Mutations In Subunits Research Articles

Loss of the E3 ubiquitin ligases UBR-5 or HECD-1 restores Caenorhabditis elegans development in the absence of SWI/SNF function

SWItch/sucrose non-fermenting (SWI/SNF) complexes are a family of chromatin remodelers that are conserved across eukaryotes. Mutations in subunits of SWI/SNF cause a multitude of different developmental disorders in humans, most of which have no current treatment options. Here, we identify an alanine-to-valine-causing mutation in the SWI/SNF subunit snfc-5 (SMARCB1 in humans) that prevents embryonic lethality in Caenorhabditis elegans nematodes harboring a loss-of-function mutation in the SWI/SNF subunit swsn-1 (SMARCC1/2 in humans). Furthermore, we found that the combination of this specific mutation in snfc-5 and a loss-of-function mutation in either of the E3 ubiquitin ligases ubr-5 (UBR5 in humans) or hecd-1 (HECTD1 in humans) can restore development to adulthood in swsn-1 loss-of-function mutants that otherwise die as embryos. Using these mutant models, we established a set of 335 genes that are dysregulated in SWI/SNF mutants that arrest their development embryonically but exhibit near wild-type levels of expression in the presence of suppressor mutations that prevent embryonic lethality, suggesting that SWI/SNF promotes development by regulating some subset of these 335 genes. In addition, we show that SWI/SNF protein levels are reduced in swsn-1; snfc-5 double mutants and partly restored to wild-type levels in swsn-1; snfc-5; ubr-5 triple mutants, consistent with a model in which UBR-5 regulates SWI/SNF levels by tagging the complex for proteasomal degradation. Our findings establish a link between two E3 ubiquitin ligases and SWI/SNF function and suggest that UBR5 and HECTD1 could be potential therapeutic targets for the many developmental disorders caused by missense mutations in SWI/SNF subunits.

The emerging pandemic recent: SARS-CoV-2

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), induces coughing, vomiting, or fever. The occurrence, duration, and severity of these symptoms vary from person to person, which makes symptoms of the COVID-19 infection ranging from those of a mild cold to death in severe cases. The cause of these differences can be found in individual characteristics of the patient. Age, former diseases of the patient, and they medications they took can such characteristics. As the SARS-CoV-2 affects the lung, diseases related to the lung such as chronic obstructive pulmonary disease, lung cancer, cystic fibrosis, pulmonary fibrosis, moderate to severe asthma, pulmonary hypertension, and pulmonary embolism are especially more dangerous to the human body when infected by the SARS-CoV-2. In this paper, through various methods including comparing the characteristics of mutant viruses and subunits of COVID-19, we aimed to describe the risk factors and deterioration of COVID-19 and the strain of SARS-CoV-2 during the COVID-19 pandemic.

Structural and Dynamic Disturbances Revealed by Molecular Dynamics Simulations Predict the Impact on Function of CCT5 Chaperonin Mutations Associated with Rare Severe Distal Neuropathies

Mutations in genes encoding molecular chaperones, for instance the genes encoding the subunits of the chaperonin CCT (chaperonin containing TCP-1, also known as TRiC), are associated with rare neurodegenerative disorders. Using a classical molecular dynamics approach, we investigated the occurrence of conformational changes and differences in physicochemical properties of the CCT5 mutations His147Arg and Leu224Val associated with a sensory and a motor distal neuropathy, respectively. The apical domain of both variants was substantially but differently affected by the mutations, although these were in other domains. The distribution of hydrogen bonds and electrostatic potentials on the surface of the mutant subunits differed from the wild-type molecule. Structural and dynamic analyses, together with our previous experimental data, suggest that genetic mutations may cause different changes in the protein-binding capacity of CCT5 variants, presumably within both hetero- and/or homo-oligomeric complexes. Further investigations are necessary to elucidate the molecular pathogenic pathways of the two variants that produce the two distinct phenotypes. The data and clinical observations by us and others indicate that CCT chaperonopathies are more frequent than currently believed and should be investigated in patients with neuropathies.

