Intracellular Protein Homeostasis Research Articles

Regenerating Chemotherapeutics through Copper-Based Nanomedicine: Disrupting Protein Homeostasis for Enhanced Tumor Therapy.

The bis-(diethyldithiocarbamate)-copper (CuET), the disulfiram (DSF)-Cu complex, has exhibited noteworthy anti-tumor property. However, its efficacy is compromised due to the inadequate oxidative conditions and the limitation of bioavailable copper. Because CuET can inactivate valosin-containing protein (VCP), a bioinformatic pan-cancer analysis of VCP is first conducted in this study to identify CuET as a promising anticancer drug for diverse cancer types. Then, based on the drug action mechanism, a nanocomposite of CuET and copper oxide (CuO) is designed and fabricated utilizing bovine serum albumin (BSA) as the template (denoted as CuET-CuO@BSA, CCB). CCB manifests peroxidase (POD)-mimicking activity to oxidize the tumor endogenous H2O2 to generate reactive oxygen species (ROS), enhancing the chemotherapy effect of CuET. Furthermore, the cupric ions released after enzymatic reaction can regenerate CuET, which markedly perturbs intracellular protein homeostasis and induces apoptosis of tumor cells. Meanwhile, CCB triggers cuproptosis by inducing the aggregation of lipoylated proteins. The multifaceted action of CCB effectively inhibits tumor progression. Therefore, this study presents an innovative CuET therapeutic strategy that creates an oxidative microenvironment in situ and simultaneously self-supply copper source for CuET regeneration through the combination of CuO nanozyme with CuET, which holds promise for application of CuET for effective tumor therapy.

History of the study of amyloidosis: from the Rokitansky's theory to the present day

In the history of amyloidosis studying the concept of liquids dyscrasia has been predominated and finally it is resulted in accepting a serum protein-precursor as a leading amyloidogenic factor in the disease pathogenesis. Consequently basic diagnostic and treatment strategy was aimed to find and eliminate this protein from the blood and this approach evidenced high effectiveness in most frequent AA and AL-amyloidosis characterized with anomaly high levels of precursors in the blood. At the same time there are less frequent and slower progressing inheritant forms of systemic amyloidosis including transthyretin induced, which are less depending on amyloidogenecity of amyloid precursor and because of that, in example, the effectiveness of transthyretin stabilizers or blockers of its synthesis is limited comparing with the precursor elimination in AA or AL. Developed in the middle of XX century a theory of local synthesis by macrophages is more preferable to describe the pathogenesis of these forms. And modern proteome analysis using give rise to confirm the key meaning of macrophage in the amyloidogenesis and proves necessity to know deeply mechanisms of macrophagial autophagia - basic tool of maintaining intracellular protein homeostasis. That is why it is difficult to hope on high effectiveness of chemical amyloid solvents in vivo, which being under macrophages regulation never could realize its chemical activities.

The Role of Ubiquitination in Osteosarcoma Development and Therapies.

The ubiquitin-proteasome system (UPS) maintains intracellular protein homeostasis and cellular function by regulating various biological processes. Ubiquitination, a common post-translational modification, plays a crucial role in the regulation of protein degradation, signal transduction, and other physiological and pathological processes, and is involved in the pathogenesis of various cancers, including osteosarcoma. Osteosarcoma, the most common primary malignant bone tumor, is characterized by high metastatic potential and poor prognosis. It is a refractory bone disease, and the main treatment modalities are surgery combined with chemotherapy. Increasing evidence suggests a close association between UPS abnormalities and the progression of osteosarcoma. Due to the complexity and pleiotropy of the ubiquitination system, each step in the ubiquitination process can be targeted by drugs. In recent years, research and development of inhibitors targeting the ubiquitin system have increased gradually, showing great potential for clinical application. This article reviews the role of the ubiquitination system in the development and treatment of osteosarcoma, as well as research progress, with the hope of improving the therapeutic effects and prognosis of osteosarcoma patients by targeting effective molecules in the ubiquitination system.

