OTUB (otubain) 1 is a human deubiquitinating enzyme that is implicated in mediating lymphocyte antigen responsiveness, but whose molecular function is generally not well defined. A structural analysis of OTUB1 shows differences in accessibility to the active site and in surface properties of the substrate-binding regions when compared with its close homologue, OTUB2, suggesting variations in regulatory mechanisms and substrate specificity. Biochemical analysis reveals that OTUB1 has a preference for cleaving Lys(48)-linked polyubiquitin chains over Lys(63)-linked polyubiquitin chains, and it is capable of cleaving NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), but not SUMO (small ubiquitin-related modifier) 1/2/3 and ISG15 (interferon-stimulated gene 15) conjugates. A functional comparison of OTUB1 and OTUB2 indicated a differential reactivity towards ubiquitin-based active-site probes carrying a vinyl methyl ester, a 2-chloroethyl or a 2-bromoethyl group at the C-terminus. Mutational analysis suggested that a narrow P1' site, as observed in OTUB1, correlates with its ability to preferentially cleave Lys(48)-linked ubiquitin chains. Analysis of cellular interaction partners of OTUB1 by co-immunoprecipitation and MS/MS (tandem mass spectrometry) experiments demonstrated that FUS [fusion involved in t(12;6) in malignant liposarcoma; also known as TLS (translocation in liposarcoma) or CHOP (CCAAT/enhancer-binding protein homologous protein)] and RACK1 [receptor for activated kinase 1; also known as GNB2L1 (guanine-nucleotide-binding protein beta polypeptide 2-like 1)] are part of OTUB1-containing complexes, pointing towards a molecular function of this deubiquitinating enzyme in RNA processing and cell adhesion/morphology.

The gene encoding the human TLS protein, also termed FUS, is located at the site of chromosomal translocations in human leukemias and sarcomas where it forms a chimeric fusion gene with one of several different genes. To identify interacting partners of TLS, we screened a yeast two-hybrid cDNA library constructed from mouse hematopoietic cells using the C-terminal region of TLS in the bait plasmid. Two cDNAs encoding members of the serine-arginine (SR) family of proteins were isolated. The first SR protein is the mouse homolog of human splicing factor SC35, and the second SR member is a novel 183-amino acid protein that we term TASR (TLS-associated serine-arginine protein). cDNA cloning of human TASR indicated that mouse and human TASR have identical amino acid sequences. The interactions between TLS and these two SR proteins were confirmed by co-transfection and immunoprecipitation studies. In vivo splicing assays indicated that SC35 and TASR influence splice site selection of adenovirus E1A pre-mRNA. TLS may recruit SR splicing factors to specific target genes through interaction with its C-terminal region, and chromosomal translocations that truncate the C-terminal region of TLS may prevent this interaction. Thus TLS translocations may alter RNA processing and play a role in malignant transformation.

p63, a transcription factor related to the p53 tumor suppressor, plays a key role in epidermal differentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (DeltaN) a transactivation domain. DeltaNp63alpha is the most widely expressed isoform, at all stages of development and in adult tissues. It supports the regenerative capacity of basal keratinocytes and its upregulation is a hallmark of human squamous carcinomas. To get insight into the complex biology of DeltaNp63alpha, we set out to identify DeltaNp63alpha interacting proteins by co-immunoprecipitation in mammalian cells and mass spectrometry analysis. A total of 49 potential DeltaNp63alpha binding proteins, including several heterogeneous ribonucleoproteins (hnRNPs), were identified. Integration of the proteomic data with a Human Coexpression Network highlighted 5 putative p63 protein interactors whose expression is significantly comodulated with p63: hnRNPA/B, hnRNPK, hnRNPQ, FUS/TLS and Keratin 5. hnRNPA/B was already described as a p63 partner, but the others were novel. Interaction of DeltaNp63alpha with hnRNPQ, hnRNPK and FUS/TLS was confirmed by reciprocal co-immunoprecipitations in human keratinocytes. The finding that DeltaNp63alpha exists in complexes with several RNA-binding proteins lays the premises for the analysis of the role of DeltaNp63alpha in mRNA metabolism and transport.