Mutations in Complex I of the Mitochondrial Electron-Transport Chain Sensitize the Fruit Fly (Drosophila melanogaster) to Ether and Non-Ether Volatile Anesthetics.

The mitochondrial electron transport chain (mETC) contains molecular targets of volatile general anesthetics (VGAs), which places carriers of mutations at risk for anesthetic complications. The ND-2360114 and mt:ND2del1 lines of fruit flies (Drosophila melanogaster) that carry mutations in core subunits of Complex I of the mETC replicate numerous characteristics of Leigh syndrome (LS) caused by orthologous mutations in mammals and serve as models of LS. ND-2360114 flies are behaviorally hypersensitive to volatile anesthetic ethers and develop an age- and oxygen-dependent anesthetic-induced neurotoxicity (AiN) phenotype after exposure to isoflurane but not to the related anesthetic sevoflurane. The goal of this paper was to investigate whether the alkane volatile anesthetic halothane and other mutations in Complex I and in Complexes II-V of the mETC cause AiN. We found that (i) ND-2360114 and mt:ND2del1 were susceptible to toxicity from halothane; (ii) in wild-type flies, halothane was toxic under anoxic conditions; (iii) alleles of accessory subunits of Complex I predisposed to AiN; and (iv) mutations in Complexes II-V did not result in an AiN phenotype. We conclude that AiN is neither limited to ether anesthetics nor exclusive to mutations in core subunits of Complex I.

Gut microbiome dysbiosis drives metabolic dysfunction in Familial dysautonomia

Familial dysautonomia (FD) is a rare genetic neurologic disorder caused by impaired neuronal development and progressive degeneration of both the peripheral and central nervous systems. FD is monogenic, with >99.4% of patients sharing an identical point mutation in the elongator acetyltransferase complex subunit 1 (ELP1) gene, providing a relatively simple genetic background in which to identify modifiable factors that influence pathology. Gastrointestinal symptoms and metabolic deficits are common among FD patients, which supports the hypothesis that the gut microbiome and metabolome are altered and dysfunctional compared to healthy individuals. Here we show significant differences in gut microbiome composition (16 S rRNA gene sequencing of stool samples) and NMR-based stool and serum metabolomes between a cohort of FD patients (~14% of patients worldwide) and their cohabitating, healthy relatives. We show that key observations in human subjects are recapitulated in a neuron-specific Elp1-deficient mouse model, and that cohousing mutant and littermate control mice ameliorates gut microbiome dysbiosis, improves deficits in gut transit, and reduces disease severity. Our results provide evidence that neurologic deficits in FD alter the structure and function of the gut microbiome, which shifts overall host metabolism to perpetuate further neurodegeneration.

