Idiopathic pulmonary fibrosis (IPF): disease pathophysiology, targets, and potential therapeutic interventions.

Idiopathic pulmonary fibrosis (IPF) is a progressive, degenerative pulmonary condition. Transforming growth factor (TGF)-ß, platelet-derived growth factor (PDGF), and tumor necrosis factor-α (TNF-α) are the major modulators of IPF that mediate myofibroblast differentiation and promote fibrotic remodeling of the lung. Cigarette smoke, asbestos fiber, drugs, and radiation are known to favor fibrotic remodeling of the lungs. Oxidative stress in the endoplasmic reticulum (ER) also leads to protein misfolding and promotes ER stress, which is predominant in IPF. This phenomenon further results in excess reactive oxygen species (ROS) aggregation, increasing oxidative stress. During protein folding in the ER, thiol groups on the cysteine residue are oxidized and disulfide bonds are formed, which leads to the production of hydrogen peroxide (H2O2) as a by-product. With the accumulation of misfolded proteins in the ER, multiple signaling cascades are initiated by the cell, collectively termed as the unfolded protein response (UPR). UPR also induces ROS production within the ER and mitochondria and promotes both pro-apoptotic and pro-survival pathways. The prevalence of post-COVID-19 pulmonary fibrosis (PCPF) is 44.9%, along with an alarming increase in "Coronavirus Disease 2019" (COVID-19) comorbidities. Fibrotic airway remodeling and declined lung function are the common endpoints of SARS-CoV-2 infection and IPF. Flavonoids are available in our dietary supplements and exhibit medicinal properties. Apigenin is a flavonoid found in plants, including chamomile, thyme, parsley, garlic, guava, and broccoli, and regulates several cellular functions, such as oxidative stress, ER stress, and fibrotic responses. In this study, we focus on the IPF and COVID-19 pathogenesis and the potential role of Apigenin in addressing disease progression.

Exploring microglia and their phenomenal concatenation of stress responses in neurodegenerative disorders.

Neuronal cells are highly functioning but also extremely stress-sensitive cells. By defending the neuronal cells against pathogenic insults, microglial cells, a unique cell type, act as the frontline cavalry in the central nervous system (CNS). Their remarkable and unique ability to self-renew independently after their creation is crucial for maintaining normal brain function and neuroprotection. They have a wide range of molecular sensors that help maintain CNS homeostasis during development and adulthood. Despite being the protector of the CNS, studies have revealed that persistent microglial activation may be the root cause of innumerable neurodegenerative illnesses, including Alzheimer's disease (AD), Parkinson's disease (PD), and Amyotrophic Lateral Sclerosis (ALS). From our vigorous review, we state that there is a possible interlinking between pathways of Endoplasmic reticulum (ER) stress response, inflammation, and oxidative stress resulting in dysregulation of the microglial population, directly influencing the accumulation of pro-inflammatory cytokines, complement factors, free radicals, and nitric oxides leading to cell death via apoptosis. Recent research uses the suppression of these three pathways as a therapeutic approach to prevent neuronal death. Hence, in this review, we have spotlighted the advancement in microglial studies, which focus on their molecular defenses against multiple stresses, and current therapeutic strategies indirectly targeting glial cells for neurodevelopmental diseases.

Structure-Based Virtual Screening and Discovery of New Bi-functional DAPK1 Inhibitors.

Recently, a new signaling complex Death-Associated Protein Kinase 1 (DAPK1)-N-methyl D-aspartate receptor subtype 2B (NR2B) engaged in the neuronal death cascade was identified where it was found that after stroke injury, N-methyl-D-aspartate glutamate (NMDA) receptors interact with DAPK1 through NR2B subunit and lead to excitotoxicity via overactivation of NMDA receptors. In this study, we used ZINC-12 database to find out potential inhibitor of DAPK1 and found some natural compounds showing good binding affinity towards DAPK1. These natural compounds showed interactions with ATP-binding site residues as well as substrate-recognition motifs. Thus, it has been concluded that the ligands those are showing interactions with both the sites could be considered as potential inhibitors for DAPK1.

