Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. We hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. We have now identified a protein complex in normal human brain that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD failed to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. We conclude that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD.

The gene MID1, the mutation of which causes X-linked Opitz G/BBB syndrome (OS, MIM 300000), encodes a microtubule-associated protein (MAP). We show that mutation of MID1 leads to a marked accumulation of the catalytic subunit of protein phosphatase 2A (PP2Ac), a central cellular regulator. PP2Ac accumulation is caused by an impairment of a newly identified E3 ubiquitin ligase activity of the MID1 protein that normally targets PP2Ac for degradation through binding to its alpha4 regulatory subunit in an embryonic fibroblast line derived from a fetus with OS. Elevated PP2Ac causes hypophosphorylation of MAPs, a pathological mechanism that is consistent with the OS phenotype.

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

One of the three structural glycoproteins of classical swine fever virus (CSFV) is E0, a disulfide-bonded homodimer that induces virus-neutralizing antibodies and occurs in a virion-bound as well as a secreted form. E0 was shown to be similar to a family of fungal and plant ribonucleases. Purified E0 from CSFV-infected cells was a potent ribonuclease specific for uridine and inhibitable by zinc ions.

E6-AP, a 100-kDa cellular protein, was originally identified through its interaction with the E6 protein of the oncogenic human papillomavirus types 16 and 18. The complex of E6-AP and E6 specifically interacts with p53 and mediates ubiquitination of p53 in concert with the E1 ubiquitin-activating enzyme and the E2 ubiquitinconjugating enzyme UbcH5. Recent results suggest that E6-AP is representative of a family of putative ubiquitinprotein ligases. Members of this family are characterized by a conserved C-terminal region, termed hect domain. In this paper, we describe the isolation of two human E2s, designated as UbcH6 and UbcH7, that in addition to UbcH5 can interact with E6-AP. UbcH6 is a novel member of an evolutionally conserved subfamily of E2s that includes UbcH5 and Saccharomyces cerevisiae UBC4. Although UbcH7 does not appear to be a member of this subfamily, UbcH7 efficiently substitutes for UbcH5 in E6-AP-dependent ubiquitination. Surprisingly, UbcH6 was only weakly active in this particular assay. In addition, UbcH5 but not UbcH6 or UbcH7 efficiently interacts with the hect protein RSP5. These results indicate that E6-AP can interact with at least two species of E2 and that different hect proteins may interact with different E2s.The ubiquitin system represents a major pathway involved in selective protein degradation (1-4). This pathway first requires the covalent attachment of ubiquitin, a highly conserved 76-amino acid protein, to defined lysine residues of substrate proteins. Ubiquitin-protein conjugates are then recognized and degraded by a specific protease complex, the 26 S proteasome. Protein ubiquitination involves three classes of enzymes. These are the ubiquitin-activating enzyme E1, 1 the ubiquitin-conjugating enzymes E2, and the ubiquitin-protein ligases E3. Ubiquitin is first activated by E1 via formation of a thioester bond between the carboxyl-terminal glycine of ubiquitin and a cysteine residue of E1. The activated ubiquitin is then transferred to one of a number of E2s preserving the high energy thioester bond. The E2s have then been thought to catalyze the final attachment of ubiquitin to a substrate protein, often in concert with E3s. E3s have been proposed to function by specifically binding to substrate proteins that are otherwise not recognized by E2s. Recent results, however, suggest that at least some E3s may also be directly involved in the final transfer of ubiquitin to a substrate protein (5).Only two genes encoding proteins with E3 activity have been cloned so far. These are UBR1 of Saccharomyces cerevisiae (6) and human E6-AP (7). E6-AP was originally identified through its interaction with the E6 oncoprotein of the cancer-associated human papillomavirus types 16 and 18 (8). The E6⅐E6-AP complex specifically binds to the tumor suppressor protein p53 and induces its ubiquitination and subsequent degradation (7, 9, 10). An essential intermediate step in E6-AP-dependent protein ubiquitination is the formation of a thioester complex between ubiquitin and E6-AP (5). Furthermor...