PL-4: INSIGHTS INTO MOLECULAR MECHANISMS OF HYPERTENSION FROM HUMAN GENETICS

Identification and investigation of rare patients with extreme forms of high and low blood pressure has led to the identification of specific genes whose mutation results in these outlier phenotypes. These rare mutations converge on genes that mediate or regulate the renal reabsorption of Na-Cl. Mutations that increase or decrease renal salt reabsorption respectively increase or decrease blood pressure. Mutations that cause hypertension include gain of function mutations in subunits of the epithelial Na+ channel (ENaC) and in any of eight genes that cause constitutive activity of ENaC by its stimulation via its major regulator, the mineralocorticoid receptor, the target of aldosterone signaling; these increase salt reabsorption in the distal nephron. Conversely, mutations that cause hypotension include loss of function mutations in ENaC subunits, but also mutations the thiazide-sensitive Na-Cl cotransporter (NCC) or a K+ channel required for cotransporter function that act in the distal convoluted tubule (DCT), or mutations in the Na-K-2Cl cotransporter as well as K+ and Cl- channels expressed in the thick ascending limb of Henle that are required for normal Na-Cl reabsorption in this nephron segment. ENaC, the Na-Cl cotransporter and the Na-K-2Cl cotransporter, as well as the mineralocorticoid receptor are all commonly used targets for treatment of hypertension and/or diuresis. In addition to these genes, genetic studies also identified a previously unrecognized pathway that regulates the balance between renal salt reabsorption and K+ secretion. Mutations in the kinases WNK1 or WNK4, or genes that mediate their degradation via ubiquitylation, CUL3 and KLHL3 cause hypertension with hyperkalemia. Activity of WNK1 and WNK4 stimulates activation of NCC promoting Na-Cl reabsorption in the DCT, reducing Na-Cl- delivery distally, thereby limiting ENaC activity and limiting K+ secretion in the distal nephron. Conversely, hyperkalemia inhibits WNK activity, promoting distal Na-Cl delivery and maximizing K+ secretion. Angiotensin II signaling promotes WNK activity, increasing Na-Cl reabsorption in the DCT and inhibiting K+ secretion, while hyperkalemia appears to inhibit WNK activity, providing distal Na+ delivery that is used to drive K+ secretion. The importance of this pathway is that it can explain the mechanism by which increased dietary K+ lowers blood pressure. Both reduced dietary salt intake and increased dietary K+ can reduce blood pressure in controlled settings, and recent large population studies have shown that increased dietary K+ and reduced Na-Cl intake not only lower blood pressure but reduce the incidence of cardiovascular disease and death.

The SWI/SNF Complex: A Frequently Mutated Chromatin Remodeling Complex in Cancer.

The switch/sucrose non-fermenting (SWI/SNF) chromatin remodeling complex is a global regulator of gene expression known to maintain nucleosome-depleted regions at active enhancers and promoters. The mammalian SWI/SNF protein subunits are encoded by 29 genes and 11-15 subunits including an ATPase domain of either SMARCA4 (BRG1) or SMARCA2 (BRM) are assembled into a complex. Based on the distinct subunits, SWI/SNF are grouped into 3 major types (subfamilies): the canonical BRG1/BRM-associated factor (BAF/cBAF), polybromo-associated BAF (PBAF), and non-canonical BAF (GBAF/ncBAF). Pan-cancer genome sequencing studies have shown that nearly 25% of all cancers bear mutations in subunits of the SWI/SNF complex, many of which are loss of function (LOF) mutations, suggesting a tumor suppressor role. Inactivation of SWI/SNF complex subunits causes widespread epigenetic dysfunction, including increased dependence on antagonistic components such as polycomb repressor complexes (PRC1/2) and altered enhancer regulation, likely promoting an oncogenic state leading to cancer. Despite the prevalence of mutations, most SWI/SNF-mutant cancers lack targeted therapeutic strategies. Defining the dependencies created by LOF mutations in SWI/SNF subunits will identify better targets for these cancers.

Abstract 1582: Proteostasis Regulators Increases the Surface Trafficking of Pathogenic Mutants in the GluN1 subunit of the NMDA Receptor

Abstract 1582: Proteostasis Regulators Increases the Surface Trafficking of Pathogenic Mutants in the GluN1 subunit of the NMDA Receptor

Escobar Syndrome in Three Patients of Two Unrelated Families

“Escobar syndrome” or “multiple pterygium syndrome” is a very rare disorder. It is characterized by congenital contractures (arthrogryposis), excessive webbing (pterygia) and scoliosis. The diagnosis is generally confirmed after birth, in the neonatal period. We describe three individuals, from two consanguineous Algerian families, affected by this syndrome. The genetic defect is a mutation of the foetal subunit of Acetyl Choline receptor.

Prevalence and characterization of quinolone-resistant Escherichia coli isolated from retail raw beef and poultry meat in Egypt.

