Both PDI and PDIp can attack the native disulfide bonds in thermally-unfolded RNase and form stable disulfide-linked complexes.

Protein disulfide isomerase (PDI) and its pancreatic homolog (PDIp) are folding catalysts for the formation, reduction, and/or isomerization of disulfide bonds in substrate proteins. However, the question as to whether PDI and PDIp can directly attack the native disulfide bonds in substrate proteins is still not answered, which is the subject of the present study. We found that RNase can be thermally unfolded at 65°C under non-reductive conditions while its native disulfide bonds remain intact, and the unfolded RNase can refold and reactivate during cooling. Co-incubation of RNase with PDI or PDIp during thermal unfolding can inactivate RNase in a PDI/PDIp concentration-dependent manner. The alkylated PDI and PDIp, which are devoid of enzymatic activities, cannot inactivate RNase, suggesting that the inactivation of RNase results from the disruption of its native disulfide bonds catalyzed by the enzymatic activities of PDI/PDIp. In support of this suggestion, we show that both PDI and PDIp form stable disulfide-linked complexes only with thermally-unfolded RNase, and RNase in the complexes can be released and reactivated dependently of the redox conditions used. The N-terminal active site of PDIp is essential for the inactivation of RNase. These data indicate that PDI and PDIp can perform thiol-disulfide exchange reactions with native disulfide bonds in unfolded RNase via formation of stable disulfide-linked complexes, and from these complexes RNase is further released.

Characterization of the estradiol-binding site structure of human pancreas-specific protein disulfide isomerase: indispensable role of the hydrogen bond between His278 and the estradiol 3-hydroxyl group.

Estradiol (E(2)), a female sex hormone, has important biological functions. Human pancreas-specific protein disulfide isomerase (PDIp), a protein folding catalyst, was recently found to be able to bind E(2). Here we report the characterization of its E(2)-binding site by using biochemical methods coupled with molecular modeling tools. Analysis of various truncated PDIp proteins showed that the b-b' fragment contains an intact E(2)-binding site that has the same binding affinity as the full-length PDIp protein, with apparent K(d) values of approximately 170 nM. Computational modeling and docking analyses revealed that the E(2)-binding site in the b-b' fragment is located in a hydrophobic pocket composed mainly of the b' domain and partially of the b domain. The hydrogen bond, formed between the 3-hydroxyl group of E(2) (donor) and PDIp's His278 (acceptor), is indispensable for its binding. By contrast, the 17ß-hydroxyl group of E(2) is of negligible importance for E(2) binding. This binding model was jointly confirmed by a series of experiments, such as selective mutation of the binding site amino acid residues and selective modification of the ligand structures.

Human pancreas-specific protein disulfide-isomerase (PDIp) can function as a chaperone independently of its enzymatic activity by forming stable complexes with denatured substrate proteins.

Members of the PDI (protein disulfide-isomerase) family are critical for the correct folding of secretory proteins by catalysing disulfide bond formation as well as by serving as molecular chaperones to prevent protein aggregation. In the present paper, we report that the chaperone activity of the human pancreas-specific PDI homologue (PDIp) is independent of its enzymatic activity on the basis of the following lines of evidence. First, alkylation of PDIp by iodoacetamide fully abolishes its enzymatic activity, whereas it still retains most of its chaperone activity in preventing the aggregation of reduced insulin B chain and denatured GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Secondly, mutation of the cysteine residues in PDIp's active sites completely abolishes its enzymatic activity, but does not affect its chaperone activity. Thirdly, the b-b' fragment of PDIp, which does not contain the active sites and is devoid of enzymatic activity, still has chaperone activity. Mechanistically, we found that both the recombinant PDIp expressed in Escherichia coli and the natural PDIp present in human or monkey pancreas can form stable complexes with thermal-denatured substrate proteins independently of their enzymatic activity. The high-molecular-mass soluble complexes between PDIp and GAPDH are formed in a stoichiometric manner (subunit ratio of 1:3.5-4.5), and can dissociate after storage for a certain time. As a proof-of-concept for the biological significance of PDIp in intact cells, we demonstrated that its selective expression in E. coli confers strong protection of these cells against heat shock and oxidative-stress-induced death independently of its enzymatic activity.

Global analysis of daily new COVID-19 cases reveals many static-phase countries including the United States potentially with unstoppable epidemic.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is hitting many countries. It is hypothesized the epidemic is differentially progressing in different countries. AIM: To investigate how the COVID-19 epidemic is going on in different countries by analyzing representative countries. METHODS: The status of COVID-19 epidemic in over 60 most affected countries was characterized. The data of daily new cases of each country were collected from Worldometer. The data of daily tests for the United States, Italy, and South Korea were collected from the Website of One World Data. Levels of daily positive COVID-19 tests in the two most affected states of the United States (New York and New Jersey) were collected from the website of the COVID Tracking Project. Statistics were analyzed using Microcal Origin software with ANOVA algorithm, and significance level was set at a P value of 0.05. RESULTS: The COVID-19 epidemic was differentially progressing in different countries. Comparative analyses of daily new cases as of April 19, 2020 revealed that 61 most affected countries can be classified into four types: Downward (22), upward (20), static-phase (12), and uncertain ones (7). In particular, the 12 static-phase countries including the United States were characterized by largely constant numbers of daily new cases in the past over 14 d. Furthermore, these static-phase countries were overall significantly lower in testing density (P = 0.016) but higher in the level of positive COVID-19 tests than downward countries (P = 0.028). These findings suggested that the testing capacity in static-phase countries was lagging behind the spread of the outbreak, i.e., daily new cases (confirmed) were likely less than daily new infections and the remaining undocumented infections were thus still expanding, resulting in unstoppable epidemic. CONCLUSION: Increasing the testing capacity and/or reducing the COVID-19 transmission are urgently needed to stop the potentially unstoppable, severing crisis in static-phase countries.

