Tumour suppressor protein sMEK1 links to IRE1 signalling pathway to modulate its activity during ER stress.

The Endoplasmic Reticulum is a pervasive, dynamic cellular organelle that performs a wide range of functions in the eukaryotic cell, including protein folding and maturation. Upon stress, ER activates an adaptive cellular pathway, namely Unfolded Protein Response, that transduces information from ER to nucleus, restoring homeostasis in the ER milieu. UPR consists of three membrane-tethered sensors; IRE1, PERK and ATF6. Among all the UPR sensors, the IRE1 branch acts as a central pathway that orchestrates several pathways to determine cell fate. However, the detailed knowledge underlying the whole process is not understood yet. Previously, we determined the sMEK1 as one of the interacting partners of IRE1. sMEK1 is a protein phosphatase, which has been indicated in a number of critical cellular functions like apoptosis, cell proliferation, and tumour suppression. In this study, we evaluated the role of sMEK1 on the IRE1 signalling pathway. Our data indicate that sMEK1 can inhibit IRE1 phosphorylation under ER stress. This inhibitory effect of sMEK1 could be reflected in its downstream effectors, Xbp1 and RIDD, which are downregulated in the presence of sMEK1. We also found that the repressing effect of sMEK1 was specific to the IRE1 signalling pathway and could be preserved even under prolonged ER stress. Our findings also indicate that sMEK1 can inhibit IRE1 and its downstream molecules under ER stress irrespective of other UPR sensors. These results help to draw the mechanistic details giving insights into different molecular connections of UPR with other pathways.

Transcriptomic Downregulation of APOE, Polymorphic Variations of APOE, Diet, Social Isolation, and Co-morbidities as Contributing Factors to Alzheimer's Disease: a Case-Control Study of Kashmiri Population.

Alzheimer's disease (AD) is the most common form of dementia, generally affecting elderly people in the age group of above 60-65 years. Amyloid deposition has been found to be a possible cause and a characteristic feature of Alzheimer's disease. Mutations, variant genotypes, or downregulation that reduce amyloid clearance or accelerate amyloid accumulation can lead to Alzheimer's disease. This study involved clinically confirmed AD patients, age matched controls of similar ethnicity, and patients who had no history of cancer or any other chronic disease. DNA and RNA extractions of samples were done as per Saguna et al. [45] and TRIzol method, respectively. Frequencies of variant genotypes were observed using the RFLP technique, whereas, for expression analysis, qPCR was performed. The association between diet, smoking status, family history, and co-morbidities was calculated using statistical tools. Expression analysis showed downregulation in more than 65% of AD cases. Hypertension and diabetes also had a significant association with AD. Allelic isoforms ε2:ε2 and ε2:ε3 tend to be less frequent among AD cases compared to controls (2.85% vs 26.15% and 11.42% vs 21.43%, respectively). Among individuals (AD cases) with ε2:ε3 and ε2:ε4, 37.5% of the patients were having severe dementia and 62.5% were having mild to moderate dementia, whereas, among individuals with ε3:ε4 and ε4:ε4, 57% were having severe dementia and 43% were having mild to moderate dementia. Besides this, all early-onset Alzheimer's patients were found to have at least one ε4 allele. The percentage of individuals with family history (cases vs controls) was 34.17% vs 3.75%, without family history 64.55% vs 95%. On comparing AD cases against controls for smoking status, the results observed are the following: chain smokers, 12.65% vs 18.75%; moderate smokers, 16.45% vs 6.25%; ex-smokers, 36.70% vs 22.50%; non-smokers, 34.17% vs 52.50%. On comparing dietary habits in AD cases against controls, the results were as follows: individuals with generally fatty diet 26.58% vs 11.25%, with mixed diet 36.70% vs 78.75%, with generally vegetarian diet 34.17% vs 10.00%, data not available 2.53% among AD cases. Family history, dietary habits, genetics, and socioeconomic status are strongly associated with the development of Alzheimer disease. Although family history or genetic makeup cannot be changed, eating habits can be changed quite easily. We simply need to go from a high-fat diet to one that is lower in fat. Regarding socioeconomic status, which includes stress of both kinds, including economic stress, stress brought on by the loss of loved ones through death or separation, and co-morbidities (hypertension and diabetes), all are manageable and even modifiable through counseling, positive behavior, and physical activity like exercise, walking, cycling, and playing games.

Quaternary structure analysis of IRE1.

