The putative propeptide of MycP1 in mycobacterial type VII secretion system does not inhibit protease activity but improves protein stability

Mycosin-1 protease(MycP1)is a serine protease anchored to the inner membrane of Mycobacterium tuberculosis,and is essential in virulence factor secretion through the ESX-1 type VII secretion system(T7SS).Bacterial physiology studies demonstrated that MycP1 plays a dual role in the regulation of ESX-1 secretion and virulence,primarily through cleavage of its secretion substrate EspB.MycP1 contains a putative N-terminal inhibitory propeptide and a catalytic triad of Asp-His-Ser,classic hallmarks of a sub-tilase family serine protease.The MycP1 propeptide was previously reported to be initially inactive and activated after prolonged incubation.In this study,we have deter-mined crystal structures of MycP1 with(MycP124-422)and without(MycP1^(63-422))the propeptide,and conducted EspB cleavage assays using the two proteins.Very high struc-tural similarity was observed in the two crystal structures.Interestingly,protease assays demonstrated positive EspB cleavage for both proteins,indicating that the putative propeptide does not inhibit protease activity.Molecu-lar dynamic simulations showed higher rigidity in regions guarding the entrance to the catalytic site in MycP124-422 than in MycP1^(63-422),suggesting that the putative propeptide might contribute to the conformational stability of the active site cleft and surrounding regions.

Vitamin D deficiency: Correlation to interleukin-17, interleukin-23 and PⅢNP in hepatitis C virus genotype 4

AIM: To assess vitamin D (Vit D) abnormalities in hepatitis C infected patients and their relationship with interleukin (IL)-17, IL-23 and N-terminal propeptide of type Ⅲ pro-collagen (PⅢNP) as immune response mediators. METHODS: The study was conducted on 50 Egyptian patients (36 male, 14 female) with hepatitis C virus (HCV) infection, who visited the Hepatology Outpatient Clinic in the Endemic Disease Hospital at Cairo University. Patients were compared with 25 ageand sexmatched healthy individuals. Inclusion criteria were based on a history of liver disease with HCV genotype 4 (HCV-4) infection (as new patients or under followup). Based on ultrasonography, patients were classified into four subgroups; 14 with bright hepatomegaly; 11 with perihepatic fibrosis; 11 with hepatic cirrhosis; and 14 with cirrhosis and hepatocellular carcinoma (HCC).Total Vit D (i.e., 25-OH-Vit D) and active Vit D [i.e., 1,25-(OH) 2 -Vit D] assays were carried out using commercial kits. IL-17, IL-23 and PⅢNP levels were assayed using enzyme linked immunosorbent assay kits, while HCV virus was measured by quantitative and qualitative polymerase chain reaction. RESULTS: Levels of Vit D and its active form were significantly lower in advanced liver disease (hepatic cirrhosis and/or carcinoma) patients, compared to those with bright hepatomegaly and perihepatic fibrosis. IL-17, IL-23 and PⅢNP levels were markedly increased in HCV patients and correlated with the progression of hepatic damage. The decrease in Vit D and active Vit D was concomitant with an increase in viral load, as well as levels of IL-17, IL-23 and PⅢNP among all subgroups of HCV-infected patients, compared to normal healthy controls. A significant negative correlation was evident between active Vit D and each of IL-17, IL-23 and PⅢNP (r = -0.679, -0.801 and -0.920 at P < 0.001, respectively). HCV-infected men and women showed no differences with respect to Vit D levels. The viral load was negatively correlated with Vit D and active Vit D (r = -0.084 and -0.846 at P < 0.001, respectively), and positively correlated with IL-17, IL-23 and PⅢNP (r = 0.951, 0.922 and 0.94 at P < 0.001, respectively). Whether the deficiency in Vit D was related to HCVinduced chronic liver disease or was a predisposing factor for a higher viral load remains to be elucidated. CONCLUSION: The negative correlations between Vit D and IL-17, IL-23 and PⅢNP highlight their involvement in the immune response in patients with HCV-4related liver diseases in Egypt.

Protein Folding Mediated by an Intramolecular Chaperone: Energy Landscape for Unimolecular Pro-Subtilisin E Maturation

Efficient and precise assembly of polypeptides into native functional states is critical for normal cellular processes. Understanding how a specific structure is encoded in the polypeptide sequence and what drives the structural progression to the native state is essential to deciphering the folding problem. Several prokaryotic and eukaryotic proteins require their propeptide-domains to function as dedicated intramolecular chaperones (IMCs). In this manuscript, we investigate the elementary steps in the IMC mediated maturation of Subtilisin E, a bacterial serine protease, and a prototype for the eukaryotic proprotein convertases (PCs). Through detailed analyses, we have attempted to define the unimolecular folding energy landscape for SbtE to understand how the stabilization of folding intermediates influences the maturation process, an aspect that is difficult to study in eukaryotic PCs. Our studies demonstrate that a rapid hydrophobic collapse precedes acquisition of tertiary structure during the folding of Pro-SbtE and results in formation of a molten-globule like intermediate. Induction of structure within the IMC stabilizes both the molten globule-like folding intermediate and the native state, and appears to expedite initial stages of folding, purely through thermodynamic stabilization of the folded state. While the induced structure does not affect the activation energies in the unimolecular folding reaction, it is detrimental to the autoproteolytic cleavage of the precursor and subsequent release and degradation of the inhibitory IMC-domain since both these stages require some degree of unfolding. Completion of Pro-SbtE maturation results in the formation of a kinetically trapped and extremely stable native state. Hence, our results suggest that the SbtE IMC appears to have evolved to be intrinsically unstructured and to bind with its cognate protease with a specific affinity that is critical for biological regulation.

Molecular assembly of recombinant chicken type II collagen in the yeast Pichia pastoris

Effective treatment of rheumatoid arthritis can be mediated by native chicken type II collagen(n CCII), recombinant peptide containing n CCII tolerogenic epitopes(CTEs), or a therapeutic DNA vaccine encoding the full-length CCOL2 A1 c DNA. As recombinant CCII(r CCII) might avoid potential pathogenic virus contamination during n CCII preparation or chromosomal integration and oncogene activation associated with DNA vaccines, here we evaluated the importance of propeptide and telopeptide domains on r CCII triple helix molecular assembly. We constructed p C-and p N-procollagen(without N-or Cpropeptides, respectively) as well as CTEs located in the triple helical domain lacking both propeptides and telopeptides, and expressed these in yeast Pichia pastoris host strain GS115(his4, Mut+) simultaneously with recombinant chicken prolyl-4-hydroxylase α and β subunits. Both p C-and p N-procollagen monomers accumulated inside P. pastoris cells, whereas CTE was assembled into homotrimers with stable conformation and secreted into the supernatants, suggesting that the large molecular weight p C-or p N-procollagens were retained within the endoplasmic reticulum whereas the smaller CTEs proceeded through the secretory pathway. Furthermore, resulting recombinant chicken type II collagen p Cα1(II) can induce collagen-induced arthritis(CIA) rat model, which seems to be as effective as the current standard n CCII. Notably, protease digestion assays showed that r CCII could assemble in the absence of C-and N-propeptides or telopeptides. These findings provide new insights into the minimal structural requirements for r CCII expression and folding.