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Despite decades of research, the pathophysiology of bipolar disorder (BD) is still not well understood. Structural brain differences have been associated with BD, but results from neuroimaging studies have been inconsistent. To address this, we performed the largest study to date of cortical gray matter thickness and surface area measures from brain magnetic resonance imaging scans of 6503 individuals including 1837 unrelated adults with BD and 2582 unrelated healthy controls for group differences while also examining the effects of commonly prescribed medications, age of illness onset, history of psychosis, mood state, age and sex differences on cortical regions. In BD, cortical gray matter was thinner in frontal, temporal and parietal regions of both brain hemispheres. BD had the strongest effects on left pars opercularis (Cohen’s d=-0.293; P=1.71 × 10-21), left fusiform gyrus (d=-0.288; P=8.25 × 10-21) and left rostral middle frontal cortex (d=-0.276; P=2.99 × 10-19). Longer duration of illness (after accounting for age at the time of scanning) was associated with reduced cortical thickness in frontal, medial parietal and occipital regions. We found that several commonly prescribed medications, including lithium, antiepileptic and antipsychotic treatment showed significant associations with cortical thickness and surface area, even after accounting for patients who received multiple medications. We found evidence of reduced cortical surface area associated with a history of psychosis but no associations with mood state at the time of scanning. Our analysis revealed previously undetected associations and provides an extensive analysis of potential confounding variables in neuroimaging studies of BD.

Degradation of misfolded or tightly regulated proteins in the endoplasmic reticulum (ER) is performed by the cytosolic ubiquitin-proteasome system and therefore requires their prior transport back to the cytosol. Here, we report on the extraction and degradation mechanism of a polytopic membrane protein. Rapid proteasomal degradation of a mutated form of the ATP-binding cassette transporter Pdr5 retained in the ER is initialized at the lumenal face of the ER membrane. Using different antibodies directed against the cytosolic tails or a lumenal loop of the transmembrane protein, it could be demonstrated that the turnover of Pdr5* demands the concerted action of both the Sec61 translocon and the ubiquitin-proteasome system. We observed a stabilization of the entire molecule within the ER membrane in yeast mutants characterized by a reduced translocation capacity or by functionally attenuated proteasomes. Moreover, no degradation intermediates were detected in any of the mutants that impede degradation of Pdr5*. Therefore, initial steps are rate-limiting for cleavage and mutations that impede downstream events prevent initiation of the process. Our data suggest that ER degradation is a mechanistically highly integrated process, requiring the combined operation of components of the degradation system acting at the lumenal face of the ER membrane, the Sec61 translocon, and the ubiquitin-proteasome system.

Studies were performed to unravel the activation and maturation mechanism of vacuolar (lysosomal) proteinases in Saccharomyces cerevisiae. In vivo and in vitro studies show that proteinase yscA and proteinase yscB are involved in the activation and processing event of pro‐carboxypeptidase yscY. Processing and activation of pro‐carboxypeptidase yscY by proteinase yscA depends on an additional factor contained in the vacuolar fraction. Comparable activation can be mimicked by sodium polyphosphate. Optimum pH for processing by this proteinase yscA‐triggered event is 5. The proteinase yscA‐triggered maturation process of pro‐carboxypeptidase yscY leads to an intermediate mol. wt form of the enzyme which is, however, fully active. Proteinase yscB transfers the intermediate mol. wt form of the original precursor to the apparently authentic, mature and active carboxypeptidase yscY. An activation and maturation scheme is devised.

