👤 Natosha L Finley

Ubiquitin chain specificity has been described for some deubiquitinases (DUBs) but lacks a comprehensive profiling in vivo. We used quantitative proteomics to compare the seven lysine-linked ubiquitin chains between wild-type yeast and its 20 DUB-deletion strains, which may reflect the linkage specificity of DUBs in vivo. Utilizing the specificity and ubiquitination heterogeneity, we developed a method termed DUB-mediated identification of linkage-specific ubiquitinated substrates (DILUS) to screen the ubiquitinated lysine residues on substrates modified with certain chains and regulated by specific DUB. Then we were able to identify 166 Ubp2-regulating substrates with 244 sites potentially modified with K63-linked chains. Among these substrates, we further demonstrated that cyclophilin A (Cpr1) modified with K63-linked chain on K151 site was regulated by Ubp2 and mediated the nuclear translocation of zinc finger protein Zpr1. The K48-linked chains at non-K151 sites of Cpr1 were mainly regulated by Ubp3 and served as canonical signals for proteasome-mediated degradation. Show less

Hypertrophic cardiomyopathy (HCM) results from mutations in genes encoding sarcomeric proteins, most often MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). A recently discovered HCM-associated 25-base pair deletion in MYBPC3 is inherited in millions worldwide. Although this mutation causes changes in the C10 domain of cMyBP-C (cMyBP-C(C10mut)), which binds to the light meromyosin (LMM) region of the myosin heavy chain, the underlying molecular mechanism causing HCM is unknown. In this study, adenoviral expression of cMyBP-C(C10mut) in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca(2+) transients, compared with wild-type cMyBP-C expression (cMyBP-C(WT)) and controls. Forty-eight hours after infection, 44% of cMyBP-C(WT) and 36% of cMyBP-C(C10mut) protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-C(C10mut) to the C-zone, whereas cMyBP-C(WT) mostly showed C-zone staining, suggesting that cMyBP-C(C10mut) could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-C(C10mut) resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-C(C10mut) displayed significantly reduced fractional shortening, sarcomere shortening, and relaxation velocities, apparently caused by defects in sarcomere function, because Ca(2+) transients were unaffected. Co-sedimentation and protein cross-linking assays confirmed that C10(mut) causes the loss of C10 domain interaction with myosin LMM. Protein homology modeling studies showed significant structural perturbation in cMyBP-C(C10mut), providing a potential structural basis for the alteration in its mode of interaction with myosin LMM. Therefore, expression of cMyBP-C(C10mut) protein is sufficient to cause contractile dysfunction in vitro. Show less

Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients. Show less

The G1 cyclin Cln3 of the yeast Saccharomyces cerevisiae is rapidly degraded by the ubiquitin-proteasome pathway. This process is triggered by p34CDC28-dependent phosphorylation of Cln3. Here we demonstrate that the molecular chaperone Ydj1, a DnaJ homolog, is required for this phosphorylation. In a ydj1 mutant at the nonpermissive temperature, both phosphorylation and degradation of Cln3 were deficient. No change was seen upon inactivation of Sis1, another DnaJ homolog. The phosphorylation defect in the ydj1 mutant was specific to Cln3, because no reduction in the phosphorylation of Cln2 or histone H1, which also requires p34CDC28, was observed. Ydj1 was required for Cln3 phosphorylation and degradation rather than for the proper folding of this cyclin, since Cln3 produced in the ydj1 mutant was fully active in the stimulation of p34CDC28 histone kinase activity. Moreover, Ydj1 directly associates with Cln3 in close proximity to the segment that is phosphorylated and signals degradation. Thus, binding of Ydj1 to this domain of Cln3 seems to be essential for the phosphorylation and breakdown of this cyclin. In a cell-free system, purified Ydj1 stimulated the p34CDC28-dependent phosphorylation of the C-terminal segment of Cln3 and did not affect phosphorylation of Cln2 (as was found in vivo). The reconstitution of this process with pure components provides evidence of a direct role for the chaperone in the phosphorylation of Cln3. Show less

Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events. Show less