IRE1 belongs to a type I transmembrane protein family harboring two functional domains, cytoplasmic domain with kinase and RNAse catalytic activity, and the luminal domain, which is involved in the sensing of unfolded proteins. IRE1 molecule undergoes dimerization in the lumenal domain, which functionally activates the catalytic C-terminal domain. IRE1 activation is directly related to transition between monomeric and dimeric forms. We have deduced two quaternary structures from the published crystal structure of IRE1. One structure with a large stable interface that requires large activation and deactivation energy to active IRE1. The other quaternary structure has low dissociation energy and is more suitable for IRE1 oligomeric transition.

The Bcl-2 family protein bid interacts with the ER stress sensor IRE1 to differentially modulate its RNase activity.

IRE1 is a transmembrane signalling protein that activates the unfolded protein response under endoplasmic reticulum stress. IRE1 is endowed with kinase and endoribonuclease activities. The ribonuclease activity of IRE1 can switch substrate specificities to carry out atypical splicing of Xbp1 mRNA or trigger the degradation of specific mRNAs. The mechanisms regulating the distinct ribonuclease activities of IRE1 have yet to be fully understood. Here, we report the Bcl-2 family protein Bid as a novel recruit of the IRE1 complex, which directly interacts with the cytoplasmic domain of IRE1. Bid binding to IRE1 leads to a decrease in IRE1 phosphorylation in a way that it can only perform Xbp1 splicing while mRNA degradation activity is repressed. The RNase outputs of IRE1 have been found to regulate the homeostatic-apoptotic switch. This study, thus, provides insight into IRE1-mediated cell survival.

The molecular mechanism and functional diversity of UPR signaling sensor IRE1.

The endoplasmic reticulum is primarily responsible for protein folding and maturation. However, the organelle is subject to varied stress conditions from time to time, which lead to the activation of a signaling program known as the Unfolded Protein Response (UPR) pathway. This pathway, upon sensing any disturbance in the protein-folding milieu sends signals to the nucleus and cytoplasm in order to restore homeostasis. One of the prime UPR signaling sensors is Inositol-requiring enzyme 1 (IRE1); an ER membrane embedded protein with dual enzyme activities, kinase and endoribonuclease. The ribonuclease activity of IRE1 results in Xbp1 splicing in mammals or Hac1 splicing in yeast. However, IRE1 can switch its substrate specificity to the mRNAs that are co-transnationally transported to the ER, a phenomenon known as Regulated IRE1 Dependent Decay (RIDD). IRE1 is also reported to act as a principal molecule that coordinates with other proteins and signaling pathways, which in turn might be responsible for its regulation. The current review highlights studies on IRE1 explaining the structural features and molecular mechanism behind its ribonuclease outputs. The emphasis is also laid on the molecular effectors, which directly or indirectly interact with IRE1 to either modulate its function or connect it to other pathways. This is important in understanding the functional pleiotropy of IRE1, by which it can switch its activity from pro-survival to pro-apoptotic, thus determining the fate of cells.

Aggregation of M3 (E376D) variant of alpha1- antitrypsin.

Alpha1-antitrypsin (α1AT) is an abundant serine-protease inhibitor in circulation. It has an important role in neutralizing the neutrophil elastase activity. Different pathogenic point mutations like Z(E342K)-α1AT have been implicated in the development of liver cirrhosis and Chronic Obstructive Pulmonary Disease (COPD), the latter being a cluster of progressive lung diseases including chronic bronchitis and emphysema. M3-α1AT (376Glu > Asp) is another variant of α1AT which so far is largely being considered as normal though increased frequency of the variant has been reported in many human diseases including COPD. We also observed increased frequency of M3-α1AT in COPD cases in Kashmiri population. The frequency of heterozygous (AC) genotype in cases and controls was 58.57% and 27.61% (odds-ratio 6.53 (2.27-15.21); p < 0.0001) respectively, while homozygous CC genotype was found to be 21.42% and 6.66% (odds-ratio 10.56 (3.63-18.64); p < 0.0001) respectively. Comparative in vitro investigations that include trypsin‒antitrypsin assay, Circular Dichroism spectroscopy and dynamic light scattering performed on wild-type (M-α1AT), M3-α1AT, and Z-α1AT proteins along with the molecular dynamics simulations revealed that M3-α1AT has properties similar to Z-α1AT capable of forming aggregates of varied size. Our maiden observations suggest that M3-α1AT may contribute to the pathogenesis of COPD and other disorders by mechanisms that warrant further investigations.

Antimutagenic activity of compounds isolated from Ajuga bracteosa Wall ex. Benth against EMS induced mutagenicity in mice.