Role of death-associated protein kinase 1 (DAPK1) in retinal degenerative diseases: an in-silico approach towards therapeutic intervention.

The Death-associated protein kinase 1 (DAPK1) has emerged as a crucial player in the pathogenesis of degenerative diseases. As a serine/threonine kinase family member, DAPK1 regulates critical signaling pathways, such as apoptosis and autophagy. In this study, we comprehensively analyzed DAPK1 interactors and enriched molecular functions, biological processes, phenotypic expression, disease associations, and aging signatures to elucidate the molecular networks of DAPK1. Furthermore, we employed a structure-based virtual screening approach using the PubChem database, which enabled the identification of potential bioactive compounds capable of inhibiting DAPK1, including caspase inhibitors and synthetic analogs. Three selected compounds, CID24602687, CID8843795, and CID110869998, exhibited high docking affinity and selectivity towards DAPK1, which were further investigated using molecular dynamics simulations to understand their binding patterns. Our findings establish a connection between DAPK1 and retinal degenerative diseases and highlight the potential of these selected compounds for the development of novel therapeutic strategies. This study provides valuable insights into the molecular mechanisms underlying DAPK1-related diseases, and offers new opportunities for the discovery of effective treatments for retinal degeneration.Communicated by Ramaswamy H. Sarma.

Genome-wide screening and identification of potential kinases involved in endoplasmic reticulum stress responses.

AIM: This study aims to identify endoplasmic reticulum stress response elements (ERSE) in the human genome to explore potentially regulated genes, including kinases and transcription factors, involved in the endoplasmic reticulum (ER) stress and its related diseases. MATERIALS AND METHODS: Python-based whole genome screening of ERSE was performed using the Amazon Web Services elastic computing system. The Kinome database was used to filter out the kinases from the extracted list of ERSE-related genes. Additionally, network analysis and genome enrichment were achieved using NDEx, the Network and Data Exchange software, and web-based computational tools. To validate the gene expression, quantitative RT-PCR was performed for selected kinases from the list by exposing the HeLa cells to tunicamycin and brefeldin, ER stress inducers, for various time points. KEY FINDINGS: The overall number of ERSE-associated genes follows a similar pattern in humans, mice, and rats, demonstrating the ERSE's conservation in mammals. A total of 2705 ERSE sequences were discovered in the human genome (GRCh38.p14), from which we identified 36 kinases encoding genes. Gene expression analysis has shown a significant change in the expression of selected genes under ER stress conditions in HeLa cells, supporting our finding. SIGNIFICANCE: In this study, we have introduced a rapid method using Amazon cloud-based services for genome-wide screening of ERSE sequences from both positive and negative strands, which covers the entire genome reference sequences. Approximately 10 % of human protein-protein interactomes were found to be associated with ERSE-related genes. Our study also provides a rich resource of human ER stress-response-based protein networks and transcription factor interactions and a reference point for future research aiming at targeted therapeutics.

COVID-19 and retinal degenerative diseases: Promising link "Kaempferol".

Coronavirus disease (COVID-19) outbreak has caused unprecedented global disruption since 2020. Approximately 238 million people are affected worldwide where the elderly succumb to mortality. Post-COVID syndrome and its side effects have popped up with several health hazards, such as macular degeneration and vision loss. It thus necessitates better medical care and management of our dietary practices. Natural flavonoids have been included in traditional medicine and have also been used safely against COVID-19 and several other diseases. Kaempferol is an essential flavonoid that has been demonstrated to influence several vital cellular signaling pathways involved in apoptosis, angiogenesis, inflammation, and autophagy. In this review, we emphasize the plausible regulatory effects of Kaempferol on hallmarks of COVID-19 and macular degeneration.

IRE1α is critical for Kaempferol-induced neuroblastoma differentiation.