Selective autophagy receptor p62 promotes antibacterial and antiviral immunity in common carp (Cyprinus carpio)

Sequestosome 1 (SQSTM1/p62) is a selective autophagy adapter protein that participates in antiviral and bacterial immune responses and plays an important regulatory role in clearing the proteins to be degraded and maintaining intracellular protein homeostasis. In this study, two p62 genes were cloned from common carp (Cyprinus carpio), namely Ccp62-1 and Ccp62-2, and conducted bioinformatics analysis on them. The results showed that Ccp62s had the same structural domain (Phox and Bem1 domain, ZZ-type zinc finger domain, and ubiquitin-associated domain) as p62 from other species. Ccp62s were widely expressed in various tissues of fish, and highly expressed in immune organs such as gills, spleen, head kidney, etc. Subcellular localization study showed that they were mainly distributed in punctate aggregates in the cytoplasm. After stimulation with Aeromonas hydrophila and spring viraemia of carp virus (SVCV), the expression level of Ccp62s was generally up-regulated. Overexpression of Ccp62s in EPC cells could inhibit SVCV replication. Upon A. hydrophila challenge, the bacterial load in Ccp62s-overexpressing group was significantly reduced, the expression levels of pro-inflammatory cytokines and interferon factors were increased, and the survival rate of the fish was improved. These results indicated that Ccp62s were involved in the immune response of common carp to bacterial and viral infections.

Feasibility of Improving Glycemic Control via Heat Therapy in individuals at risk for Alzheimer’s Disease (FIGHT-AD): a pilot study

Impaired glycemic control increases the risk for type 2 diabetes and Alzheimer’s Disease (AD). Mechanisms such as impaired energy metabolism, cellular bioenergetic function, reduced intracellular protein homeostasis, and inflammation are all potential contributors to AD pathology. Heat therapy, via hot water immersion, has been shown to improve blood glucose regulation, insulin resistance, and inflammation. Given the potential contribution of these factors to brain health, heat therapy could offer immense clinical benefit to individuals at risk for AD. We performed a pilot study to test the feasibility of utilizing heat therapy in older individuals at risk for AD due to metabolic risk. The study intervention involved two pre-visits which included a cognitive function screen, sleep, diet, and physical activity questionnaires, as well as a glucose tolerance test and MRI scan. The intervention consisted of four weeks of heat therapy (3 visits/wk) for a total of 12 visits, followed by two post-intervention study visits identical to the pre-study visits. A total of 18 individuals (9 male, 9 female), average age 71.1 ± 3.9 y completed the intervention. Participants demonstrated metabolic risk based on average HbA1c values of 6.6 ± 0.67, Body Mass Index (BMI) of 29.1 ± 5.4, and 15 of the 18 participants were on hypertensive medications. Our cohort was highly diverse, with 33% underrepresented minority group representation. We had 96% participant adherence for the study (8 missed visits out of 216 total visits), and 1 study related Adverse Event (mild dizziness/nausea) that was mild. Overall, the research participants responded to a post-intervention survey saying they enjoyed participating in the study and it was not a burden on their schedules. In addition, we obtained preliminary data demonstrating a significant effect of the heat therapy intervention on body mass index, mean arterial pressure, and cerebral perfusion (p<0.05), and a trend for improved self-reported sleep quality (Pittsburgh Sleep Quality Index; p=0.06). Future studies of longer duration are needed to provide a comprehensive understanding of the impact of heat therapy on glucose homeostasis and brain health. NIH P30 AG035982 (KU ADRC), pilot funds to PCG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Protein quality control systems in the endoplasmic reticulum and the cytosol coordinately prevent alachlor-induced proteotoxic stress in Saccharomyces cerevisiae