Characterization of the estradiol-binding site structure of human protein disulfide isomerase (PDI).

BACKGROUND: Earlier studies showed that 17ß-estradiol (E(2)), an endogenous female sex hormone, can bind to human protein disulfide isomerase (PDI), a protein folding catalyst for disulfide bond formation and rearrangement. This binding interaction can modulate the intracellular levels of E(2) and its biological actions. However, the structure of PDI's E(2)-binding site is still unclear at present, which is the focus of this study. METHODOLOGY/PRINCIPAL FINDINGS: The E(2)-binding site structure of human PDI was studied by using various biochemical approaches coupled with radiometric receptor-binding assays, site-directed mutagenesis, and molecular computational modeling. Analysis of various PDI protein fragments showed that the [(3)H]E(2)-binding activity is not associated with the single b or b' domain but is associated with the b-b' domain combination. Computational docking analyses predicted that the E(2)-binding site is located in a hydrophobic pocket composed mainly of the b' domain and partially of the b domain. A hydrogen bond, formed between the 3-hydroxyl group of E(2) and His256 of PDI is critical for the binding interaction. This binding model was jointly confirmed by a series of detailed experiments, including site-directed mutagenesis of the His256 residue coupled with selective modifications of the ligand structures to alter the binding interaction. CONCLUSIONS/SIGNIFICANCE: The results of this study elucidated the structural basis for the PDI-E(2) binding interaction and the reservoir role of PDI in modulating the intracellular E(2) levels. The identified PDI E(2)-binding site is quite different from its known peptide binding sites. Given that PDI is a potential therapeutic target for cancer chemotherapy and HIV prevention and that E(2) can inhibit PDI activity in vitro, the E(2)-binding site structure of human PDI determined here offers structural insights which may aid in the rational design of novel PDI inhibitors.

Human pancreas-specific protein disulfide isomerase homolog (PDIp) is an intracellular estrogen-binding protein that modulates estrogen levels and actions in target cells.

Earlier studies showed that protein disulfide isomerase (PDI), a well-known protein folding catalyst, can bind estrogens. Whether other PDI homologs can also bind estrogens, and if so, what are the biological functions of this unique property are not known at present and thus are the subjects of our present investigation. Here we report that, of the six representative PDI homologs examined (human PDI, PDIp, ERp57, ERp72, PDIA6 and rat PDIr), only the human pancreas-specific PDI homolog (PDIp) had a similar binding affinity for radiolabeled 17beta-estradiol (E(2)) as did PDI, with apparent K(d) values of 1.5+/-0.3 and 1.5+/-0.2microM, respectively. However, PDIp and PDI had distinctly different binding preference for several estrogen analogs. Moreover, we found that PDIp could serve as a high-capacity intracellular E(2)-binding protein and could modulate the intracellular concentrations of E(2) in cultured mammalian cells as well as in human pancreatic tissue. The PDIp-bound E(2) in a cell could be released following a drop in the extracellular E(2) concentrations, and the released E(2) could then augment estrogen receptor-mediated transcriptional activity. Notably, the estrogen receptor alpha and beta were also found to be expressed in rodent and human pancreatic tissues where high levels of PDIp were detected. Altogether, these data show that, in addition to its well-documented function as a protein folding catalyst, PDIp can also serve as an effective modulator of the cellular levels and biological actions of endogenous estrogens in certain target sites (such as the pancreas) where estrogen receptors and PDIp are co-present.

Pancreas-specific protein disulfide isomerase has a cell type-specific expression in various mouse tissues and is absent in human pancreatic adenocarcinoma cells: implications for its functions.

Members of the protein disulfide isomerase (PDI) family play a critical role in catalyzing the formation of disulfide bonds in secretory proteins, and most of these enzymes have a wide tissue distribution. However, the pancreas-specific PDI homolog was previously suggested to be exclusively expressed in the pancreas (thus commonly referred to as PDIp). In the present study, we found that PDIp was also highly expressed in several other tissues in mice, including the stomach, cecum, ileum, adrenal glands, epididymis, and prostate. Notably, in the digestive organs, such as the stomach and pancreas, very high levels of PDIp were selectively expressed in the digestive enzyme-secreting cells (e.g., gastric chief cells and pancreatic acinar cells). This observation suggests that PDIp may function as a protein-folding catalyst for secretory digestive enzymes. In ileum, PDIp was exclusively expressed in Paneth cells. In addition, high levels of PDIp expression were also detected in normal human pancreas, but its expression was mostly absent in human pancreatic duct adenocarcinoma and pancreatic cancer cell lines. The absence of PDIp expression in pancreatic adenocarcinoma may serve as an additional biomarker for pancreatic cancer.