Comparative study of mature and zymogen mite cysteine protease stability and pH unfolding

Background

Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes.

Methods

To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1.

Results

We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen.

General significance

Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis.     

Relationship between propeptide pH unfolding and inhibitory ability during ProDer p 1 activation mechanism

The major allergen Der p 1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The propeptide of Der p 1 exhibits a specific fold that makes it unique in the CA1 propeptide family. In this study, we investigated the activation steps involved in the maturation of the recombinant protease Der p 1 expressed in Pichia pastoris and the interaction of the full-length and truncated soluble propeptides with their parent enzyme in terms of activity inhibition and BIAcore interaction analysis. According to our results, the activation of protease Der p 1 is a multistep mechanism that is characterized by at least two intermediates. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (K_D = 7 nM) at neutral pH. This inhibition is pH dependent. It decreases from pH 7 to pH 4 and can be related to conformational changes of the propeptide characterized by an increase of its flexibility and formation of a molten globule state. Our results indicate that activation of the zymogen at pH 4 is a compromise between activity preservation and propeptide unfolding.     

Activation mechanism of recombinant Der p 3 allergen zymogen: contribution of cysteine protease Der p 1 and effect of propeptide glycosylation

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence T(P1)R.     

Recombinant Der p 1 and Der f 1 exhibit cysteine protease activity but no serine protease activity

Although mite major group 1 allergens, Der p 1 and Der f 1, were first isolated as cysteine proteases, some studies reported that natural Der p 1 exhibits mixed cysteine and serine protease activity. Clarifying whether the serine protease activity originates from Der p 1 or is due to contamination is important for distinguishing between the pathogenic proteolytic activities of group 1 allergens and mite-derived serine proteases. Recombinant mite group 1 allergens would be useful tool for addressing this issue, because they are completely free from contamination by mite serine proteases. Recombinant Der p 1 and Der f 1, and highly purified natural forms exhibited only cysteine protease activity. However, commercially available natural forms exhibited both activities, but the two activities were eluted into different fractions in size-exclusion column chromatography. The substrate specificity associated with the serine protease activity was similar to that of Der f 3. These results indicate that the serine protease activity does not originate from group 1 allergens.     

Crystal Structure of Der f 7, a Dust Mite Allergen from Dermatophagoides farinae

Background

Der f 7 is the group 7 allergen from the dust mite Dermatophagoides farinae, homologous to the major allergen Der p 7 from D. pteronyssinus. Monoclonal antibody that bind to residues Leu48 and Phe50 was found to inhibit IgE binding to residue Asp159, which is important for the cross-reactivity between Der f 7 and Der p 7.

Methodology/Principal Findings

Here, we report the crystal structure of Der f 7 that shows an elongated and curved molecule consisting of two anti-parallel β-sheets – one 4-stranded and the other 5-stranded – that wrap around a long C-terminal helix. The overall fold of Der f 7 is similar to Der p 7 but key difference was found in the β1–β2 loop region. In Der f 7, Leu48 and Phe50 are in close proximity to Asp159, explaining why monoclonal antibody binding to Leu48 and Phe50 can inhibit IgE binding to Asp159. Both Der f 7 and Der p 7 bind weakly to polymyxin B via a similar binding site that is formed by the N-terminal helix, the 4-stranded β-sheet and the C-terminal helix. The thermal stability of Der f 7 is significantly lower than that of Der p 7, and the stabilities of both allergens are highly depend on pH.

Conclusion/Significance

Der f 7 is homologous to Der p 7 in terms of the amino acid sequence and overall 3D structure but with significant differences in the region proximal to the IgE epitope and in thermal stability. The crystal structure of Der f 7 provides a basis for studying the function and allergenicity of this group of allergens.     

In vitro characterization of ligand-induced oligomerization of the S. cerevisiae G-protein coupled receptor,Ste2p

Background

The S. cerevisiae α-factor receptor, Ste2p, is a G-protein coupled receptor that plays key roles in yeast signaling and mating. Oligomerization of Ste2p has previously been shown to be important for intracellular trafficking, receptor processing and endocytosis. However the role of ligand in receptor oligomerization remains enigmatic.

