A functional genomic approach, based on systematic data gathering, was used to characterize a family of proteins containing a tripartite motif (TRIM). A total of 37 TRIM genes/proteins were studied, 21 of which were novel. The results demonstrate that TRIM proteins share a common function: by means of homo-multimerization they identify speci®c cell compartments.

The TRIM/RBCC proteins are defined by the presence of the tripartite motif composed of a RING domain, one or two B-box motifs and a coiled-coil region. These proteins are involved in a plethora of cellular processes such as apoptosis, cell cycle regulation and viral response. Consistently, their alteration results in many diverse pathological conditions. The highly conserved modular structure of these proteins suggests that a common biochemical function may underlie their assorted cellular roles. Here, we review recent data indicating that some TRIM/RBCC proteins are implicated in ubiquitination and propose that this large protein family represents a novel class of 'single protein RING finger' ubiquitin E3 ligases.

X-linked recessive chondrodysplasia punctata (CDPX) is a congenital defect of bone and cartilage development characterized by aberrant bone mineralization, severe underdevelopment of nasal cartilage, and distal phalangeal hypoplasia. A virtually identical phenotype is observed in the warfarin embryopathy, which is due to the teratogenic effects of coumarin derivatives during pregnancy. We have cloned the genomic region within Xp22.3 where the CDPX gene has been assigned and isolated three adjacent genes showing highly significant homology to the sulfatase gene family. Point mutations in one of these genes were identified in five patients with CDPX. Expression of this gene in COS cells resulted in a heat-labile arylsulfatase activity that is inhibited by warfarin. A deficiency of a heat-labile arylsulfatase activity was demonstrated in patients with deletions spanning the CDPX region. These data indicate that CDPX is caused by an inherited deficiency of a novel sulfatase and suggest that warfarin embryopathy might involve drug-induced inhibition of the same enzyme.

BackgroundThe TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection.ResultsHere we defined the entire human TRIM family and also identified the TRIM sets of other vertebrate (mouse, rat, dog, cow, chicken, tetraodon, and zebrafish) and invertebrate species (fruitfly, worm, and ciona). By means of comparative analyses we found that, after assembly of the tripartite motif in an early metazoan ancestor, few types of C-terminal domains have been associated with this module during evolution and that an important increase in TRIM number occurred in vertebrate species concomitantly with the addition of the SPRY domain. We showed that the human TRIM family is split into two groups that differ in domain structure, genomic organization and evolutionary properties. Group 1 members present a variety of C-terminal domains, are highly conserved among vertebrate species, and are represented in invertebrates. Conversely, group 2 is absent in invertebrates, is characterized by the presence of a C-terminal SPRY domain and presents unique sets of genes in each mammal examined. The generation of independent sets of group 2 genes is also evident in the other vertebrate species. Comparing the murine and human TRIM sets, we found that group 1 and 2 genes evolve at different speeds and are subject to different selective pressures.ConclusionWe found that the TRIM family is composed of two groups of genes with distinct evolutionary properties. Group 2 is younger, highly dynamic, and might act as a reservoir to develop novel TRIM functions. Since some group 2 genes are implicated in innate immune response, their evolutionary features may account for species-specific battles against viral infection.

Proteins of the Myc and Mad family are involved in transcriptional regulation and mediate cell differentiation and proliferation. These molecules share a basic-helix-loop-helix leucine zipper domain (bHLHZip) and bind DNA at the E box (CANNTG) consensus by forming heterodimers with Max. We report the isolation, characterization and mapping of a human gene and its mouse homolog encoding a new member of this family of proteins, named Rox. Through interaction mating and immunoprecipitation techniques, we demonstrate that Rox heterodimerizes with Max and weakly homodimerizes. Interestingly, bandshift assays demonstrate that the Rox-Max heterodimer shows a novel DNA binding specificity, having a higher affinity for the CACGCG site compared with the canonical E box CACGTG site. Transcriptional studies indicate that Rox represses transcription in both human HEK293 cells and yeast. We demonstrate that repression in yeast is through interaction between the N-terminus of the protein and the Sin3 co-repressor, as previously shown for the other Mad family members. ROX is highly expressed in quiescent fibroblasts and expression markedly decreases when cells enter the cell cycle. Moreover, ROX expression appears to be induced in U937 myeloid leukemia cells stimulated to differentiate with 12-O-tetradecanoylphorbol-13-acetate. The identification of a novel Max-interacting protein adds an important piece to the puzzle of Myc/Max/Mad coordinated action and function in normal and pathological situations. Furthermore, mapping of the human gene to chromosome 17p13.3 in a region that frequently undergoes loss of heterozygosity in a number of malignancies, together with the biochemical and expression features, suggest involvement of ROX in human neoplasia.

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Opitz syndrome (OS) is a multiple congenital anomaly manifested by abnormal closure of midline structures. The gene responsible for the X-linked form of this disease, MID1, encodes a protein (midin) that contains a RING, two B-boxes, a coiled-coil (the so-called tripartite motif) and an RFP-like domain. The tripartite motif is characteristic of a family of proteins, named the B-box family, involved in cell proliferation and development. Since the subcellular compartmentalization and the ability to form multiprotein structures both appear to be crucial for the function of this family of proteins, we have studied these properties on the wild-type and mutated forms of midin. We found that endogenous midin is associated with microtubules throughout the cell cycle, co-localizing with cytoplasmic fibres in interphase and with the mitotic spindle and midbodies during mitosis and cytokinesis. Immunoprecipitation experiments demonstrated the ability of the tripartite motif to mediate midin homodimerization, consistent with the evidence, obtained by gel filtration analysis, that midin exists in the form of large protein complexes. Functional characterization of altered forms of midin, resulting from mutations found in OS patients, revealed that association with microtubules is compromised, while the ability to homodimerize and form multiprotein complexes is retained. We suggest that midin is involved in the formation of multiprotein structures acting as anchor points to microtubules and that impaired association with these cytoskeletal structures causes OS developmental defects.

Opitz syndrome (OS) is a human genetic disease characterized by deformities such as cleft palate that are attributable to defects in embryonic development at the midline. Gene mapping has identified OS mutations within a protein called Mid1. Wild-type Mid1 predominantly colocalizes with microtubules, in contrast to mutant versions of Mid1 that appear clustered in the cytosol. Using yeast two-hybrid screening, we found that the ␣4-subunit of protein phosphatases 2A͞4͞6 binds Mid1. Epitope-tagged ␣4 coimmunoprecipitated endogenous or coexpressed Mid1 from COS7 cells, and this required only the conserved C-terminal region of ␣4. Localization of Mid1 and ␣4 was influenced by one another in transiently transfected cells. Mid1 could recruit ␣4 onto microtubules, and high levels of ␣4 could displace Mid1 into the cytosol. Metabolic 32 P labeling of cells showed that Mid1 is a phosphoprotein, and coexpression of full-length ␣4 decreased Mid1 phosphorylation, indicative of a functional interaction. Association of green fluorescent protein-Mid1 with microtubules in living cells was perturbed by inhibitors of MAP kinase activation. The conclusion is that Mid1 association with microtubules, which seems important for normal midline development, is regulated by dynamic phosphorylation involving MAP kinase and protein phosphatase that is targeted specifically to Mid1 by ␣4. Human birth defects may result from environmental or genetic disruption of this regulatory cycle.