Alachlor, a widely used chloroacetanilide herbicide for controlling annual grasses in crops, has been reported to rapidly trigger protein denaturation and aggregation in the eukaryotic model organism Saccharomyces cerevisiae. Therefore, this study aimed to uncover cellular mechanisms involved in preventing alachlor-induced proteotoxicity. The findings reveal that the ubiquitin-proteasome system (UPS) plays a crucial role in eliminating alachlor-denatured proteins by tagging them with polyubiquitin for subsequent proteasomal degradation. Exposure to alachlor rapidly induced an inhibition of proteasome activity by 90 % within 30 min. The molecular docking analysis suggests that this inhibition likely results from the binding of alachlor to β subunits within the catalytic core of the proteasome. Notably, our data suggest that nascent proteins in the endoplasmic reticulum (ER) are the primary targets of alachlor. Consequently, the unfolded protein response (UPR), responsible for coping with aberrant proteins in the ER, becomes activated within 1 h of alachlor treatment, leading to the splicing of HAC1 mRNA into the active transcription activator Hac1p and the upregulation of UPR gene expression. These findings underscore the critical roles of the protein quality control systems UPS and UPR in mitigating alachlor-induced proteotoxicity by degrading alachlor-denatured proteins and enhancing the protein folding capacity of the ER.

The Role of Chaperone-Mediated Autophagy in Tissue Homeostasis and Disease Pathogenesis.

Chaperone-mediated autophagy (CMA) is a selective proteolytic pathway in the lysosomes. Proteins are recognized one by one through the detection of a KFERQ motif or, at least, a KFERQ-like motif, by a heat shock cognate protein 70 (Hsc70), a molecular chaperone. CMA substrates are recognized and delivered to a lysosomal CMA receptor, lysosome-associated membrane protein 2A (LAMP-2A), the only limiting component of this pathway, and transported to the lysosomal lumen with the help of another resident chaperone HSp90. Since approximately 75% of proteins are reported to have canonical, phosphorylation-generated, or acetylation-generated KFERQ motifs, CMA maintains intracellular protein homeostasis and regulates specific functions in the cells in different tissues. CMA also regulates physiologic functions in different organs, and is then implicated in disease pathogenesis related to aging, cancer, and the central nervous and immune systems. In this minireview, we have summarized the most important findings on the role of CMA in tissue homeostasis and disease pathogenesis, updating the recent advances for this Special Issue.

Hsp70 ameliorates sleep deprivation-induced anxiety-like behavior and cognitive impairment in mice

BackgroundMany neurobehavioral processes, including psychomotor, cognitive, and affection are negatively impacted by sleep deprivation (SD), which may be harmful to a person's physical and mental health. Heat shock proteins (Hsps) have been demonstrated to play a protective role in a number of neurodegenerative diseases and are essential for maintaining intracellular protein homeostasis, but their roles in SD remain elusive. MethodsA mouse SD model was constructed using a modified multi-platform water environment method. The cognitive function was tested by novel object recognition test and Y-maze test, and anxiety-like behaviors were assessed by open field test (OFT). Protein expression was determined by Western blotting assay and ELISA assay. ResultsWe found that SD could profoundly enhance anxiety levels and impair cognitive function in mice. SD also reduced the expression levels of p-cAMP-response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) and increased microglial activation and neuroinflammatory response in the hippocampus of mice. The intranasal injection of human recombinant Hsp70 protein could alleviate SD-induced anxiety and cognitive impairment, as well as restore pCREB and BDNF levels and reduce microglia-induced neuroinflammation in the hippocampus of SD mice. ConclusionsHsp70 treatment might serve as a potential treatment for mitigating SD-related unfavorable symptoms.

TCH-165 attenuates cardiac ischaemia/reperfusion injury by balancing mitochondrial dynamics via increasing proteasome activity

The proteasome is the main complex responsible for maintaining intracellular protein homeostasis, impairment of which is associated with cardiac ischaemia/reperfusion (I/R) injury. The small molecule TCH-165 has been found to activate the 20S proteasome to remove disordered proteins in multiple myeloma and glioblastoma. However, the preventive effect of TCH-165 against I/R-mediated cardiac impairment in mice remains largely unknown. Here, a cardiac I/R model was established in mice. Heart function was assessed with echocardiography. Cardiac infarction, myocyte death, and superoxide level were evaluated by 2,3,5-triphenyltetrazolium chloride (TTC)-Evans blue staining, terminal deoxynucleotidyl transferase-mediated dUTP nick and labelling (TUNEL) assay and immunostaining, respectively. Our results showed that TCH-165 treatment markedly ameliorated I/R-mediated cardiac dysfunction and decreased the infarct size, apoptosis, and superoxide levels. Mechanistically, TCH-165 increased immunoproteasome subunit expression/activity, increasing pro-fission protein dynamin-1-like protein (DNM1L, also known as DRP1) degradation and the expression of the pro-fusion proteins mitofusin 1/2 (Mfn1/2) and thereby leading to mitochondrial fission/fusion balance. In vitro experiments confirmed that inhibition of proteasome activity by epoxomicin abolished the protective effect of TCH-165 against hypoxia/reoxygenation (H/R)-induced increases in cardiomyocyte apoptosis, superoxide production and mitochondrial fission. In summary, TCH-165 is a newly discovered inducer of immunoproteasome activity that exerts a preventive effect against cardiac I/R damage by targeting Drp1 degradation, indicating that it may be as a potential therapeutic candidate for ischaemic heart disease.