Methods

Using functional recombinant forms of purified Ste2p, atomic force microscopy, dynamic light scattering and chemical crosslinking are applied to investigate the role of ligand in Ste2p oligomerization.

Results

Atomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of α-factor nearly double that of Ste2p alone. This observation is supported by complementary dynamic light scattering measurements which indicate a ligand-induced increase in the polydispersity of the Ste2p hydrodynamic radius. Finally, chemical cross-linking of HEK293 plasma membranes presenting recombinant Ste2p indicates α-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis.

Conclusions

α-factor induces oligomerization of Ste2p in vitro and in membrane.

General significance

These results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo.     

A novel locus for body mass index on 5p15.2: a meta-analysis of two genome-wide association studies

Objective

Genetic factors play an important role in modulating the vulnerability to body mass index (BMI). The purpose of this study is to identify novel genetic variants for BMI using genome-wide association (GWA) meta-analysis.

Methods

PLINK software was used to perform meta-analysis of two GWA studies (the FUSION and Marshfield samples) of 5218 Caucasian individuals with BMI. A replication study was conducted using the SAGE sample with 762 individuals.

Results

Through meta-analysis we identified 33 SNPs associated with BMI with p < 10^− 4. The most significant association was observed with rs2967951 (p = 1.19 × 10^− 6) at 5p15.2 within ROPN1L gene. Two additional SNPs within ROPN1L and 5 SNPs within MARCH6 (the top SNP was rs2607292 with 4.27 × 10^− 6) further supported the association with BMI on 5p15.2 (p < 1.8 × 10^− 5). Conditional analysis on 5p15.2 could not distinguish the effects of ROPN1L and MARCH6. Several SNPs within MARCH6 and ROPN1L were replicated in the SAGE sample (p < 0.05).

Conclusion

We identified a novel locus for BMI. These findings offer the potential for new insights into the pathogenesis of BMI and obesity and will serve as a resource for replication in other populations to elucidate the potential role of these genetic variants in BMI and obesity.     

Simvastatin induced HCT116 colorectal cancer cell apoptosis through p38MAPK-p53-survivin signaling cascade

Background

Statins, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors with cholesterol-lowering properties, were recently shown to exhibit anti-cancer effects. However, the molecular mechanism underlying statin-induced cancer cell death remains to be elucidated. Elevated level of survivin is often found over-expressed in human cancers and has been implicated in the progression of tumorigenesis. Given its central role in cell division and action as an apoptosis suppressor, survivin represents a potential molecular target in cancer management.

Methods

In this study, we explored the underlying mechanisms in simvastatin-induced HCT116 colorectal cancer cell apoptosis.

Results

Simvastatin decreased cell viability and induced cell apoptosis in HCT116 cells. These results are associated with the modulation of p21^cip/Waf1 and survivin. Survivin knockdown using survivin siRNAs also decreased cell viability and induced cell apoptosis. Simvastatin's actions on p21^cip/Waf1, survivin and apoptosis were reduced in p53 null HCT116 cells. Simvastatin caused an increase in p53 phosphorylation and acetylation. In addition, simvastatin activated p38 mitogen-activated protein kinase (p38MAPK), whereas an inhibitor of p38MAPK signaling abrogated simvastatin's effects of increasing p53 and p21^cip/Waf1 promoter luciferase activity. Cell viability and survivin promoter luciferase activity in the presence of simvastatin were also restored by p38MAPK inhibitor. Furthermore, Sp1 binding to the survivin promoter region decreased while p53 and p63 binding to the promoter region increased after simvastatin exposure.

Conclusions

Simvastatin activates the p38MAPK-p53-survivin cascade to cause HCT116 colorectal cancer cell apoptosis.

General significance

This study delineates, in part, the underlying mechanisms of simvastatin in decreasing survivin and subsequent colorectal cancer cell apoptosis.     

