Species-specific protein-protein interactions govern the humanization of the 20S proteasome in yeast.

Yeast and humans share thousands of genes despite a billion years of evolutionary divergence. While many human genes can functionally replace their yeast counterparts, nearly half of the tested shared genes cannot. For example, most yeast proteasome subunits are "humanizable," except subunits comprising the ß-ring core, including ß2c (HsPSMB7, a constitutive proteasome subunit). We developed a high-throughput pipeline to humanize yeast proteasomes by generating a large library of Hsß2c mutants and screening them for complementation of a yeast ß2 (ScPup1) knockout. Variants capable of replacing ScPup1 included (1) those impacting local protein-protein interactions (PPIs), with most affecting interactions between the ß2c C-terminal tail and the adjacent ß3 subunit, and (2) those affecting ß2c proteolytic activity. Exchanging the full-length tail of human ß2c with that of ScPup1 enabled complementation. Moreover, wild-type human ß2c could replace yeast ß2 if human ß3 was also provided. Unexpectedly, yeast proteasomes bearing a catalytically inactive HsPSMB7-T44A variant that blocked precursor autoprocessing were viable, suggesting an intact propeptide stabilizes late assembly intermediates. In contrast, similar modifications in human ß2i (HsPSMB10), an immunoproteasome subunit and the co-ortholog of yeast ß2, do not enable complementation in yeast, suggesting distinct interactions are involved in human immunoproteasome core assembly. Broadly, our data reveal roles for specific PPIs governing functional replaceability across vast evolutionary distances.

Comparative analysis of mRNA transcripts of HT-29 cell line expressed in identical quantities for pathogenic E. coli strains UM146 and UM147 with control Escherichia coli Nissle 1917.

Aim of study was comparative analysis of mRNA transcripts of HT-29 cell line, expressed in identical quantities for the combination of pathogenic and non-pathogenic Escherichia coli strains. HT-29 confluent monolayers infection with two pathogenic E. coli strains UM146 and UM147 resulted in two sets of mRNA transcripts that were identical with RNA transcripts obtained for non-pathogenic one strain E. coli Nissle 1917. In this study genome-wide experiments were conducted using expression microarray-system. Only one common mRNA transcript coding for CCDC65 gene was equally expressed by HT-29 cells after incubation challenge with three different E. coli strains used. This gene and its bacterial analogue are important in the ciliary or flagellar motility, respectively. Altogether, 78 and 81 HT-29 mRNA transcripts for E. coli UM146 and E. coli UM147 had identical RNA quantity in comparison to the response obtained for non-pathogenic E. coli Nissle 1917 interactions with HT-29 monolayers. Specific analysis using REACTOME and agriGO terms enrichment data-mining tools as well as word-cloud analysis allowed for identification the most important processes characteristic during HT-29 cell line infections for each pathogenic E. coli strain used. The importance of results may contribute to recognition of those processes during bacterial infections that are identical with processes arising from human interaction with non-pathogenic strains that belong to the same bacterial species.

Trypsin-Like Activity in Oral Cavity Is Associated with Risk of Fever Onset in Older Residents of Nursing Homes: An 8-Month Longitudinal Prospective Cohort Pilot Study.

This study aimed to evaluate the association between trypsin-like activity in the oral cavity and the onset of fever in independent older residents of nursing homes. Independent older residents aged ≥ 65 years in 10 nursing homes were included in this study, which was conducted in Kitakyushu, Japan. For 8 months, follow-up dates on which the body temperatures of participants were more than 37.2 °C were noted. Trypsin-like activity in the oral cavity was detected by ADCHECK® with five-grade evaluation at baseline. Data from 53 independent participants with median age 89.0 (67­102) years were available for analysis. ADCHECK® scores were associated with fever days (r = 0.312, p = 0.029). The average periods until the onset of fever in participants with ADCHECK® Scores 1 and 2, Score 3, and Scores 4 and 5 were 6.6 ± 0.5, 5.0 ± 0.7, and 4.1 ± 1.0 months, respectively. ADCHECK® Scores 4 and 5 signified a higher risk of fever compared to ADCHECK® Scores 1 and 2 (hazards ratio 5.9, 95% confidence interval 1.4­23.9, p = 0.013), adjusted for possible confounders. We concluded that trypsin-like activity in the oral cavity was associated with the risk of fever in independent older residents of nursing homes.

Hydrogen Peroxide-Induced Oxidative Stress Activates Proteasomal Trypsin-Like Activity in Human U373 Glioma Cells.

Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal, a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. The major proteolytic system for modified protein degradation is the ubiquitin-proteasome pathway. However, our previous studies using U937 human leukemic cells showed that 4-hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, U373 human glioma cells were cultured in the presence of hydrogen peroxide (H2O2) to investigate the relationships of proteasome and/or cathepsin G activities and H2O2-induced GAPDH degradation. Treatment of cells with H2O2 for 5 h in culture decreased GAPDH activity as well as its protein concentration in a concentration-dependent manner. Two proteasomal activities (peptidylglutamyl-peptide hydrolase and chymotrypsin-like hydrolase activities) and cathepsin G activity were decreased by H2O2 treatment in a concentration-dependent manner, but proteasomal trypsin-like hydrolase activity increased with cell exposure to high H2O2 concentrations. Among the protease inhibitors examined here, H2O2-induced activation of trypsin-like activity and GAPDH degradation were inhibited by the proteasome inhibitor lactacystin. Furthermore, H2O2-induced activation of trypsin-like activity was also inhibited by another proteasome inhibitor MG-132. These results suggested that proteasomal trypsin-like activity played an important role in eliminating oxidatively modified GAPDH formed in these cells during H2O2 exposure.

Proteasome as a Molecular Target of Microcystin-LR.

Proteasome degrades proteins in eukaryotic cells. As such, the proteasome is crucial in cell cycle and function. This study proved that microcystin-LR (MC-LR), which is a toxic by-product of algal bloom, can target cellular proteasome and selectively inhibit proteasome trypsin-like (TL) activity. MC-LR at 1 nM can inhibit up to 54% of the purified 20S proteasome TL activity and 43% of the proteasome TL activity in the liver of the cyprinid rare minnow (Gobiocypris rarus). Protein degradation was retarded in GFP-CL1-transfected PC-3 cells because MC-LR inhibited the proteasome TL activity. Docking studies indicated that MC-LR blocked the active site of the proteasome ß2 subunit; thus, the proteasome TL activity was inhibited. In conclusion, MC-LR can target proteasome, selectively inhibit proteasome TL activity, and retard protein degradation. This study may be used as a reference of future research on the toxic mechanism of MC-LR.

Enhanced rate of degradation of basic proteins by 26S immunoproteasomes.

Immunoproteasomes are alternative forms of proteasomes specialized in the generation of MHC class I antigenic peptides and important for efficient cytokine production. We have identified a new biochemical property of 26S immunoproteasomes, namely the ability to hydrolyze basic proteins at greatly increased rates compared to constitutive proteasomes. This enhanced degradative capacity is specific for basic polypeptides, since substrates with a lower content in lysine and arginine residues are hydrolyzed at comparable rates by constitutive and immunoproteasomes. Crucially, selective inhibition of the immunoproteasome tryptic subunit ß2i strongly reduces degradation of basic proteins. Therefore, our data demonstrate the rate limiting function of the proteasomal trypsin-like activity in controlling turnover rates of basic protein substrates and suggest new biological roles for immunoproteasomes in maintaining cellular homeostasis by rapidly removing a potentially harmful excess of free histones that can build up under different pathophysiological conditions.

Protein damage, repair and proteolysis.

Proteins are continuously affected by various intrinsic and extrinsic factors. Damaged proteins influence several intracellular pathways and result in different disorders and diseases. Aggregation of damaged proteins depends on the balance between their generation and their reversal or elimination by protein repair systems and degradation, respectively. With regard to protein repair, only few repair mechanisms have been evidenced including the reduction of methionine sulfoxide residues by the methionine sulfoxide reductases, the conversion of isoaspartyl residues to L-aspartate by L-isoaspartate methyl transferase and deglycation by phosphorylation of protein-bound fructosamine by fructosamine-3-kinase. Protein degradation is orchestrated by two major proteolytic systems, namely the lysosome and the proteasome. Alteration of the function for both systems has been involved in all aspects of cellular metabolic networks linked to either normal or pathological processes. Given the importance of protein repair and degradation, great effort has recently been made regarding the modulation of these systems in various physiological conditions such as aging, as well as in diseases. Genetic modulation has produced promising results in the area of protein repair enzymes but there are not yet any identified potent inhibitors, and, to our knowledge, only one activating compound has been reported so far. In contrast, different drugs as well as natural compounds that interfere with proteolysis have been identified and/or developed resulting in homeostatic maintenance and/or the delay of disease progression.