The goal of this study was to look at quinolone-resistant (QR) Escherichia coli (E. coli) from retail beef and poultry meat in Egypt by looking at the QR mechanisms in the resistant strains. In total, 120 samples of raw poultry meat (n = 60) and beef meat (n = 60) were purchased from Mansoura retail stores between January and March 2021, and evaluated microbiologically for E. coli. Then, an antimicrobial sensitivity test was applied to all isolates. The prevalence of QR E. coli with concern for the QR determinants, including quinolone resistance-determining regions (QRDRs) mutations, the plasmid-mediated quinolone resistance gene (PMQR), and the efflux pump activity were determined. The total prevalence of E. coli was 34.2% (41/120). Noticeably, the prevalence of E. coli in poultry meat (40%, 24/60) was higher than that of beef (28%, 17/60). All strains were assessed for their antimicrobial susceptibility using the disc diffusion technique; the highest rate of resistance (100%) was displayed to clindamycin and cefuroxime, followed by ampicillin (97.6%), doxycycline (92.7%), amoxicillin-clavulanate (92.7%), nalidixic acid (NA) (80.5%), sulfamethoxazole/trimethoprim (70.7%), chloramphenicol (63.4%), gentamicin, and azithromycin (58.5% each). Multiple antimicrobial resistance (strains resistant to three or more antimicrobial classes) was displayed by 97.6% of E. coli isolates. Regarding QR, 37 isolates could resist at least one of the examined quinolones. Regarding PMQR genes, qnrS was determined in 70% (7/10) of QR E. coli, while qnrA, qnrB, and qnrD were not identified. While the mutations determined regions of QR in the resistant E. coli isolates, S83L was the most prevalent in gyrase subunit A either alone or combined with D87N and D87Y, and three isolates of QR E. coli isolates revealed a topoisomerase IV subunit mutation harboring S80I. 20% of the isolates displayed efflux activity, as NA showed a considerable difference between its zones of inhibition. The high prevalence of antimicrobial-resistant E. coli, with concern for QR strains harboring different resistance mechanisms in poultry meat and beef, threatens the public's health. Thus, standard manufacturing procedures and adequate hygiene conditions must be followed in all phases of meat preparation, production, and consumption, and public knowledge should be improved.

B” and C subunits of PP2A regulate the levels of reactive oxygen species and superoxide dismutase activities in Arabidopsis

The serine-threonine protein phosphatases PP2A regulate many cellular processes, however their role in oxidative stress responses and defence is less known. We show the involvement of its C (catalytic) and B” (a regulatory) subunits. The c3c4 (C subunit) and fass (B”) subunit mutants and Col wt of Arabidopsis were used. Controls and treatments with the PP2A inhibitor microcystin-LR (MCY-LR) and reactive oxygen species (ROS) inducer diquat (DQ) were employed. ROS levels of primary roots were largely genotype dependent and both C and B” subunit mutants had increased sensitivity to MCY-LR and DQ indicating the involvement of these subunits in oxidative stress induction. Superoxide dismutases (SOD), mainly the Cu/Zn-SOD isoform, as key enzymes involved in ROS scavenging are also showing altered (mostly increased) activities in both c3c4 and fass mutants and have opposite relations to ROS induction. This indicates that the two types of subunits involved have partially different regulatory roles. In relation to this, control and MCY-LR/DQ treated B” subunit mutants were proven to have altered levels of phosphorylation of histone H2AX. γH2AX, the phosphorylated form indicates double stranded DNA damage during oxidative stress. Overall we point out the probable pivotal role of several PP2A subunits in the regulation of oxidative stress responses in plants and pave the way for future research to reveal the signaling pathways involved.

Analysis of compound heterozygous and homozygous mutations found in peripheral subunits of human respiratory Complex I, NDUFS1, NDUFS2, NDUFS8 and NDUFV1, by modeling in the E. coli enzyme