Multiple roles of glucose-6-phosphatases in pathophysiology: State of the art and future trends

Background

The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate to glucose and inorganic phosphate. The enzyme is a part of a multicomponent system that includes several integral membrane proteins; the catalytic subunit (G6PC) and transporters for glucose-6-phosphate, inorganic phosphate and glucose. The G6PC gene family presently includes three members, termed as G6PC, G6PC2, and G6PC3. Although the three isoforms show a moderate amino acid sequence homology, their membrane topology and catalytic site are very similar. The isoforms are expressed differently in various tissues. Mutations in all three genes have been reported to be associated with human diseases.

Scope of review

The present review outlines the biochemical features of the G6PC gene family products, the regulation of their expression, their role in the human pathology and the possibilities for pharmacological interventions.

Major conclusions

G6PCs emerge as integrators of extra- and intracellular glucose homeostasis. Beside the well known key role in blood glucose homeostasis, the members of the G6PC family seem to play a role as sensors of intracellular glucose and of intraluminal glucose/glucose-6-phosphate in the endoplasmic reticulum.

General significance

Since mutations in the three G6PC genes can be linked to human pathophysiological conditions, the better understanding of their functioning in connection with genetic alterations, altered expression and tissue distribution has an eminent importance.     

p38 MAPK and ERK1/2 pathways are involved in the pro-apoptotic effect of notoginsenoside Ft1 on human neuroblastoma SH-SY5Y cells

Aims

This study aims to investigate the effect and the mechanisms of notoginsenoside Ft1, a natural compound exclusively found in P. notoginseng, on the proliferation and apoptosis of human neuroblastoma SH-SY5Y cells.

Main methods

CCK-8 assay was used to assess the cell proliferation. Flow cytometry was performed to measure the cell cycle distribution and cell apoptosis. Hoechst 33258 staining was conducted to confirm the morphological changes of apoptotic cells. Protein expression was detected by western blot analysis and caspase 3 activity was measured by colorimetric assay kit.

Key findings

Among the saponins examined, Ft1 showed the best inhibitory effect on cell proliferation of SH-SY5Y cells with IC₅₀ of 45 μM. Ft1 not only arrested the cell cycle at S, G₂/M stages, but also promoted cell apoptosis, which was confirmed by Hoechst 33258 staining. Further studies demonstrated that Ft1 up-regulated the protein expressions of cleaved caspase 3, phospho-p53, p21, and cyclin B1, but down-regulated that of Bcl-2. Moreover, Ft1 enhanced the phosphorylation of ERK1/2, JNK and p38 MAPK. However, the phosphorylation of Jak2 and p85 PI3K was reduced by Ft1. Inhibitors of p38 MAPK and ERK1/2 but not JNK abrogated the up-regulated protein expressions of cleaved caspase 3, p21 and down-regulated protein expression of Bcl-2 as well as elevated caspase 3 activity induced by Ft1.

Significance

Ft1 arrested the proliferation and elicited the apoptosis of SH-SY5Y cells possibly via p38 MAPK and ERK1/2 pathways, which indicates the potential therapeutic effect of it on human neuroblastoma.     

Involvement of ASK1–p38 pathway in the pathogenesis of diabetes triggered by pancreatic ß cell exhaustion

Background

Diabetes mellitus is characterized by high blood glucose levels. Pancreatic ß cell death contributes to type 1 and type 2 diabetes. Akita mice, which harbor a human permanent neonatal diabetes-linked mutation (Cys96Tyr) in the insulin gene, are well established as an animal model of diabetes caused by pancreatic ß cell exhaustion. Mutant Insulin 2 protein (Ins2^C96Y) induces endoplasmic reticulum (ER) stress and pancreatic ß cell death in Akita mice, although the molecular mechanism of Ins^C96Y-induced cell death remains unclear.

Methods

We investigate the mechanisms of Ins2^C96Y-induced pancreatic ß cell death in vitro and in vivo, using p38 inhibitor (SB203580), MIN6 cell (pancreatic ß cell line), Akita mice and apoptosis signal-regulating kinase 1 (ASK1) knockout mice.

