Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s. To identify proteins that can interact with the human E2 UbcH7, a yeast twohybrid screen was performed. Two proteins were identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the presence of RING finger and in between RING fingers (IBR) domains. No other overt structural similarity was observed between the two proteins. In vitro binding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar that both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway.The primary role of the protein ubiquitinylation pathway is the targeting of intracellular substrate proteins for degradation (1). In this process, ubiquitin is first activated in an ATPdependent step forming a thioester bond with an ubiquitinactivating enzyme (E1). Ubiquitin is then transferred to an ubiquitin-conjugating enzyme (E2), 1 retaining the high energy thioester bond. Thereafter, the E2, alone or in conjunction with an ubiquitin-protein ligase (E3), catalyzes the final attachment of ubiquitin to the target protein (2). Ubiquitin itself can then serve as a ubiquitinylation substrate, resulting in the generation of polyubiquitinylated proteins possibly with the aid of an E4 (3). Finally, ubiquitinylated proteins are recognized and degraded by the 26 S proteasome. It has been proposed that the selection of an individual protein for proteasomal degradation via the ubiquitin pathway requires a unique combination of an E2 and E3. In S. cerevisiae, some 13 E2s or E2-related proteins have been identified, and many more are present in higher eukaryotes (2). E2s are characterized by a conserved catalytic domain of approximately 150 amino acid residues. Despite their functional redundancy, individual E2s appear to be involved in different cellular processes and, therefore, in the ubiquitinylation of different substrate proteins. The distinct substrate specificity of E2s is at least in part explained by the observation that different E2s interact with different E3s.Four classes of E3 have been identified to date; in contrast to E2s, they exhibit no overt sequence homology. These are yeast UBR1 and its mammalian homologues (4), mammalian E6-AP

Ubiquitin-conjugating enzymes (E2s) are essential components of the post-translational protein ubiquitination pathway, mediating the transfer of activated ubiquitin to substrate proteins. We have identified a human gene, UBE2L3, localized on Chromosome (Chr) 22q11. 2-13.1, encoding an E2 almost identical to that encoded by the recently described human L-UBC (UBE2L1) gene present on Chr 14q24.3. Using chromosome-specific vectorette PCR, we have determined the intron/exon structure of UBE2L3. In contrast to the intronless UBE2L1 gene, the coding sequence of UBE2L3 is interrupted by three large introns. UBE2L3-derived mRNA appears to be the predominant species in most tissues rather than the transcript from UBE2L1 or another homologous gene UBE2L2, which maps to Chr 12q12. We also present additional evidence that these genes are members of a larger multigene family. The primary sequence of the protein encoded by UBE2L3 is identical to partial peptide sequence derived from the rabbit E2 'E2-F1,' suggesting that we have identified the human homolog of this protein. This latter E2 has been demonstrated to participate in transcription factor NF-kappaB maturation, c-fos degradation, and human papilloma virus-mediated p53 degradation in vitro.

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We have modified the automated differential display reverse transcription polymerase chain reaction technique (DDRT-PCR) such that a single fluorescently labeled universal primer (d(F)CTCACG-GATCCGTCGATTTT) is used in all PCRs together with a selection of arbitrary primers. We term this fluorescent detection procedure FDDRT-PCR. Anchoring primers of general structure dTGGTCTCACGGATCCTCGA-(T)12 VN (where N can be any deoxynucleoside and V can be any deoxynucleoside other than thymidine) are used for the RT step, and the universal primer together with selected arbitrary primers are then used for the PCR amplification. Advantages of this approach are: (i) the fluorescently labeled universal primer is a constant feature in every PCR, so that changes in banding profile are highly likely to reflect the incorporation of different arbitrary 10-mer primers; (ii) artifacts that result from arbitrary 10-mer to arbitrary 10-mer primer amplifications are not observed by fluoresence detection on an automated gene scanner because such products are not fluorescently labeled; (iii) sample throughput and ease of data handling are increased when compared with the conventional radioactive/manual approach and (iv) using a single fluorescently labeled primer in all PCRs is highly cost-effective.

We have identified a novel ubiquitin conjugating enzyme gene, L-UBC, which maps to human Chromosome (Chr) 14q24.3. This is also the location of the major early onset familial Alzheimer's disease gene (FAD3). L-UBC encodes a protein that demonstrates homology to the yeast ubiquitin conjugating enzyme, UBC-4, and human UbcH5. Their functions are to ubiquitinate specific proteins targeted for degradation. The protein also exhibits very strong homology to a rabbit protein, E2-F1, which mediates p53 degradation driven by papilloma virus E6 protein in vitro. The accumulation of specific proteins that have undergone aberrant processing in neurofibrillary tangles and amyloid plaques is the classic pathological feature in brains of Alzheimer's disease patients. Abnormal ubiquitination has previously been suggested to play a role in the etiology of Alzheimer's disease. This gene therefore represents a plausible candidate gene for FAD3.

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Mutations of the presenilin 1 (PS-i) gene at the Alzheimer's disease (AD) FAD3 locus on chromosome i4q24.3 are responsible for the majority of familial early-onset AD. As genes responsible for familial forms of AD are obvious candidates for further investigation in 'sporadic" disease, we performed a molecular analysis of PS-i transcripts extracted from brain tissues of a series of histologically confirmed cases of "sporadic" AD (n = 10) and also from histologically "normal" (non-Alzheimer) age-matched brain controls (n = 5). No sequence changes in the PS-i coding sequence were detected after analysis by reverse transcription-PCR. This suggests that the frequency of mutations in the PS-i (S182) coding region in "sporadic" Alzheimer's disease is very low. However, we demonstrated that the PS-i gene is highly variably spliced. One splice variant involves the 5' untranslated region of the PS-i gene only and hence encodes for normal PS-i. Six further splice variants involve coding regions ofthe PS-i gene and result in truncated proteins lacking specific transmembrane domains. Most of these variants do not coincide with recognized sites of introns in the PS-i gene. One of these variants, resulting in the loss of transmembrane domain TM-Vll, was found only in an AD patient.

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