Respiratory Complex I (NADH:ubiquinone oxidoreductase) is composed of 45 subunits, seven mitochondrially-encoded and 38 imported. Mutations in the nuclearly-encoded subunits have been regularly discovered in humans in recent years, and many lead to cardiomyopathy, Leigh Syndrome, and early death. From the literature, we have identified mutations at 17 different sites and constructed 31 mutants in a bacterial model system. Many of these mutations, found in NDUFS1, NDUFS2, NDUFS8, and NDUFV1, map to subunit interfaces, and we hypothesized that they would disrupt assembly of Complex I. The mutations were constructed in the homologous E. coli genes, nuoG, nuoCD, nuoI and nuoF, respectively, and expressed from a plasmid containing all Complex I genes. Membrane vesicles were prepared and rates of deamino-NADH oxidase activity measured, which indicated a range of reduced activity. Some mutants were also analyzed using recently developed assays of assembly, time-delayed expression, and co-immunoprecipitation, which showed that assembly was disrupted. With compound heterozygotes, we determined which mutation was more deleterious. Construction of alanine mutations allowed us to distinguish between phenotypes that were caused by loss of the original amino acid or introduction of the mutant residue.

DGCR8 Microprocessor Subunit Mutation and Expression Deregulation in Thyroid Lesions.

DGCR8 emerged recently as miRNAs biogenesis pathway protein with a highlighted role in thyroid disease. This study aimed to characterize this miRNA biogenesis component, in particular the p.(E518K) mutation and DGCR8 expression in a series of thyroid lesions. The series of thyroid lesions was genotyped for the c.1552G>A p.(E518K) mutation. When frozen tissue was available, DGCR8 mRNA expression was analysed by qPCR. Formalin-fixed paraffin-embedded tissues were studied for DGCR8 immunoexpression. We present for the first time the p.(E518K) mutation in a case of poorly differentiated thyroid carcinoma and present the deregulation of DGCR8 expression at mRNA level in follicular-patterned tumours. The obtained data solidify DGCR8 as another important player of miRNA-related gene mutations in thyroid tumorigenesis, particularly in follicular-patterned thyroid tumours.

Analysis of Human Clinical Mutations of Mitochondrial ND1 in a Bacterial Model System for Complex I.

The most common causes of mitochondrial dysfunction and disease include mutations in subunits and assembly factors of Complex I. Numerous mutations in the mitochondrial gene ND1 have been identified in humans. Currently, a bacterial model system provides the only method for rapid construction and analysis of mutations in homologs of human ND1. In this report, we have identified nine mutations in human ND1 that are reported to be pathogenic and are located at subunit interfaces. Our hypothesis was that these mutations would disrupt Complex I assembly. Seventeen mutations were constructed in the homologous nuoH gene in an E. coli model system. In addition to the clinical mutations, alanine substitutions were constructed in order to distinguish between a deleterious effect from the introduction of the mutant residue and the loss of the original residue. The mutations were moved to an expression vector containing all thirteen genes of the E. coli nuo operon coding for Complex I. Membrane vesicles were prepared and rates of deamino-NADH oxidase activity and proton translocation were measured. Samples were also tested for assembly by native gel electrophoresis and for expression of NuoH by immunoblotting. A range of outcomes was observed: Mutations at four of the sites allow normal assembly with moderate activity (50−76% of wild type). Mutations at the other sites disrupt assembly and/or activity, and in some cases the outcomes depend upon the amino acid introduced. In general, the outcomes are consistent with the proposed pathogenicity in humans.

Economic Disparities in the Outcomes of Sickle Cell Disease-Related Admissions over a Decade