Neuroblastoma is an embryonic malignancy that arises out of the neural crest cells of the sympathetic nervous system. It is the most common childhood tumor known for its spontaneous regression via the process of differentiation. The induction of differentiation using small molecules such as retinoic acid is one of the therapeutic strategies to treat the residual disease. In this study, we have reported the effect of kaempferol (KFL) in inducing differentiation of neuroblastoma cells in vitro. Treatment of neuroblastoma cells with KFL reduced the proliferation and enhanced apoptosis along with the induction of neuritogenesis. Analysis of the expression of neuron-specific markers such as ß-III tubulin, neuron-specific enolase, and N-myc downregulated gene 1 revealed the process of differentiation accompanying KFL-induced apoptosis. Further analysis to understand the molecular mechanism of action showed that the effect of KFL is mediated by the activation of the endoribonuclease activity of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum-resident transmembrane protein. In silico docking analysis and biochemical assays using recombinant human IRE1α confirm the binding of KFL to the ATP-binding site of IRE1α, which thereby activates IRE1α ribonuclease activity. Treatment of cells with the small molecule STF083010, which specifically targets and inhibits the endoribonuclease activity of IRE1α, showed reduced expression of neuron-specific markers and curtailed neuritogenesis. The knockdown of IRE1α using plasmid-based shRNA lentiviral particles also showed diminished changes in the morphology of the cells upon KFL treatment. Thus, our study suggests that KFL induces differentiation of neuroblastoma cells via the IRE1α -XBP1 pathway.

Identification of FDA-approved drugs as novel allosteric inhibitors of human executioner caspases.

The regulation of apoptosis is a tightly coordinated process and caspases are its chief regulators. Of special importance are the executioner caspases, caspase-3/7, the activation of which irreversibly sets the cell on the path of death. Dysregulation of apoptosis, particularly an increased rate of cell death lies at the root of numerous human diseases. Although several peptide-based inhibitors targeting the homologous active site region of caspases have been developed, owing to their non-specific activity and poor pharmacological properties their use has largely been restricted. Thus, we sought to identify FDA-approved drugs that could be repurposed as novel allosteric inhibitors of caspase-3/7. In this study, we virtually screened a catalog of FDA-approved drugs targeting an allosteric pocket located at the dimerization interface of caspase-3/7. From among the top-scoring hits we short-listed 5 compounds for experimental validation. Our enzymatic assays using recombinant caspase-3 suggested that 4 out of the 5 drugs effectively inhibited caspase-3 enzymatic activity in vitro with IC50 values ranging ~10-55 µM. Structural analysis of the docking poses show the 4 compounds forming specific non-covalent interactions at the allosteric pocket suggesting that these molecules could disrupt the adjacently-located active site. In summary, we report the identification of 4 novel non-peptide allosteric inhibitors of caspase-3/7 from among FDA-approved drugs.

Identification of ASB7 as ER stress responsive gene through a genome wide in silico screening for genes with ERSE.

The endoplasmic reticulum (ER) not only performs its basic function of regulating calcium homeostasis, lipid biosynthesis, folding, modifying and transporting proteins but also plays a decisive role in regulating multiple cellular processes ranging from cell growth and differentiation to apoptosis and autophagy. Disturbances in ER homeostasis initiate the unfolded protein response (UPR) implicated in the pathogenesis of many human diseases. Drugging the UPR components for therapeutic interventions has received considerable attention. The purpose of this study is to identify genes that are previously unsuspected to be regulated under ER stress. Because ER stress-inducible gene expression is majorly regulated under ERSE elements, we screened human genome by adopting an in silico approach using ERSE elements (I, II, III) as probes and identified 337 candidate genes. Having knowledge of the importance of E3 ubiquitin ligase in the ERAD machinery; we validated our preliminary search by focusing on one of the hits i.e. ASB7 gene that encodes E3 ubiquitin ligase. In HeLa cells, we found that pharmacological induction of ER stress led to an increase in the expression of ASB7 with simultaneous activation of UPR pathways. Although knockdown of ASB7 expression leads to significant reduction in GRP78 and CHOP mRNA levels, it did not protect cells from ER stress-induced cell death. Also, an up-regulation in the expression of pro-inflammatory genes like TNF-α and IL-1ß in ASB7 knockdown cells was observed under ER stress. Collectively, our findings suggest that ASB7 is regulated under ER stress and this study also identifies several other genes that could apparently be regulated under ER stress.