A novel genetically encodable tagging system for visualizing translation dynamics in living cells.

Many diseases, such as cancers and Alzheimer's disease, are caused by disrupted intracellular protein homeostasis. Understanding how proteins are translated is critical for the development of therapeutics that can help maintain protein homeostasis and therefore fight diseases. Over the past six years, the use of repeat epitope tags combined with complementary fluorescent intrabodies, such as the SunTag or the spaghetti monster imaging systems, has made it possible to visualize translation dynamics at the single mRNA level in real time. By applying these tagging systems, many mechanisms of translation have been uncovered. However, the lack of comparable and orthogonal tagging systems has made applications that require multicolor imaging challenging. To address this challenge, we developed a novel genetically encodable tagging system. For this, we grafted complementary determining regions or loops identified through deep mining of the Protein Data Bank to a stable single-chain variable fragment (scFv) scaffold that folds correctly in the reduced intracellular environment. After the loop grafting, the binding of the wildtype scFv was rescued in living cells. Our FRAP data suggests the recovery halftime of this tagging system is ∼5 min, significantly longer than our recently developed anti-HA and anti-FLAG frankenbodies, even though the epitope tag is about the same length. We believe these favorable properties will make this novel tagging system a useful new tool in the expanding live-cell imaging toolbox.

The functions of IRE1α in neurodegenerative diseases: Beyond ER stress.

Inositol-requiring enzyme 1 α (IRE1α) is a type I transmembrane protein that resides in the endoplasmic reticulum (ER). IRE1α, which is the primary sensor of ER stress, has been proven to maintain intracellular protein homeostasis by activating X-box binding protein 1 (XBP1). Further studies have revealed novel physiological functions of the IRE1α, such as its roles in mRNA and protein degradation, inflammation, immunity, cell proliferation and cell death. Therefore, the function of IRE1α is not limited to its role in ER stress; IRE1α is also important for regulating other processes related to cellular physiology. Furthermore, IRE1α plays a key role in neurodegenerative diseases that are caused by the phosphorylation of Tau protein, the accumulation of α-synuclein (α-syn) and the toxic effects of mutant Huntingtin (mHtt). Therefore, targeting IRE1α is a valuable approach for treating neurodegenerative diseases and regulating cell functions. This review discusses the role of IRE1α in different cellular processes, and emphasizes the importance of IRE1α in neurodegenerative diseases.

Exosomal DNAJB11 promotes the development of pancreatic cancer by modulating the EGFR/MAPK pathway

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with invasive and metastatic characteristics and poor prognosis. Intracellular protein homeostasis is associated with invasion and metastasis of pancreatic cancer, but the specific molecular mechanism remains unclear. Our previous studies have revealed that DNAJB11, a key protein in protein homeostasis, is secreted by exosomes in the supernatant of dissociated pancreatic cancer cells with high metastasis. The results from transcriptome sequencing and co-immunoprecipitation (Co-IP)-based liquid chromatography with tandem mass spectrometry (LC–MS/MS) showed that depletion of DNAJB11 levels could increase HSPA5 expression and induce endoplasmic reticulum stress through the PRKR-like endoplasmic reticulum kinase signaling pathway in pancreatic cancer cells. Furthermore, exosomal DNAJB11 promoted cell development of PC cells in vitro and in vivo. In addition, exosomal DNAJB11 could regulate the expression of EGFR and activate the downstream MAPK signaling pathway. Clinical blood samples were collected to evaluate the potential of exosome DNAJB11 as a diagnostic biomarker and therapeutic target for the treatment of pancreatic cancer. This study could provide a new theoretical basis and potential molecular targets for the treatment of pancreatic cancer.