Background: According to the CDC, Sickle Cell Disease (SCD) occurs in 1 out of every 365 African American births and 1 out of every 16,300 Hispanic American births. It is an inherited disorder caused secondary to a mutation in the hemoglobin beta subunit and has complex phenotypes. The disease is characterized by recurrent hemolysis, vaso-occlusion leading to pain episodes, and chronic organ damage due to ischemia. A comprehensive care management team is not well established for this disease. There is sparse information regarding the trends in outcomes of SCD admissions in the past decade where rapid advances have been made in treatment including the evolving hematopoietic stem cell transplant. In this study, we sought to analyze the trends and outcomes of SCD admissions in the United States from 2011- 2019 and the influence of socioeconomic status. Methods: The National Inpatient Sample (NIS) database was queried to identify adult patients admitted with a principal diagnosis of Sickle Cell Disease using the International Classification of Disease (ICD-9) and ICD-10 codes. Trends for primary in-hospital outcomes including mortality, length of stay (LOS), and total hospitalization charges (THC) were assessed. The impact of economic status on these outcomes was also studied. Statistics were performed using STATA and multivariate linear and logistic regression analysis was used to adjust for confounders. Results: The rate of hospitalizations for SCD remained stable from 2010 to 2019. Women constituted 53.99% and the majority of patients were young adults (86.62%) and Black (90.36%) as expected. Many patients belonged to the low-income quartile group and were admitted to hospitals with a large bed size and urban teaching hospitals. The overall mortality rate among adult SCD hospitalizations from 2010 to 2019 decreased by an odds ratio of 0.96 (p=0.008) after adjusting for age, sex, and race and is reassuring. However, on subgroup analysis, this trend was replicated for those belonging to a higher income quartile group whereas there was no statistically significant decrease in the mortality rate trend for those belonging to the lower income quartile group. After adjusting for confounders there was an increase in the total charges in all subgroups (p<0.001). The mean LOS decreased for the total population and most subgroups (p<0.001). Conclusion: Despite the advances in the understanding of SCD and its treatment, its benefits have not reached all the people affected. Access to healthcare and the treatment costs associated could be one among the possible reasons why the lower income quartile group have not yet benefited. Meaningful progress in healthcare is not achievable unless these economic disparities are addressed. It is imperative to develop economic policies to address these inequities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Proteasome Regulatory Atpases PSMC2 and PSMC6 Modulate Proteosome Inhibitor Sensitivity and Correlate with Survival in Multiple Myeloma

Background: Multiple myeloma (MM) cells synthesize and secrete massive amounts of immunoglobulins and thus are exquisitely sensitive to therapeutics that disrupt proteostasis. The 26S proteasome complex is a 2.5-MDa holoenzyme comprised of >32 individual subunits responsible for the bulk of intracellular protein degradation. 26S proteasomes are comprised of barrel-shaped proteolytic core complex, the 20S core particle (CP), capped at either or both ends by 19S regulatory particles (RPs), which confer ATPase activity and specificity for ubiquitinated proteins. Six distinct ATPases (PSMC1-6) form a heterohexameric ring-like structure that rests atop the 20S CP's α-subunits and is responsible for unfolding and shuttling target proteins for degradation within the 20S CP. The proteasome inhibitor (PI) bortezomib (BTZ) is a potent, selective, and reversible drug that dramatically improved the prognosis of multiple myeloma (MM) patients and remains the backbone of induction therapy for transplant-eligible new diagnosis MM (NDMM), but response to therapy remains highly variable. Here, we predicted that gene expression analysis of publically available datasets could identify components within the ubiquitin-proteasome system (UPS) that areprognostic in patients treated with BTZ, suggesting potential new targets in MM. Methods: We interrogated gene expression profiles (GEP) from the randomized, phase 3 Assessment of Proteasome Inhibition for Extending Remissions (APEX) study that compared BTZ with high-dose dexamethasone (DEX) in RRMM patients who had received 1-3 prior therapies. APEX datasets served as a test cohort to identify genes that influenced survival in patients treated with BTZ. Patients consented to genomic analyses of pretreatment tumor samples. CD138+ cells were isolated from BM aspirates and analyzed by RNA microarrays to generate GEP datasets. We correlated gene expression with response to BTZ or DEX and overall survival (OS). We then validated individual genes identified from the APEX analysis using the MM Research Foundation (MMRF) CoMMpass study, a longitudinal observational analysis of the clinical and molecular data for >1000 NDMM patients receiving various standard approved treatments. Finally, we used shRNA to knockdown PSMC2 in RPMI8226 and ARH77 cells and tested their sensitivity to BTZ and carfilzomib (CFZ). Results: Analysis of the APEX dataset revealed that increased expression of genes PSMC2 and PSMC6, members of the heterohexameric ATPase ring of the 19S RP, correlated with poorer OS (PSMC2- Figure 1A, PSMC6- Median OS 320 versus 620 days for upper and lower tertile PSMC6, respectively). Using the CoMMpass database, increased PSMC2 expression also correlated with a worse OS (Figure 1B), while increased PSMC6 correlated with worse OS as well as progression free survival (PFS) (OS- HR= 2.29 p=0.0014, PFS- HR=1.63 p=0.0109). PSMC6 displayed greater expression in 50 patients above the median RNA expression (Fragments per Kb transcript per million mapped reads (FPKM)= 1 to 100) while PSMC2 expression was elevated in 400 patients above the median (FPKM= 25 to 100). shRNA-mediated PSMC2 knockdown (KD) increased PI susceptibility in ARH77 and the RPMI8226 MM cells. In ARH77 cells, the BTZ LD50 decreased from 11 nM to 2.5 nM and the CFZ LD50 decreased from 11 to 9 nM in control and KD cells, respectively. In RPMI8226 cells, the BTZ LD50 decreased from 30 nM to 20 nM and the CFZ LD50 decreased from 28 to 17 nM in control and KD cells, respectively. Conclusion: Taken together, our results indicate that increased PSMC2 and PSMC6 correlates with OS in MM in both patients treated with BTZ in the APEX and with a variety of standard therapies in the CoMMpass database. Analysis in this validation cohort extends the clinical significance of PSMC2/PSMC6 beyond the landmark APEX trial and suggests they may inform survival in patients treated with modern triplet anti-myeloma therapies. In addition to the prognostic importance across our test and validation datasets, point mutations in each ATPase subunit have been associated with PI resistance. Consistent with these findings, PSMC2 KD increased sensitivity to PI therapy. Controlled inhibition or degradation of proteasome 19S ATPase subunits using small molecules, PROTACs or RNA-based modalities now provides an opportunity to therapeutically target proteostasis in MM and other malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EPCO-43. CHROMATIN REMODELERS LOST IN TRANSLATION

