

Characterization of SKM1, a Saccharomyces cerevisiae gene encoding a novel Ste20/PAK-like protein kinase

Ste20/PAK serine/threonine protein kinases have been suggested as playing essential roles in cell signalling and morphogenesis as potential targets of Cdc42 and Rac GTPases. We have isolated and characterized the Saccharomyces cerevisiae SKM1 gene, which codes for a novel member of this family of protein kinases. The amino acid sequence analysis of Skm1p revealed the presence of a PH domain and a putative p21-binding domain near its amino terminus, suggesting its involvement in cellular signalling or cytoskeletal functions. However, deletion of SKM1 produced no detectable phenotype under standard laboratory conditions. Moreover, disruption of each of the two other S. cerevisiae Ste20/PAK-like kinase-encoding genes, STE20 and CLA4 , in skm1 backgrounds, showed that Skm1p is not redundant with Ste20p or Cla4p. Interestingly, overexpression of SKM1 led to morphological alterations, indicating a possible role for this protein in morphogenetic control. Furthermore, overproduction of Skm1p lacking its N-terminus caused growth arrest. This effect was also seen when similarly truncated versions of Ste20p or Cla4p were overexpressed. We further observed that overproduction of this C-terminal fragment of Skm1p complements the mating defect of a ste20 mutant strain. These results suggest that the N-terminal domains of S. cerevisiae Ste20/PAK-like protein kinases share a negative regulatory function and play a role in substrate specificity.     

Characterization of fortilin, a novel antiapoptotic protein

Apoptosis is meticulously controlled in living organisms. Its dysregulation has been shown to play a key role in a number of human diseases, including neoplastic, cardiovascular, and degenerative disorders. Bcl-2 family member proteins and inhibitors of apoptosis proteins are two major negative regulators of apoptosis. We report here the characterization of novel antiapoptotic protein, fortilin, which we identified through yeast two-hybrid library screening. Sequence analysis of fortilin revealed it to be a 172-amino acid polypeptide highly conserved from mammals to plants. Fortilin is structurally unrelated to either Bcl-2 family member proteins or inhibitors of apoptosis proteins. Northern blot analysis showed the fortilin message to be ubiquitous in normal tissue but especially abundant in the liver, kidney, and small intestine. Western blot analysis using anti-fortilin antibody showed more extensive expression in cancerous cell lines (H1299, MCF-7, and A549) than in cell lines derived from normal tissue (HEK293). Immunocytochemistry using HeLa cells transiently expressing FLAG-tagged fortilin and immunohistochemistry using human breast ductal carcinoma tissue and anti-fortilin antibody both showed that fortilin is predominantly localized in the nucleus. Functionally, the transient overexpression of fortilin in HeLa cells prevented them, in a dose-dependent fashion, from undergoing etoposide-induced apoptosis. Consistently, U2OS cells stably expressing fortilin protected the cells from cell death induced by etoposide over various concentrations and durations of exposure. In addition, fortilin overexpression inhibited caspase-3-like activity as assessed by the cleavage of fluorogenic substrate benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)coumarin. Furthermore, the antisense depletion of fortilin from breast cancer cell line MCF-7 was associated with massive cell death. These data suggest that fortilin represents a novel antiapoptotic protein involved in cell survival and apoptosis regulation.     

AhSSK1, a novel SKP1-like protein that interacts with the S-locus F-box protein SLF

The S-locus F-box (SLF/SFB) protein, recently identified as the pollen determinant of S-RNase-based self-incompatibility (SI) in Solanaceae, Scrophulariaceae and Rosaceae, has been proposed to serve as the subunit of an SCF (SKP1-CUL1-F-box) ubiquitin ligase and to target its pistil counterpart S-RNase during the SI response. However, the underlying mechanism is still in dispute, and the putative SLF-binding SKP1-equivalent protein remains unknown. Here, we report the identification of AhSSK1, Antirrhinum hispanicumSLF-interacting SKP1-like1, using a yeast two-hybrid screen against a pollen cDNA library. GST pull-down assays confirmed the SSK1-SLF interaction, and showed that AhSSK1 could connect AhSLF to a CUL1-like protein. AhSSK1, despite having a similar secondary structure to other SKP1-like proteins, appeared quite distinctive in sequence and unique in a phylogenetic analysis, in which no SSK1 ortholog could be predicted in the sequenced genomes of Arabidopsis and rice. Thus, our results suggest that the pollen-specific SSK1 could be recruited exclusively as the adaptor of putative SCF(SLF) in those plants with S-RNase-based SI, providing an important clue to dissecting the function of the pollen determinant.     