Results

The expression of Ins^C96Y activated the ASK1–p38 pathway. Deletion of ASK1 mitigated Ins^C96Y-induced pancreatic ß cell death and delayed the onset of diabetes in Akita mice. Moreover, p38 inhibitor suppressed Ins^C96Y-induced MIN6 cell death.

Conclusions

These findings suggest that ER stress-induced ASK1–p38 activation, which is triggered by the accumulation of Ins^C96Y, plays an important role in the pathogenesis of diabetes.

General significance

Pancreatic ß cell death caused by insulin overload appears to be involved in the pathogenesis of type 1 and type 2 diabetes. Inhibition of the ASK1–p38 pathway may be an effective therapy for various types of diabetes.     

Domestic Mite Antigens in Floor and Airborne Dust at Workplaces in Comparison to Living Areas: A New Immunoassay to Assess Personal Airborne Allergen Exposure

Objectives

Allergens produced by domestic mites (DM) are among the most common allergic sensitizers and risk factors for asthma. To compare exposure levels between workplaces and living areas a new assay able to measure airborne DM antigen concentrations was developed.

Methods

At workplaces and in living areas, 213 floor dust samples and 92 personal inhalable dust samples were collected. For sensitive quantification of DM antigens, a new enzyme immunoassay (EIA) based on polyclonal antibodies to Dermatophagoides farinae extract was developed. Reactivity of five house dust mite and four storage mite species was tested. All dust samples were tested with the new EIA and with the Der f 1 and Der p 1-EIAs (Indoor Biotechnologies, UK) which detect major allergens from D. farinae and D. pteronyssinus by monoclonal antibodies. Samples below the detection limit in the DM-EIA were retested in an assay variant with a fluorogenic substrate (DM-FEIA).

Results

The newly developed DM-EIA detects antigens from all nine tested domestic mite species. It has a lower detection limit of 200 pg/ml of D.farinae protein, compared to 50 pg/ml for the DM-FEIA. DM antigens were detected by DM-EIA/FEIA in all floor dust and 80 (87%) of airborne samples. Der f 1 was found in 133 (62%) floor dust and in only 6 airborne samples, Der p 1 was found in 70 (33%) of floor samples and in one airborne sample. Der f 1 and DM concentrations were highly correlated. DM-antigens were significantly higher in inhalable airborne samples from textile recycling, bed feather filling, feed production, grain storage and cattle stables in comparison to living areas.

Conclusions

A new sensitive EIA directed at DM antigens was developed. DM antigen quantities were well correlated to Der f 1 values and were measurable in the majority (87%) of airborne dust samples. Some workplaces had significantly higher DM antigen concentrations than living areas.     

Aeroallergen Der p 2 induces apoptosis of bronchial epithelial BEAS-2B cells via activation of both intrinsic and extrinsic pathway

Background

Excessive apoptosis of airway epithelium is reported to induce airway remodeling and inhibited airway epithelium repair is highly associated with development of asthma and chronic obstructive pulmonary disease. Der p 2 is a major allergen derived from Dermatophagoides pteronyssinus and commonly causes airway hypersensitiveness and asthma; however, the connection between Der p 2 and epithelial apoptosis remains unclear. This study was aimed to explore whether Der p 2 induces apoptosis of airway epithelial cells and the underlying mechanisms.

Results

Our results showed that recombinant Der p 2 (rDP2) inhibited cell growth and induced apoptosis of human bronchial epithelial cell BEAS-2B. Further investigation revealed that rDP2 increased intracellular reactive oxygen species, level of cytosolic cytochrome c and cleavage of caspase-9 and caspase-3. rDP2 also induced activation of p38 mitogen-activated protein kinase (P38) and c-Jun N-terminal kinase (JNK), and triggered proapoptotic signals including decrease of Bcl-2, increase of Bax and Bak, and upregulation of Fas and Fas ligand. In parallel, rDP2 inhibited glycogen synthase kinase 3beta and consequently enhanced degradation of cellular (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP). Involvement of toll-like receptor (TLR)2 in rDP2-induced apoptosis was also demonstrated using specific small inhibitory RNA.

