Z (Glu342 --> Lys) and S(iiyama) (Ser53 --> Phe) genetic variations of human alpha1-antitrypsin (alpha1-AT) cause a secretion blockage in the hepatocytes, leading to alpha1-AT deficiency in the plasma. Using in vitro folding analysis, we have shown previously that these mutations interfere with the proper folding of polypeptides. To understand the fundamental cause for the secretion defect of the Z and S(iiyama) variants of alpha1-AT, we investigated in vivo folding and stability of these variant alpha1-AT using the secretion system of yeast Saccharomyces cerevisiae. Various thermostable mutations suppressing the folding block of the Z variant in vitro corrected the secretion defect as well as the intracellular degradation in the yeast secretion system. Significantly, the extent of suppression in the secretion defect of Z protein was proportional to the extent of suppression in the folding defect, assuring that the in vivo defect associated with the Z variant is primarily derived from the folding block. In contrast, the folding and secretion efficiency of S(iiyama) was not much improved by the same mutations. In addition, none of the rarely secreted S(iiyama) alpha1-AT carrying the stabilizing mutations for the wild type and Z variant were active. It appears that the major defect in S(iiyama) variant is the loss of stability in contrast to the kinetic block of folding in the Z variant.

Reversible posttranslational modifications on specific amino acid residues can efficiently regulate protein functions. O-Phosphorylation is the prototype and analogue to the rapidly emerging mechanism of regulation known as S-glutathionylation. The latter is being recognized as a potentially widespread form of modulation of the activities of redox-sensitive thiol proteins, especially those involved in signal transduction pathways and translocation. The abundance of reduced glutathione in cells and the ready conversion of sulfenic acids and S-nitroso derivatives to S-glutathione mixed disulfides support the notion that reversible S-glutathionylation is likely to be the preponderant mode of redox signal transduction. The glutaredoxin enzyme has served as a focal point and important tool for evolution of this regulatory mechanism because of its characterization as a specific and efficient catalyst of protein-SSG de-glutathionylation (akin to phosphatases). Identification of specific mechanisms and enzyme(s) that catalyze formation of protein-SSG intermediates, however, is largely unknown and represents a prime objective for furthering understanding of this evolving mechanism of cellular regulation. Several proteomic approaches, including the use of cysteine-reactive fluorescent and radiolabel probes, have been developed to detect arrays of proteins whose cysteine residues are modified in response to oxidants, thus identifying them as potential interconvertible proteins to be regulated by redox signaling (glutathionylation). Specific criteria were used to evaluate current data on cellular regulation via S-glutathionylation. Among many proteins under consideration, actin, protein tyrosine phosphatase-1B, and Ras stand out as the best current examples for establishing this regulatory mechanism.

Mutations in ␣-synuclein are known to be associated with Parkinson's disease (PD). The coexistence of this neuronal protein with ubiquitin and proteasome subunits in Lewy bodies in sporadic disease suggests that alterations of ␣-synuclein catabolism may contribute to the pathogenesis of PD. The degradation pathway of ␣-synuclein has not been identified nor has the kinetics of this process been described. We investigated the degradation kinetics of both wild-type and A53T mutant 6XHis-tagged ␣-synuclein in transiently transfected SH-SY5Y cells. Degradation of both isoforms followed firstorder kinetics over 24 h as monitored by the pulse-chase method. However, the t1 ⁄2 of mutant ␣-synuclein was 50% longer than that of the wild-type protein (p < 0.01). The degradation of both recombinant proteins and endogenous ␣-synuclein in these cells was blocked by the selective proteasome inhibitor ␤-lactone (40 M), indicating that both wild-type and A53T mutant ␣-synuclein are degraded by the ubiquitin-proteasome pathway. The slower degradation of mutant ␣-synuclein provides a kinetic basis for its intracellular accumulation, thus favoring its aggregation.