Characterization, evolution and tissue-specific expression of AmphiCalbin, a novel gene encoding EF-hand calcium-binding protein in amphioxus Branchiostoma belcheri

An amphioxus full-length cDNA, AmphiCalbin, encoding a novel EF-hand calcium-binding protein （EFCaBP）, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri. It consists of 1321 bp with a 636 bp open reading frame encoding a protein of 211 amino acids with a molecular mass of approximately 24.5 kDa. The phylogenetic analysis offers two interesting inferences. First, AmphiCalbin clusters with a group of unnamed EFCaBPs that are differentiated from other identified EFCaBPs. Second, AmphiCalbin falls at the base of the vertebrate unnamed EFCaBPs clade, probably representing their prototype. This is also corroborated by the fact that AmphiCalbin has an exon-intron organization identical to that of vertebrate unnamed EFCaBP genes. Both tissue-section in situ hybridization and whole-mount in situ hybridization prove a tissue-specific expression pattern of AmphiCalbin, with high levels of expression in the digestive system and gonads. It is proposed that AmphiCalbin might play a role in the digestive system and gonads. These observations lay the foundation for further understanding of the function of the unnamed EFCaBPs.     

NABP1, a novel RORgamma-regulated gene encoding a single-stranded nucleic-acid-binding protein

RORgamma2 (retinoid-related orphan receptor gamma2) plays a critical role in the regulation of thymopoiesis. Microarray analysis was performed in order to uncover differences in gene expression between thymocytes of wild-type and RORgamma-/- mice. This analysis identified a novel gene encoding a 22 kDa protein, referred to as NABP1 (nucleic-acid-binding protein 1). This subsequently led to the identification of an additional protein, closely related to NABP1, designated NABP2. Both proteins contain an OB (oligonucleotide/oligosaccharide binding) motif at their N-terminus. This motif is highly conserved between the two proteins. NABP1 is highly expressed in the thymus of wild-type mice and is greatly suppressed in RORgamma-/- mice. During thymopoiesis, NABP1 mRNA expression is restricted to CD4+CD8+ thymocytes, an expression pattern similar to that observed for RORgamma2. These observations appear to suggest that NABP1 expression is regulated either directly or indirectly by RORgamma2. Confocal microscopic analysis showed that the NABP1 protein localizes to the nucleus. Analysis of nuclear proteins by size-exclusion chromatography indicated that NABP1 is part of a high molecular-mass protein complex. Since the OB-fold is frequently involved in the recognition of nucleic acids, the interaction of NABP1 with various nucleic acids was examined. Our results demonstrate that NABP1 binds single-stranded nucleic acids, but not double-stranded DNA, suggesting that it functions as a single-stranded nucleic acid binding protein.     

Isolation and characterization of a novel F-box protein Pof10 in fission yeast

The SCF complex is a type of ubiquitin-protein ligase (E3) that consists of invariable components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Using a yeast two-hybrid system, we isolated six proteins that interact with Schizosaccharomyces pombe Skp1. Among them, Pof10 is a novel F-box protein consisting of 662 amino acids, harboring the F-box domain required for the binding to Skp1 and followed by four WD40 repeats. Overexpression of Pof10 in fission yeast resulted in loss of viability with marked morphological changes that are similar to those in pop1 mutant yeast. Coexpression of Skp1 with Pof10 prevented the lethality, suggesting that the lethality from Pof10 overexpression results from the sequestration of Skp1 from other F-box proteins including Pop1. Whereas most F-box proteins show rapid turnover, Pof10 has a remarkably long half-life in vivo and has been shown to be localized predominantly in cytoplasm. These results suggest that the stable F-box protein Pof10 might target abundant cytoplasmic proteins for degradation in fission yeast.