CDDO and ATRA Instigate Differentiation of IMR32 Human Neuroblastoma Cells.

Neuroblastoma is the most common solid extra cranial tumor in infants. Improving the clinical outcome of children with aggressive tumors undergoing one of the multiple treatment options has been a major concern. Differentiating neuroblastoma cells holds promise in inducing tumor growth arrest and treating minimal residual disease. In this study, we investigated the effect of partial PPARγ agonist 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) on human neuroblastoma IMR32 cells. Our results demonstrate that treatment with low concentration of CDDO and particularly in combination with all trans retinoic acid (ATRA) induced neurite outgrowth, increased the percentage of more than two neurites bearing cells, and decreased viability in IMR32 cells. These morphological changes were associated with an increase in expression of bonafide differentiation markers like ß3-tubulin and Neuron Specific Enolase (NSE). The differentiation was accompanied by a decrease in the expression of MYCN whose amplification is known to contribute to the pathogenesis of neuroblastoma. MYCN is known to negatively regulate NMYC downstream-regulated gene 1 (NDRG1) in neuroblastomas. MYCN down-regulation induced by CDDO correlated with increased expression of NDRG1. CDDO decreased Anaplastic Lymphoma Kinase (ALK) mRNA expression without affecting its protein level, while ATRA significantly down-regulated ALK. Antagonism of PPARγ receptor by T0070907 meddled with differentiation inducing effects of CDDO as observed by stunted neurite growth, increased viability and decreased expression of differentiation markers. Our findings indicate that IMR32 differentiation induced by CDDO in combination with ATRA enhances, differentiation followed by cell death via cAMP-response-element binding protein (CREB) independent and PPARγ dependent signaling mechanisms.

Autophagy: The spotlight for cellular stress responses.

Autophagy is an essential cellular mechanism which plays "housekeeping" role in normal physiological processes including removing of long lived, aggregated and misfolded proteins, clearing damaged organelles, growth regulation and aging. Autophagy is also involved in a variety of biological functions like development, cellular differentiation, defense against pathogens and nutritional starvation. The integration of autophagy into these biological functions and other stress responses is determined by the transcriptional factors that undertake the regulatory mechanism. This review discusses the machinery of autophagy, the molecular web that connects autophagy to various stress responses like inflammation, hypoxia, ER stress, and various other pathologic conditions. Defects in autophagy regulation play a central role in number of diseases, including neurodegenerative diseases, cancer, pathogen infection and metabolic diseases. Similarly, inhibiting autophagy would contribute in the treatment of cancer. However, understanding the biology of autophagy regulation requires pharmacologically active compounds which modulate the autophagy process. Inducers of autophagy are currently receiving considerable attention as autophagy upregulation may be a therapeutic benefit for certain neurodegenerative diseases (via removal of protein aggregates) while the inhibitors are being investigated for the treatment of cancers. Both induction and inhibition of autophagy have been proven to be beneficial in the treatment of cancer. This dual role of autophagy in cancers is now getting uncovered by the advancement in the research findings and development of effective autophagy modulators.

Exploring putative inhibitors of Death Associated Protein Kinase 1 (DAPK1) via targeting Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN) substrate recognition motifs.