Ajuga bracteosa Wall ex. Benth. (Lamiaceae) has been reported to possess many biological activities including antibacterial, antifungal, antispasmodic and antioxidant activity but there is no report as such on its mutagenic and/or anti-mutagenic activity. The aim of the present study was to isolate compounds from the methanol extract of the aerial parts of Ajuga bracteosa and determine their anti-mutagenic activity against the mutagen, EMS in animal model mice. The study was undertaken in order to corroborate the traditional use of the plant Ajuga bracteosa. The compounds were isolated from the methanol extract of the aerial parts of Ajuga bracteosa using silica gel column chromatography. Structural elucidation of the isolated compounds was done using spectral data analysis and comparison with literature. High performance liquid chromatography (HPLC) was used for the qualitative and quantitative determination of the isolated compounds in the crude methanol extract. The isolated compounds and standard drug were evaluated in vivo for antimutagenic activity against EMS induced mutagenicity taking mice as model organism by micronucleus and chromosomal aberration tests. Four major compounds were identified as 1) 14, 15-dihydroajugapitin 2) ß- Sitosterol 3) Stigmasterol and 4) 8-O-acetylharpagide. A quick and sensitive HPLC method was developed for qualitative and quantitative determination of three isolated marker compounds from Ajuga bracteosa. 14, 15-dihydroajugapitin reduced the micronuclei by 85.10%, followed by ß- Sitosterol (72.3%) while as 8-O-acetylharpagide reduced the micronuclei by 46%. It is therefore evident from the present study that the plant contains rich source of anticancer and antimutagenic drugs.

SERPINA1 Hepatocyte-Specific Promoter Polymorphism Associate with Chronic Obstructive Pulmonary Disease in a Study of Kashmiri Ancestry Individuals.

PURPOSE: Different mutations in coding and non-coding sequences of the SERPINA1 gene have been implicated in the pathogenesis of COPD. However, - 10T/C mutation in the hepatocyte-directed promoter region has not been associated with COPD pathogenesis so far. Here, we report an increased frequency of - 10C genotype that is associated with decreased levels of serum alpha1-antitrypsin (α1AT) in COPD patients. METHODS: The quantification of serum α1AT was done by ELISA, the phenol-chloroform method was used for DNA extraction, PCR products were directly sequenced. The IBM SPSS Statistics v21 software was used for statistical analyses of the data. RESULTS: The mean serum α1AT level was found to be 1.203+0.239 and 3.162+0.160 g/L in COPD cases and in control, respectively. The - 10C allele is associated with an increased risk of COPD [OR, 3.50 (95%CI, 1.86-6.58); p < 0.001]. The combined variant genotype (TT+CC) was significantly found associated with an increased risk of COPD [OR, 3.20 (95% CI, 1.47-6.96); p = 0.003]. A significant association of the family history with COPD (overall p value= 0.0331) suggests that genetics may play an important role in the pathogenesis of COPD. CONCLUSION: The polymorphism associated with hepatocyte-specific promoter region (- 10T/C) is likely to be associated with the pathogenesis of COPD. It is quite possible that the change of the base in the hepatocyte-specific promoter of the SERPINA1 gene can modulate its strength, thereby driving the reduced expression of α1AT.

Alpha-1-antitrypsin deficiency: Genetic variations, clinical manifestations and therapeutic interventions.

Alpha-1-antitrypsin (AAT) is an acute phase secretory glycoprotein that inhibits neutrophil proteases like elastase and is considered as the archetype of a family of structurally related serine-protease inhibitors termed serpins. Serum AAT predominantly originates from liver and increases three to five fold during host response to tissue injury and inflammation. The AAT deficiency is unique among the protein-misfolding diseases in that it causes target organ injury by both loss-of-function and gain-of-toxic function mechanisms. Lack of its antiprotease activity is associated with premature development of pulmonary emphysema and loss-of-function due to accumulation of resultant aggregates in chronic obstructive pulmonary disease (COPD). This' in turn' markedly reduces the amount of AAT that is available to protect lungs against proteolytic attack by the enzyme neutrophil elastase. The coalescence of AAT deficiency, its reduced efficacy, and cigarette smoking or poor ventilation conditions have devastating effect on lung function. On the other hand, the accumulation of retained mutant proteins in the endoplasmic reticulum of hepatocytes in a polymerized form rather than secreted into the blood in its monomeric form is associated with chronic liver disease and predisposition to hepatocellular carcinoma (HCC) by gain- of- toxic function. Liver injury resulting from this gain-of-toxic function mechanism in which mutant AAT retained in the ER initiates a series of pathologic events, eventually culminating at liver cirrhosis and HCC. Here in this review, we underline the structural, genetic, polymorphic, biochemical and pathological advances made in the field of AAT deficiency and further comprehensively emphasize on the therapeutic interventions available for the patient.