Abstract The chromatin remodeler complexes mammalian SWI/SNF (SWItch/Sucrose Non-Fermentable) are altered in more than 40% of cancers and are involved in therapeutic resistance. In children,SMARCB1 is mutated in 95% of rhabdoid tumors, and the remaining 5% present mutations in SMARCA4. These tumors are typically identified in children younger than three years old and have a very poor prognosis with a survival under one year following diagnosis. SMARCB1 expression is also lost in more than 90% of epithelial sarcomas, a rare and aggressive tumor affecting young adults, and in 50% of pediatric chordomas. Additional mSWI/SNF subunits are also highly mutated in childhood cancers such as ARID1A and ARID1B in neuroblastomas, the most common solid tumor in children, and the ARID subunits and SMARCA4 in medulloblastomas, the most common brain cancer in children. To date, mSWI/SNF roles in normal and cancer cells are still largely undetermined and focus on nuclear functions. However, our assessment of genetic dependencies and drug sensitivity screens revealed that cancer cells mutated for mSWI/SNF are genetically dependent and exclusively sensitive to translation factors and translation pathway inhibitors. We demonstrate that mSWI/SNF complexes are extensively localized in the cytoplasm and that they interact with the translation initiation machinery. Furthermore, short-term inhibition, depletion and cancer mutations of specific subunits alter protein synthesis and increase sensitivity to translation inhibitors. Most mutations in these complexes being considered loss of function, these results suggest a potential therapeutic opportunity for diseases presenting mutations in these complexes. In conclusion, our work demonstrates the chromatin remodelers mSWI/SNF have direct roles in translation that will not only be relevant for the molecular understanding of cancer but also lead to new therapeutic opportunities.