Maize HEAT UP-REGULATED GENE 1 plays vital roles in heat stress tolerance.

Increasing temperature is one of the major threats to maize growth and yield globally. Under heat stress conditions, intracellular protein homeostasis is seriously disturbed, leading to accumulation of abnormally folded proteins, especially in the endoplasmic reticulum (ER). Molecular chaperones are vital players in the renaturation process and in preventing protein aggregation. However, heat stress tolerance-associated chaperones are not well documented in maize. Here, we characterized the biological roles of HEAT UP-REGULATED GENE 1 (ZmHUG1) in maize. ZmHUG1 encodes a heat-inducible holdase-type molecular chaperone localized in the ER. Knockout mutant of ZmHUG1 exhibited remarkably enhanced sensitivity to heat stress. Accordingly, the zmhug1 mutant showed severe ER stress under high temperature. MAIZE PRENYLATED RAB ACCEPTOR 1.C1 (ZmPRA1.C1) was identified as a client of ZmHUG1, and heat-induced aggregation of ZmPRA1.C1 was accelerated in the zmhug1 mutant. Furthermore, the expression of ZmHUG1 was rapidly transactivated by ER stress sensor BASIC LEUCINE ZIPPER DOMAIN 60 (bZIP60) when heat stress occurred. This study reveals a ZmHUG1-based thermo-protective mechanism in maize.

Sorting nexins: role in the regulation of blood pressure.

Sorting nexins (SNXs) are a family of proteins that regulate cellular cargo sorting and trafficking, maintain intracellular protein homeostasis, and participate in intracellular signaling. SNXs are also important in the regulation of blood pressure via several mechanisms. Aberrant expression and dysfunction of SNXs participate in the dysregulation of blood pressure. Genetic studies show a correlation between SNX gene variants and the response to antihypertensive drugs. In this review, we summarize the progress in SNX-mediated regulation of blood pressure, discuss the potential role of SNXs in the pathophysiology and treatment of hypertension, and propose novel strategies for the medical therapy of hypertension.

Stankiewicz-Isidor syndrome: expanding the clinical and molecular phenotype

PurposeHaploinsufficiency of PSMD12 has been reported in individuals with neurodevelopmental phenotypes, including developmental delay/intellectual disability (DD/ID), facial dysmorphism, and congenital malformations, defined as Stankiewicz-Isidor syndrome (STISS). Investigations showed that pathogenic variants in PSMD12 perturb intracellular protein homeostasis. Our objective was to further explore the clinical and molecular phenotypic spectrum of STISS. MethodsWe report 24 additional unrelated patients with STISS with various truncating single nucleotide variants or copy-number variant deletions involving PSMD12. We explore disease etiology by assessing patient cells and CRISPR/Cas9-engineered cell clones for various cellular pathways and inflammatory status. ResultsThe expressivity of most clinical features in STISS is highly variable. In addition to previously reported DD/ID, speech delay, cardiac and renal anomalies, we also confirmed preaxial hand abnormalities as a feature of this syndrome. Of note, 2 patients also showed chilblains resembling signs observed in interferonopathy. Remarkably, our data show that STISS patient cells exhibit a profound remodeling of the mTORC1 and mitophagy pathways with an induction of type I interferon-stimulated genes. ConclusionWe refine the phenotype of STISS and show that it can be clinically recognizable and biochemically diagnosed by a type I interferon gene signature.

