Sse1, Hsp110 chaperone of yeast, controls the cellular fate during endoplasmic reticulum stress.

Sse1 is a cytosolic Hsp110 molecular chaperone of yeast, Saccharomyces cerevisiae. Its multifaceted roles in cellular protein homeostasis as a nucleotide exchange factor (NEF), as a protein-disaggregase and as a chaperone linked to protein synthesis (CLIPS) are well documented. In the current study, we show that SSE1 genetically interacts with IRE1 and HAC1, the endoplasmic reticulum-unfolded protein response (ER-UPR) sensors implicating its role in ER protein homeostasis. Interestingly, the absence of this chaperone imparts unusual resistance to tunicamycin-induced ER stress which depends on the intact Ire1-Hac1 mediated ER-UPR signaling. Furthermore, cells lacking SSE1 show inefficient ER-stress-responsive reorganization of translating ribosomes from polysomes to monosomes that drive uninterrupted protein translation during tunicamycin stress. In consequence, the sse1Δ strain shows prominently faster reversal from ER-UPR activated state indicating quicker restoration of homeostasis, in comparison to the wild-type (WT) cells. Importantly, Sse1 plays a critical role in controlling the ER-stress-mediated cell division arrest, which is escaped in sse1Δ strain during chronic tunicamycin stress. Accordingly, sse1Δ strain shows significantly higher cell viability in comparison to WT yeast imparting the stark fitness following short-term as well as long-term tunicamycin stress. These data, all together, suggest that cytosolic chaperone Sse1 is an important modulator of ER stress response in yeast and it controls stress-induced cell division arrest and cell death during overwhelming ER stress induced by tunicamycin.

Spectrum of Primary Immunodeficiency Disorders in Hospitalized Children: Multicentric Data From Kolkata.

OBJECTIVE: To evaluate the incidence and types of primary immunodeficiency diseases (PIDs) in hospitalized children with infection. METHODS: This prospective study was conducted in five tertiary-care facilities in Kolkata over two consecutive years between November 1, 2018 and October 31, 2020. We included all children aged upto 12years who were hospitalized and screened them for PID. Children were screened for suspected IPD using Jeffrey Modell Foundation (JMF) Criteria; any child who satisfied at least 2 out of 10 warning signs was further evaluated for PIDs. RESULTS: Out of 33,204 hospital admissions, 50 children satisfied JMF criteria. Out of 50 children screened during the study period, 27 were finally diagnosed with an underlying PID, with a prevalence of 1 in 1000 hospitalized children. Majority (37.03%) of them had antibody deficiency followed by phagocytic defect (33.3%). Chronic granulomatous disease was the commonest PID followed by common variable immunodeficiency. Around 62.97% children presented with respiratory infections and overall Acinetobacter baumannii was the commonest isolated organism. CONCLUSION: Our study presents the first cohort of PID from eastern India. A methodical step-wise clinical and diagnostic approach can facilitate early diagnosis and timely therapeutic interventions.

A new way to mitigate pathological Tau?

In this issue of Neuron, Wang et al.1 show that the RNA-binding protein G3BP2 interacts with Tau in human neurons and in brains of patients with Alzheimer's disease (AD), suggesting a new role for G3BP2 with implications for therapeutic sequestration of Tau in neurodegenerative diseases.

Treatments against Polymorphosal discrepancies in Glioblastoma Multiforme.

Glioblastoma (GB) are aggressive tumors that obstruct normal brain function. While the skull cannot expand in response to cancer growth, the growing pressure in the brain is generally the first sign. It can produce more frequent headaches, unexplained nausea or vomiting, blurred peripheral vision, double vision, a loss of feeling or movement in an arm or leg, and difficulty speaking and concentrating; all depend on the tumor's location. GB can also cause vascular thrombi, damaging endothelial cells and leading to red blood cell leakage. Latest studies have revealed the role of single nucleotide polymorphisms (SNPs) in developing and spreading cancers such as GB and breast cancer. Many discovered SNPs are associated with GB, particularly in great abundance in the promoter region, creating polygenetic vulnerability to glioma. This study aims to compile a list of some of the most frequent and significant SNPs implicated with GB formation and proliferation.

Stress Responses Elicited by Misfolded Proteins Targeted to Mitochondria.

The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and discrete protein quality control (PQC) machineries resulting inherent differences in their protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to these sub-mitochondrial compartments, we took misfolding and aggregation-prone stressor proteins and fused it to well described signal sequences to specifically target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, by employing transcriptomics and proteomics, we report a comprehensive stress response elicited by stressor proteins specifically targeted to mitochondrial matrix or IMS. A general response to proteotoxic stress by mitochondria-targeted misfolded proteins is mitochondrial fragmentation, and an adaptive abrogation of mitochondrial respiration with concomitant upregulation of glycolysis. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that stress-imparted by bipartite signal sequence-fused stressor proteins to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.