Novel variants of SERPIN1A gene: Interplay between alpha1-antitrypsin deficiency and chronic obstructive pulmonary disease.

Alpha1-antitrypsin (AAT) is one of the major circulating anti-protease whose levels in circulation are raised during excessive amount of proteases, especially neutrophil elastase (NE) released during the course of inflammation. Proteolytic attack of NE on peripheral organs, more exclusively on lung parenchyma has severe consequence that may precipitate pulmonary emphysema. Normally, human body has its own molecular and physiological mechanisms to synthesize and regulate the production of anti-protease like AAT to mitigate the extent of inflammatory damage. AAT coded by serine-protease inhibitor (SERPINA1) is predominantly expressed in hepatocytes and to some extent by macrophages, monocytes, lung tissue etc. The observation that persons with AAT deficiency developed chronic obstructive pulmonary disease (COPD) and early-onset of emphysema proposed a role for pathways connecting AAT in pathogenesis. Extensive studies have been done till now to bridge a connection between numerous genetic polymorphisms of SERPINA1 gene and the early onset of COPD. Here in this review, we have comprehensively discussed some of the variants of SERPINA1 gene discovered till date and their association with the exacerbation of obstructive pulmonary disease.

Natural osmolytes alleviate GRP78 and ATF-4 levels: Corroboration for potential modulators of unfolded protein response.

AIMS: Osmolytes are small organic molecules which play a significant role in maintaining functional homeostasis of proteins under extreme hostile stresses. Any imbalance to cell homeostasis leads to Endoplasmic Reticulum stress (ER-stress) to which a set of cellular responses both at transcriptional and translational level are initialed for restoration of cellular homeostasis called Unfolded Protein Response (UPR). In the present study we evaluated the role of Sarcosine, Betaine, Hydroxyectoine and Ectoine as potential modulators of UPR. ER-stress was induced by Tunicamycin, a prototypic experimental ER-stress inducer. MAIN METHODS: The endogenous cellular levels of UPR markers Glucose-Regulated Protein 78 (GRP78) and Activating Transcription Factor-4 (ATF-4) were evaluated in presence and absence of these osmolytes after inducing UPR with tunicamycin. As a prelude to this, IC50 values of these osmolytes were determined by using cell viability assays like MTT and Trypan Blue exclusion assay. KEY FINDINGS: We found that these osmolytes in a dose-dependent manner increased the rate of restoration of homeostasis as was evident by the decreased endogenous levels of GRP78 and ATF-4. SIGNIFICANCE: These natural osmolytes can thus be useful in therapeutic intervention to mitigate the pathophysiological state resulting from ER-stress.

Splicing functions and global dependency on fission yeast slu7 reveal diversity in spliceosome assembly.

The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7(+) (spslu7(+)) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3' splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3' splice site distance, intron length, and the impact of its A/U content at the 5' end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1(+), a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly.

Context dependent splicing functions of Bud31/Ycr063w define its role in budding and cell cycle progression.

The yeast Bud31 protein, a Prp19 complex (NTC) member, aids spliceosome assembly and thus promotes efficient pre-mRNA splicing. The bud31 null cells show mild budding abnormalities at optimal growth temperatures and, at higher temperatures, have growth defects with aberrant budding. Here we have assessed cell cycle transitions which require Bud31. We find Bud31 facilitates passage through G1-S regulatory point (Start) but is not needed for G2-M transition or for exit from mitosis. To co-relate Bud31 functions in cell division with splicing, we studied the splicing status of transcripts that encode proteins involved in budding. We find Bud31 promotes efficient splicing of only some of these pre-mRNAs, for example, ARP2 and SRC1. Wild type cells have a long and a short isoform of SRC1 mRNA and protein, out of which the shorter mRNA splice variant is predominant. bud31Δ cells show inefficient SRC1 splicing and entirely lack the shorter SRC1 spliced mRNA isoform. Yeast PRP17, another NTC sub-complex member, is also required for G1-S and G2-M cell cycle transitions. We examined genetic interactions between BUD31 and PRP17. While both factors were needed for efficient cell cycle dependent gene expression, our data indicate that distinct pre-mRNAs depend on each of these non-essential splicing factors.