Conclusions

Our findings indicate that rDP2 suppresses cell growth and trigger apoptosis of BEAS-2B cells, which may attribute to induction of both intrinsic and extrinsic pathway via TLR2 and P38/JNK signaling and c-FLIP degradation. It suggests that Der p 2 may aggravate respiratory disorders through enhancement of apoptosis and the consequent airway injury.

     

A chondroitin synthase-1 (ChSy-1) missense mutation in a patient with neuropathy impairs the elongation of chondroitin sulfate chains initiated by chondroitin N-acetylgalactosaminyltransferase-1

Background

Previously, we identified two missense mutations in the chondroitin N-acetylgalactosaminyltransferase-1 gene in patients with neuropathy. These mutations are associated with a profound decrease in chondroitin N-acetylgalactosaminyltransferase-1 enzyme activity. Here, we describe a patient with neuropathy who is heterozygous for a chondroitin synthase-1 mutation. Chondroitin synthase-1 has two glycosyltransferase activities: it acts as a GlcUA and a GalNAc transferase and is responsible for adding repeated disaccharide units to growing chondroitin sulfate chains.

Methods

Recombinant wild-type chondroitin synthase-1 enzyme and the F362S mutant were expressed. These enzymes and cells expressing them were then characterized.

Results

The mutant chondroitin synthase-1 protein retained approximately 50% of each glycosyltransferase activity relative to the wild-type chondroitin synthase-1 protein. Furthermore, unlike chondroitin polymerase comprised of wild-type chondroitin synthase-1 protein, the non-reducing terminal 4-O-sulfation of GalNAc residues synthesized by chondroitin N-acetylgalactosaminyltransferase-1 did not facilitate the elongation of chondroitin sulfate chains when chondroitin polymerase that consists of the mutant chondroitin synthase-1 protein was used as the enzyme source.

Conclusions

The chondroitin synthase-1 F362S mutation in a patient with neuropathy resulted in a decrease in chondroitin polymerization activity and the mutant protein was defective in regulating the number of chondroitin sulfate chains via chondroitin N-acetylgalactosaminyltransferase-1. Thus, the progression of peripheral neuropathies may result from defects in these regulatory systems.

General significance

The elongation of chondroitin sulfate chains may be tightly regulated by the cooperative expression of chondroitin synthase-1 and chondroitin N-acetylgalactosaminyltransferase-1 in peripheral neurons and peripheral neuropathies may result from synthesis of abnormally truncated chondroitin sulfate chains.     

Characterization of a gene encoding prohibitin in silkworm, Bombyx mori

Background

Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. However, its molecular roles are largely unknown.

Methods

To better understand the function of prohibitin protein in silkworm (BmPHB), its coding sequence was isolated from a cDNA library of silkworm pupae. An His-tagged BmPHB fusion protein was expressed in Escherichia coli Rosetta (DE3) and purified with affinity and reversed-phase chromatography. Purified rBmPHB was used to generate anti-BmPHB polyclonal antibody. The subcellular localization of BmPHB was analysed by immunohistochemistry.

Results

BmPHB gene has an ORF of 825 bp, encoding a predicted peptide with 274 amino acid residues. Immunostaining indicate that prohibitin is expressed in nucleus and predominately in cytoplasm. Western blot analyses indicated that, in the fifth instar larva, BmPHB was expressed descendingly in gonad, malpighian tubule, trachea, fatty body, intestine, and head. However, no expression was detected in larva's silk gland and epidermis. In addition, BmPHB was expressed in the nascent egg, larva and pupa, but not in the moth.

Conclusions

The expression of BmPHB gene presents differential characteristic in different stage and tissues. It may play important roles in the development of silkworm.

General significance

Studies on prohibitin have been still restricted to a few specific insects and insect cell lines such as Drosophila, Acyrthosiphon pisum and mosquito cell lines, not yet in silkworm. This is a first characterization of prohibitin in silkworm, B. mori.