The biosynthesis and processing of the vacuolar (lysosomal) proteinase yscB was followed in vivo and in vitro. In vitro transcription–translation of the cloned proteinase yscB gene results in a high mol. wt precursor protein (HMr‐precursor) of Mr = 73,000. In vivo a precursor of identical mol. wt is found in sec6l mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum (ER). In contrast to N‐glycosylated intermediate mol. wt forms of proteinase yscB, the HMr‐precursor of the enzyme is not N‐glycosylated, indicating its appearance outside the ER. sec18 Mutant cells, wild type for translocation of proteins into the ER but blocked in the delivery step of secretory proteins to the Golgi apparatus, accumulate a lower mol. wt precursor (LMr‐precursor) of proteinase yscB of Mr = 41,500. Processing of the HMr‐precursor of proteinase yscB to the LMr‐precursor requires proteolytic cleavage which is independent of the proteinase yscA gene product, a protein known to be involved in the processing event of the LMr‐precursor of proteinase yscB to the mature enzyme of Mr = 33,000. A LMr‐precursor of proteinase yscB of Mr = 42,000 accumulates in proteinase yscA‐deficient mutant cells (allele pep4‐3). This form is enzymatically active. Incubation in vitro of the LMr‐precursor of proteinase yscB with mature proteinase yscA leads to further activation and to processing of the enzyme yielding the mature 33,000 Mr protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated beta-subunits, we investigated the specificities and contributions of the different beta-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active beta-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with beta5/Pre2 cleaving after hydrophobic, beta2/Pup1 after basic, and beta1/Pre3 after acidic residues, but with the exception that beta2/Pup1 and beta1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the "branched chain amino acid-preferring" component are associated with beta5/Pre2, beta1/Pre3, or beta2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a non-fermentable carbon source leads to selective and rapid degradation of fructose-1,6-bisphosphatase. This so called catabolite inactivation of the enzyme is brought about by the ubiquitin-proteasome system. To identify additional components of the catabolite inactivation machinery, we isolated three mutant strains, gid1, gid2, and gid3, defective in glucose-induced degradation of fructose-1,6-bisphospha-tase. All mutant strains show in addition a defect in catabolite inactivation of three other gluconeogenic enzymes: cytosolic malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase. These findings indicate a common mechanism for the inactivation of all four enzymes. The mutants were also impaired in degradation of short-lived N-end rule substrates, which are degraded via the ubiquitin-proteasome system. Site-directed mutagenesis of the amino-terminal proline residue yielded fructose-1,6-bisphosphatase forms that were no longer degraded via the ubiquitin-proteasome pathway. All amino termini other than proline made fructose-1,6-bisphosphatase inaccessible to degradation. However, the exchange of the amino-terminal proline had no effect on the phosphorylation of the mutated enzyme. Our findings suggest an essential function of the amino-terminal proline residue for the degradation process of fructose-1,6-bisphosphatase. Phosphorylation of the enzyme was not necessary for degradation to occur.

Proteasomes are highly complex proteases responsible for selective protein degradation in the eukaryotic cell. 26 S proteasomes consist of two regulatory 19 S cap complexes and the 20 S proteasome, which acts as the proteolytic core module. We isolated six mutants of the yeast Saccharomyces cerevisiae containing mutations in the 20 S proteasome beta-type subunit Pre3. Three mutations (pre3-2, pre3-3, and pre3-5) which reside at the active site cleft of the Pre3 subunit solely caused reduction of the proteasomal peptidylglutamyl peptide-hydrolyzing activity but did not lead to detectable defects in protein degradation nor to any other phenotype. However, the pre3-2 mutation strengthened phenotypes induced by other 20 S proteasomal mutations, indicating that the peptidylglutamyl peptide-hydrolyzing activity has to fulfill some rescue functions. The other three mutations (pre3-1, pre3-4, and pre3-6) are located at diverse sites of the Pre3 protein and caused multiple defects in proteasomal peptide cleaving activities. pre3-1 and pre3-6 mutants exhibited significant defects in proteasomal protein degradation; they accumulated ubiquitinated proteins and stabilized defined substrate proteins as, e.g. fructose-1,6-bisphosphatase. In addition, pre3-1 and pre3-6 mutant cells exhibited pleiotropic phenotypes as temperature sensitivity and cell cycle-related effects.

The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different alpha-type subunits and two inner rings made of 7 different beta-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform beta-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal beta-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal beta-type subunits is exerted by active subunits and occurs in the fully assembled particle. This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.

Proteinase A and proteinase B, two vacuolar enzymes in Saccharomyces cerevisiae, are synthesized as larger precursors with apparent molecular weights of approximately 52,000 and 42,000, respectively. These precursor molecules are processed to their mature forms of 42,000 molecular weight for proteinase A and 33,000 molecular weight for proteinase B. In the presence of tunicamycin, an inhibitor of the synthesis of protein-asparagine linked carbohydrate moieties, two smaller molecular forms each of precursor and mature proteinase A were synthesized, indicating that proteinase A contains N-linked carbohydrate which is apparently not required for processing. Tunicamycin interferes also with the glycosylation of the proteinase B precursor, whereas no unglycosylated mature proteinase B could be detected.

A new peptidase, which we call proteinase yscE, was purified from the yeast Saccharomyces cerevisiae. The enzyme cleaves the synthetic substrates Cbz-Gly-Gly-Leu-4-nitroanilide, Cbz-Ala-Ala-Leu-4-nitroanilide, and Suc-Phe-Leu-Phe-4-nitroanilide (Cbz and Suc are defined as benzyloxycarbonyl and succinyl, respectively) at the 4-nitroanilide bond and exhibits a slight activity against [3H]methylcasein. Optimum pH for cleavage of the chromogenic substrates is found to be in the range of 8.2 to 8.6. The purified enzyme has an apparent Stokes radius of Rs = 75.2 A as judged by gel chromatography and is composed of subunits. Mercurials were found to be strong inhibitors of the enzyme activity.