Autism associated mutations in β2 subunit of voltage-gated calcium channels constitutively activate gene expression

Membrane depolarization triggers gene expression through voltage-gated calcium channels (VGCC) in a process called Excitation-transcription (ET) coupling. Mutations in the channel subunits α11.2, or β2d, are associated with neurodevelopmental disorders such as ASD. Here, we found that two mutations S143F and G113S within the rat Cavβ2a corresponding to autistic related mutations Cavβ2dS197F and Cavβ2dG167S in the human Cavβ2d, activate ET-coupling via the RAS/ERK/CREB pathway. Membrane depolarization of HEK293 cells co-expressing α11.2 and α2δ with Cavβ2aS143F or Cavβ2aG113S triggers constitutive transcriptional activation, which is correlated with facilitated channel activity. Similar to the Timothy-associated autistic mutation α11.2G406R, constitutive gene activation is attributed to a hyperpolarizing shift in the activation kinetics of Cav1.2. Pulldown of RasGRF2 and RhoGEF by wt and the Cavβ2a autistic mutants is consistent with Cavβ2/Ras activation in ET coupling and implicates Rho signaling as yet another molecular pathway activated by Cavα11.2/Cavβ2 . Facilitated spontaneous channel activity preceding enhanced gene activation via the Ras/ERK/CREB pathway, appears a general molecular mechanism for Ca2+ channel mediated ASD and other neurodevelopmental disorders.

A variant-proof SARS-CoV-2 vaccine targeting HR1 domain in S2 subunit of spike protein

The emerging SARS-CoV-2 variants, commonly with many mutations in S1 subunit of spike (S) protein are weakening the efficacy of the current vaccines and antibody therapeutics. This calls for the variant-proof SARS-CoV-2 vaccines targeting the more conserved regions in S protein. Here, we designed a recombinant subunit vaccine, HR121, targeting the conserved HR1 domain in S2 subunit of S protein. HR121 consisting of HR1–linker1–HR2–linker2–HR1, is conformationally and functionally analogous to the HR1 domain present in the fusion intermediate conformation of S2 subunit. Immunization with HR121 in rabbits and rhesus macaques elicited highly potent cross-neutralizing antibodies against SARS-CoV-2 and its variants, particularly Omicron sublineages. Vaccination with HR121 achieved near-full protections against prototype SARS-CoV-2 infection in hACE2 transgenic mice, Syrian golden hamsters and rhesus macaques, and effective protection against Omicron BA.2 infection in Syrian golden hamsters. This study demonstrates that HR121 is a promising candidate of variant-proof SARS-CoV-2 vaccine with a novel conserved target in the S2 subunit for application against current and future SARS-CoV-2 variants.

Evaluation of Rpoβ Gene and Its Various Mutants in Multidrug-Resistant Tuberculosis Cases by GeneXpert Method.

India is one of the countries in the world which contribute to the global burden of multidrug-resistant tuberculosis (MDR-TB). GeneXpert is a method recommended by the World Health Organization (WHO) that uses five overlapping probes (Probe A to E) to detect mutation in the beta subunit of ribonucleic acid (RNA) polymerase gene (Rpoβ)responsible for rifampicin resistance in Mycobacterium tuberculosis (MTB). All the pulmonary and extrapulmonary samples received in tuberculosis (TB) laboratory for testing of MTBfrom January 2018 to December 2020 were analyzed for bacillary load and rifampicin resistance was identified by analyzing the number of missing probes. During the study period, a total of 10,021 samples were tested for MDR-TB. Out of those samples, 2674 samples were positive for MTB of which 2321 were pulmonary samples and 353 were extra-pulmonary samples. Rifampicin resistance was detected in 385 pulmonary samples and 63 extrapulmonary samples. These samples were further differentiated according to the bacillary load. The highest number of mutations was observed in Probe E followed by Probe B, Probe A, and Probe D, and the lowest was observed in Probe C. Also, mutations were associated when all probes were present or a few combinations of probes were missing. GeneXpert assay is a rapid molecular method that detected MTB and rifampicin resistance with a two-hour turnaround. It uses molecular beacons to detect mutation in the Rpoβ gene. This study can be useful in analyzing the prevalence and epidemiology of MTB in a particular demographic area and also the addition of a few more probes can enhance the identification of the mutation in other codons and therefore better therapeutic interventions can be developed accordingly.