Recently, a new signaling complex Death Associated Protein Kinase 1 (DAPK1) ̶ N-methyl-D-aspartate receptor subtype 2B (NMDAR2B or NR2B) engaged in the neuronal death cascade was identified and it was found that after stroke injury, N-methyl-D-aspartate glutamate (NMDA) receptors interact with DAPK1 through NR2B subunit and lead to excitotoxicity via over-activation of NMDA receptors. An acute brain injury, such as stroke, is a serious life-threatening medical condition which occurs due to poor blood supply to the brain and further leads to neuronal cell death. During a stroke, activated DAPK1 migrates towards the extra-synaptic site and binds to NR2B subunit of NMDA receptor. It is this DAPK1-NR2B interaction that arbitrates the pathological processes like apoptosis, necrosis, and autophagy of neuronal cells observed in stroke injury, hence we aimed to inhibit this vital interaction to prevent neuronal damage. In the present study, using PubChem database, we applied an integrative approach of virtual screening and molecular dynamic simulations and identified a potential lead compound 11 that interrupts DAPK1-NR2B interaction by competing with both ATP and substrate for their binding sites on DAPK1. This inhibitor was found potent and considerably selective to DAPK1 as it made direct contact with the ATP binding sites as well as substrate recognition motifs: Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN). Further in vitro and in vivo experiments are demanded to validate the efficacy of compound 11 nevertheless, it can be considered as suitable starting point for designing DAPK1 inhibitors.

Death Associated Protein Kinase 1 (DAPK1): A Regulator of Apoptosis and Autophagy.

Death-Associated Protein Kinase 1 (DAPK1) belongs to a family of five serine/threonine (Ser/Thr) kinases that possess tumor suppressive function and also mediate a wide range of cellular processes, including apoptosis and autophagy. The loss and gain-of-function of DAPK1 is associated with various cancer and neurodegenerative diseases respectively. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms involved in DAPK1-mediated autophagy/apoptosis. In the present review, we have discussed the structural information and various cellular functions of DAPK1 in a comprehensive manner.

A molecular web: endoplasmic reticulum stress, inflammation, and oxidative stress.

Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER) is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded-protein response (UPR) through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS). Toxic accumulation of ROS within ER and mitochondria disturbs fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways have been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease, and others. In this review, we have discussed the UPR signaling pathways, and networking between ER stress-induced inflammatory pathways, oxidative stress, and mitochondrial signaling events, which further induce or exacerbate ER stress.

In silico identification of genetic variants in glucocerebrosidase (GBA) gene involved in Gaucher's disease using multiple software tools.

Gaucher's disease (GD) is an autosomal recessive disorder caused by the deficiency of glucocerebrosidase, a lysosomal enzyme that catalyses the hydrolysis of the glycolipid glucocerebroside to ceramide and glucose. Polymorphisms in GBA gene have been associated with the development of Gaucher disease. We hypothesize that prediction of SNPs using multiple state of the art software tools will help in increasing the confidence in identification of SNPs involved in GD. Enzyme replacement therapy is the only option for GD. Our goal is to use several state of art SNP algorithms to predict/address harmful SNPs using comparative studies. In this study seven different algorithms (SIFT, MutPred, nsSNP Analyzer, PANTHER, PMUT, PROVEAN, and SNPs&GO) were used to predict the harmful polymorphisms. Among the seven programs, SIFT found 47 nsSNPs as deleterious, MutPred found 46 nsSNPs as harmful. nsSNP Analyzer program found 43 out of 47 nsSNPs are disease causing SNPs whereas PANTHER found 32 out of 47 as highly deleterious, 22 out of 47 are classified as pathological mutations by PMUT, 44 out of 47 were predicted to be deleterious by PROVEAN server, all 47 shows the disease related mutations by SNPs&GO. Twenty two nsSNPs were commonly predicted by all the seven different algorithms. The common 22 targeted mutations are F251L, C342G, W312C, P415R, R463C, D127V, A309V, G46E, G202E, P391L, Y363C, Y205C, W378C, I402T, S366R, F397S, Y418C, P401L, G195E, W184R, R48W, and T43R.

Synthetic triterpenoid cyano enone of methyl boswellate activates intrinsic, extrinsic, and endoplasmic reticulum stress cell death pathways in tumor cell lines.