A previous study led to the discovery of new proteinases in yeast (Achstetter, T., Ehmann, C., and Wolf, D. H. (1981) Arch. Biochem. Biophys. 207, 445-454). The search for proteolytic enzymes active in the neutral pH range has been extended. Studies were done on a mutant lacking four well-known proteinases involved in protein degradation, the two endoproteinases A and B and the two carboxypeptidases Y and S. Twenty-nine chromogenic peptides (amino terminally blocked peptidyl-4-nitroanilides) as well as [3H]methylcasein were used as substrates in this search. For the detection of endoproteolytic activity using chromogenic peptide substrates two versions of the assay were used. In one system the direct cleavage of the 4-nitroanilide bond was measured. In the second, the cleavage of the chromogenic peptide at some site other than the 4-nitroanilide bond was measured. Both variations led to the discovery of multiple proteinase activities. Regulation of these proteolytic activities under different growth conditions of cells was observed. Proteolytic activity on [3H]methylcasein was also found. Ion-exchange chromatography and gel filtration were used for the reproducible separation of the multiple proteolytic activities.

A newly recognized peptidase, designated proteinase yscD, was purified from the yeast Saccharomyces cerevisiae. The enzyme cleaves the Pro-Phe bond of the synthetic peptide substrate Bz-Pro-Phe-Arg-4-nitroanilide and the Ala-Ala bond of Ac-Ala-Ala-Pro-Ala-4-nitroanilide, Ac-Ala-Ala-Pro-Phe-4-nitroanilide, and MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide with high efficiency (Bz-, Ac-, and MeO-Suc are defined as benzoyl, acetyl, and methoxy-succinyl, respectively). [3H]Methylcasein does not serve as a substrate. Optimum pH for cleavage of Bz-Pro-Phe-Arg-4-nitroanilide is in the range of 6.5 to 7; for Ac-Ala-Ala-Pro-Ala-4-nitroanilide the range is between 5.75 and 6. For MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide the pH optimum was found to be 5.5. The purified enzyme has an apparent Stokes radius of Rs = 37.9 A as judged by gel chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a molecular weight of approximately 83,000 for the enzyme. Mercurials and EDTA were found to be potent inhibitors of proteinase yscD activity.

Mutants deficient in the vacuolar (lysosomal) endopeptidases proteinase yscA and proteinase yscB of the yeast Saccharomyces cerevisiaeexhibit a drastically reduced protein degradation rate under nutritional stress conditions. The differentiation process of sporulation is considerably disturbed by the absence of the two endopeptidases. Also under vegetative growth conditions and under conditions of false protein synthesis, the two vacuolar endopeptidases exhibit some effect on protein degradation, which is, however, much less pronounced as found under starvation conditions. Proteinase yscA deficiency leads to rapid cell death when glucose-grown cells starve for nitrogen or other nutrients. Whereas overall protein degradation is affected in the endopeptidase mutants, degradation of two distinct false proteins analyzed is not altered in the absence of proteinase yscA and proteinase yscB. Also catabolite inactivation and degradation of fructose-1,6-bisphosphatase is not affected to a greater extent in the endopeptidase-deficient strains.

Catabolite inactivation of fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis, is due to phosphorylation and subsequent degradation in the yeast Saccharomyces cerevisiae. The degradation process of the enzyme had been shown to depend on the action of the proteasome. Here we report that components of the ubiquitin pathway target FBPase to proteolysis. Upon glucose addition to yeast cells cultured on nonfermentable carbon sources FBPase is ubiquitinated in vivo. A multiubiquitin chain containing isopeptide linkages at Lys48 of ubiquitin is attached to FBPase. Formation of a multiubiquitin chain is a prerequisite for the degradation of FBPase. Catabolite degradation of FBPase is dependent on the ubiquitin-conjugating enzymes Ubc1, Ubc4, and Ubc5. The 26 S proteasome is involved in the degradation process.

Mutants of Saccharomyces cerevisiae lacking dipeptidyl aminopeptidase yscV were isolated from a strain already defective in dipeptidyl aminopeptidase yscIV, an enzyme with overlapping substrate specificity. The mutants were identified by a staining technique with the chromogenic substrate Ala-Pro-4-methoxy-beta-naphthylamide to screen colonies for the absence of the enzyme. One of the mutants had a thermolabile activity, indicating that it contained a structural gene mutation. The 53 mutants analyzed fell into one complementation group that corresponded to the yscV structural gene, DAP2. The defect segregated 2:2 in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive. Diploids heterozygous for DAP2 displayed gene dosage effects with respect to yscV enzyme activity. The absence of dipeptidyl aminopeptidase yscV or the combined loss of both dipeptidyl aminopeptidases yscIV and yscV did not affect mitotic growth under rich or poor growth conditions. In contrast to the dipeptidyl aminopeptidase yscIV lesion (ste13), which leads to alpha sterility because strains secrete incompletely processed forms of the alpha-factor pheromone, the dipeptidyl aminopeptidase yscV lesion did not affect mating, and strains produced fully active alpha-factor pheromone. dap2 mutants did not show any obvious phenotype under a variety of conditions tested.