Molecular cloning, characterization and expression patterns of HSP70 and HSP60 in the pufferfish (Takifugu rubripes)

Takifugu rubripes is a commercially important fish in the Chinese aquaculture industry due to its excellent meat quality. In eukaryotic cells, heat shock proteins (HSPs) play a crucial role in maintaining intracellular protein homeostasis. They play significant roles in response to adverse environmental stresses in aquatic animals. In this study, we used bioinformatics to comprehensively compare two different full-length cDNA sequences at the molecular level in the HSP family of T. rubripes. We performed various physicochemical studies such as race cloning and RT-qPCR, and found that TrHSP60 (575 amino acids) has a larger polypeptide, followed by TrHSP70–13 (441 amino acids). Homology and phylogenetic studies showed that these sequences have the greatest homology to Takifugu bimaculatus. The mRNA of the two genes were constitutively expressed in all tested tissues of untreated T. rubripes, including brain, muscle, kidney, liver, spleen, gill, and intestine, with highest expression levels in the intestine. Three experimental groups were established in this study, the H1 group (20 °C), H2 group (24 °C) and H3 group (28 °C), in order to analyze the expressed levels of TrHSP70 in T. rubripes. To understand the role of HSPs in temperature stress in T. rubripes, we analyzed the expression levels of HSPs in T. rubripes at different temperatures. The results showed that the mRNA expression of the HSP gene were influenced by temperature stress. Gene expression of TrHSP70 and TrHSP60 were mainly up-regulated at 12 h and 48 h in the H1 group, respectively. Meanwhile, gene expression of TrHSP70 was highest at 48 h in the H3 group and TrHSP60 reached was lowest at 24 h in the H3 group. The results showed that the two genes were constitutively expressed genes, which play an important role in maintaining normal physiological function and stress responses. The results show that TrHSP70 and TrHSP60 are constitutively expressed genes, which plays important role in maintaining normal physiological function and coping with stress.

Cisd2 Preserves the Youthful Pattern of the Liver Proteome during Natural Aging of Mice.

Cisd2 (CDGSH iron sulfur domain 2) is a pro-longevity gene that extends the lifespan and health span of mice, ameliorates age-associated structural damage and limits functional decline in multiple tissues. Non-alcoholic fatty liver disease (NAFLD), which plays an important role in age-related liver disorders, is the most common liver disease worldwide. However, no medicines that can be used to specifically and effectively treat NAFLD are currently approved for this disease. Our aim was to provide pathological and molecular evidence to show that Cisd2 protects the liver from age-related dysregulation of lipid metabolism and protein homeostasis. This study makes four major discoveries. Firstly, a persistently high level of Cisd2 protects the liver from age-related fat accumulation. Secondly, proteomics analysis revealed that Cisd2 ameliorates age-related dysregulation of lipid metabolism, including lipid biosynthesis and β-oxidation, in mitochondria and peroxisomes. Thirdly, Cisd2 attenuates aging-associated oxidative modifications of proteins. Finally, Cisd2 regulates intracellular protein homeostasis by maintaining the functionality of molecular chaperones and protein synthesis machinery. Our proteomics findings highlight Cisd2 as a novel molecular target for the development of therapies targeting fatty liver diseases, and these new therapies are likely to help prevent subsequent malignant progression to cirrhosis and hepatocellular carcinoma.

Structural and functional dissection of reovirus capsid folding and assembly by the prefoldin-TRiC/CCT chaperone network

Intracellular protein homeostasis is maintained by a network of chaperones that function to fold proteins into their native conformation. The eukaryotic TRiC chaperonin (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) facilitates folding of a subset of proteins with folding constraints such as complex topologies. To better understand the mechanism of TRiC folding, we investigated the biogenesis of an obligate TRiC substrate, the reovirus σ3 capsid protein. We discovered that the σ3 protein interacts with a network of chaperones, including TRiC and prefoldin. Using a combination of cryoelectron microscopy, cross-linking mass spectrometry, and biochemical approaches, we establish functions for TRiC and prefoldin in folding σ3 and promoting its assembly into higher-order oligomers. These studies illuminate the molecular dynamics of σ3 folding and establish a biological function for TRiC in virus assembly. In addition, our findings provide structural and functional insight into the mechanism by which TRiC and prefoldin participate in the assembly of protein complexes.

Single nucleotide polymorphisms (SNPs) in endoplasmic reticulum (ER) stress response genes to predict first-line treatment outcome in patients (pts) with metastatic colorectal cancer (mCRC): Data from the MAVERICC and FIRE-3 trials.