We explored the effect of a novel synthetic triterpenoid compound cyano enone of methyl boswellates (CEMB) on various prostate cancer and glioma cancer cell lines. CEMB displayed concentration-dependent cytotoxic activity with submicromolar lethal dose 50% (LD(50)) values in 10 of 10 tumor cell lines tested. CEMB-induced cytotoxicity is accompanied by activation of downstream effector caspases (caspases 3 and 7) and by upstream initiator caspases involved in both the extrinsic (caspase 8) and intrinsic (caspase 9) apoptotic pathways. By using short interfering RNAs (siRNA), we show evidence that knockdown of caspase 8, DR4, Apaf-1, and Bid impairs CEMB-induced cell death. Similar to other proapoptotic synthetic triterpenoid compounds, CEMB-induced apoptosis involved endoplasmic reticulum stress, as shown by partial rescue of tumor cells by siRNA-mediated knockdown of expression of genes involved in the unfolded protein response such as IRE1α, PERK, and ATF6. Altogether, our results suggest that CEMB stimulates several apoptotic pathways in cancer cells, suggesting that this compound should be evaluated further as a potential agent for cancer therapy.

Novel systems biology insights using antifibrotic approaches for diabetic kidney disease.

Although several interventions slow the progression of diabetic nephropathy, current therapies do not halt progression completely. Recent preclinical studies suggested that pirfenidone (PFD) prevents fibrosis in various diseases, but the mechanisms underlying its antifibrotic action are incompletely understood. To explore the therapeutic potential of PFD, we studied the PFD-treated db/db diabetic mouse kidney by liquid chromatography-tandem mass spectrometry proteomics. A total of 21 proteins unique to PFD-treated diabetic kidneys were identified. Analysis of gene ontology and protein-protein interactions of these proteins suggested that PFD may regulate RNA translation. Two key proteins involved in mRNA translation initiation and elongation were further evaluated and found to be regulated by PFD at the level of phosphorylation. In conclusion, insights from combining proteomics and bioinformatics improve the likelihood of rapid advancement of novel clinical therapies focused on reducing inflammation and fibrosis for diabetic complications.

PMAP: databases for analyzing proteolytic events and pathways.

The Proteolysis MAP (PMAP, http://www.proteolysis.org) is a user-friendly website intended to aid the scientific community in reasoning about proteolytic networks and pathways. PMAP is comprised of five databases, linked together in one environment. The foundation databases, ProteaseDB and SubstrateDB, are driven by an automated annotation pipeline that generates dynamic 'Molecule Pages', rich in molecular information. PMAP also contains two community annotated databases focused on function; CutDB has information on more than 5000 proteolytic events, and ProfileDB is dedicated to information of the substrate recognition specificity of proteases. Together, the content within these four databases will ultimately feed PathwayDB, which will be comprised of known pathways whose function can be dynamically modeled in a rule-based manner, and hypothetical pathways suggested by semi-automated culling of the literature. A Protease Toolkit is also available for the analysis of proteases and proteolysis. Here, we describe how the databases of PMAP can be used to foster understanding of proteolytic pathways, and equally as significant, to reason about proteolysis.

Confirmation of human protein interaction data by human expression data.

BACKGROUND: With microarray technology the expression of thousands of genes can be measured simultaneously. It is well known that the expression levels of genes of interacting proteins are correlated significantly more strongly in Saccharomyces cerevisiae than those of proteins that are not interacting. The objective of this work is to investigate whether this observation extends to the human genome. RESULTS: We investigated the quantitative relationship between expression levels of genes encoding interacting proteins and genes encoding random protein pairs. Therefore we studied 1369 interacting human protein pairs and human gene expression levels of 155 arrays. We were able to establish a statistically significantly higher correlation between the expression levels of genes whose proteins interact compared to random protein pairs. Additionally we were able to provide evidence that genes encoding proteins belonging to the same GO-class show correlated expression levels. CONCLUSION: This finding is concurrent with the naive hypothesis that the scales of production of interacting proteins are linked because an efficient interaction demands that involved proteins are available to some degree. The goal of further research in this field will be to understand the biological mechanisms behind this observation.