103 Background: ER stress, triggered by the disruption of intracellular protein homeostasis, promotes the activation of a highly conserved adaptive program called unfolded protein response (UPR). UPR signaling has been reported to contribute to tumor initiation and progression, as well as microenvironment remodeling and chemoresistance in multiple cancer types, including CRC. Several studies point to a direct role of the UPR in tumor angiogenesis and an interaction with EGFR signaling. We therefore hypothesized that genetic variants in ER stress response genes may predict first-line treatment outcome in mCRC pts. Methods: The impact on outcome of 17 functional SNPs in 8 core genes of the ER stress response pathway ( IRE1, PERK, ATF6, XBP1, CHOP, GRP78, GADD34, ATF4) was analyzed on a total of 560 pts enrolled in two independent randomized first-line trials: MAVERICC (FOLFIRI/bevacizumab, [bev] n = 163; FOLFOX6/bev, n = 161), and FIRE-3 (FOLFIRI/bev, n = 107; FOLFIRI/cetuximab [cet], n = 129). Genomic DNA from blood samples of pts was genotyped through the OncoArray, a custom array manufactured by Illumina. The association between SNPs and clinical outcomes was evaluated using Cox regression and log-rank tests. The SNP*treatment interaction was assessed in both trials. Results: IRE1 rs16947383 any T allele variant was associated with shorter progression free survival (PFS) and overall survival (OS) compared to the C/C genotype in pts treated with FOLFIRI/bev in the MAVERICC trial both in uni- and multivariate analysis: 10.5 vs 14.1 months for PFS, adjusted hazard ratio (HR) 1.83, 95% confidence interval (CI) 1.12-2.99, adjusted P-value = 0.02; 23.8 vs 38.4 months for OS, adjusted HR 2.12, 95%CI 1.1-4.09, adjusted P-value = 0.02. The association with OS was validated in the FIRE-3 FOLFIRI/bev arm where any T allele carriers showed significantly shorter OS compared to the C/C genotype in both uni- and multivariate analysis: 21.1 vs 44.2 months, adjusted HR 2.06, 95%CI 1.01-4.18, adjusted P-value = 0.05. This effect was not observed in MAVERICC FOLFOX6 and FIRE-3 cet cohorts. A significant interaction was found between IRE1 rs16947383 and treatment on OS in both trials: P = 0.04 in MAVERICC (FOLFIRI vs FOLFOX6), and P = 0.03 in FIRE-3 (bev vs cet). In the MAVERICC FOLFOX6/bev arm, XBP1 rs2239815 and PERK rs6731022 SNPs were associated with shorter PFS and OS, while GADD34 rs610308 with longer OS, in multivariable analysis ( P < 0.05). Conclusions: Our results provide evidence that germline polymorphisms in ER stress response genes may have a predictive value in mCRC pts receiving first-line bev-based treatment and contribute to modulate anti-VEGF efficacy.

Accurate, automatic annotation of peptidases with hotpep-protease

Peptidases are essential for intracellular protein processing, signaling and homeostasis, physiological processes and for digestion of food. Moreover, peptidases are important biotechnological enzymes used in processes such as industrial food processing, leather manufacturing and the washing industry. Identification of peptidases is a crucial step in their characterization but peptidase annotation is not a trivial task due to their large sequence diversity.In the present study short, conserved sequence profiles were generated for all peptidase families with more than four members in the comprehensive Merops peptidase database. The sequence profiles were combined with the Homology to Peptide Pattern (Hotpep) method for automatic annotation of peptidases. This method is a stand-alone software that annotates protease sequences to Merops family and subgroup and is suitable for large-scale sequence analysis. Compared to the Mammalian Degradome Database Hotpep-protease had an accuracy of 92% and a sensitivity of 96% for annotation of the human degradome. Annotation by commonly used methods (Blast and conserved domains) had an accuracy of 69% and a sensitivity of 78%. For fungal genomes, there were large differences between annotation with Hotpep-protease, Blast- and Hidden Markov Model-based annotation and the Merops annotation, which confirms the difficulty of large-scale peptidase annotation. Manual annotation indicated that Hotpep-protease had a positive prediction rate of 0.90 compared to a positive prediction rate of 0.67 for Blast search. Hence, Hotpep-protease is highly accurate method for fast and accurate annotation of peptidases.