Conserved and divergent chaperoning effects of Hsp60/10 chaperonins on protein folding landscapes.

The GroEL/ES chaperonin cavity surface charge properties, especially the negative charges, play an important role in its capacity to assist intracavity protein folding. Remarkably, the larger fraction of GroEL/ES negative charges are not conserved among different bacterial species, resulting in a large variation in negative-charge density in the GroEL/ES cavity across prokaryotes. Intriguingly, eukaryotic GroEL/ES homologs have the lowest negative-charge density in the chaperonin cavity. This prompted us to investigate if GroEL's chaperoning mechanism changed during evolution. Using a model in vivo GroEL/ES substrate, we show that the ability of GroEL/ES to buffer entropic traps in the folding pathway of its substrate was partially dependent upon the negative-charge density inside its cavity. While this activity of GroEL/ES was found to be essential for Escherichia coli, it has been perfected in some organisms and diminished in others. However, irrespective of their charges, all the tested homologs retained their ability to regulate polypeptide chain collapse and remove enthalpic traps from folding pathways. The ability of these GroEL/ES homologs to buffer mutational variations in a model substrate correlated with their negative-charge density. Thus, Hsp60/10 chaperonins in different organisms may have changed to accommodate a different spectrum of mutations on their substrates.

Mechanistic Underpinnings of Morphology Transition in Electrodeposition under the Application of Pulsatile Potential.

We quantitatively investigate the role of voltage fluctuation in terms of different waveforms on the electrodeposition dynamics and morphology for varying electrolyte concentrations. Dependent on the electrolyte concentration, a wide range of morphologies ranging from highly branched dendrites to comparatively closed packed electrodeposits has been captured. We mechanistically map the deposition dynamics by image analysis and demonstrate the highly porous dendritic dynamics to be independent of external perturbation. Additionally, comparatively closed packed morphological features show significant sensitivity toward the frequency and nature of the waveforms. The results provide fundamental insights into the correlation between the time scales of voltage fluctuation and growth dynamics. We comprehensively analyze the effect of the waveform nature on the average deposition height and show sinusoidal fluctuation to be preferred over square and pulse for metal batteries for lower deposition heights.

Integrating an ER Stress Reporter for Monitoring Genome-Wide UPR-ER in Budding Yeast.

Genetic interaction studies have been instrumental in understanding and organizing cellular pathways. This has been helpful in identifying and arranging genes according to pathways, identifying novel pathways, ascribing gene function, and providing information regarding redundant and antagonistic pathways. Synthetic Genetic Array (SGA) uses growth to identify genome scale gene interaction networks. While this has provided most of the genetic interaction data available, SGA coupled to other reporters have the potential to identify components of pathways that specifically affect the probed reporter. The method described here utilizes SGA principles to understand conserved elements of endoplasmic reticulum (ER) homeostasis in the presence and absence of ER stress. The use of a fluorescent reporter of ER stress allows quantitative measurements and provides a handle to measure the proteostasis capacity of the ER in a high-throughput manner. The integration of such a fluorescent reporter in the background of different mutant/deletion strains is sufficient to identify genetic modules in a high-throughput manner.

The rise in cases of mucormycosis, candidiasis and aspergillosis amidst COVID19.

The Coronavirus outbreak globally has changed the medical system and also led to a shortage of medical facilities in both developing and underdeveloped countries. The COVID19 disease, being novel in nature along with high infectivity and frequent mutational rate, has been termed to be fatal across the globe. The advent of infection by SARS-CoV-2 has brought a myriad of secondary complications and comorbidities resulting in additional challenges to the health care system induced by novel therapeutic procedures. The emerging variant with respect to the Indian subcontinent and the associated genetic mutations have worsened the situation at hand. Proper clinical management along with epidemiological studies and clinical presentations in scientific studies and trials is necessary in order to combat the simultaneous waves of emerging strains. This article summarizes three of the major fungal outbreaks in India namely mucormycosis, candidiasis and aspergillosis, and elaborates their subtypes, pathogenesis, symptoms and treatment and detection techniques. A detail of future therapeutics under consideration are also elaborated along with a general hypothesis on how COVID19 is related to immunological advances leading to major widespread fungal infection in the country. The factors that contribute in promoting virus proliferation and invasive fungal infections include cell-mediated immunity, associated immunocompromised conditions and treatment protocols that slows down immune mechanisms. To better comprehend a fungal or bacterial outbreak, it is very important to conduct audits mediated through multicenter national and state research teams for recognizing patterns and studying current cases of fungal infection in both healthy and comorbid groups of COVID19 patients.

COVID-19 and mucormycosis syndemic: double health threat to a collapsing healthcare system in India.

In addition to the overwhelming and uncontrollable second wave of COVID-19 in India, the country is also dealing with an outbreak of mucormycosis, a deadly fungal infection, which is affecting thousands of COVID-19 patients. With the increasing number of cases of mucormycosis and a fatality rate of 50%, many Indian states and union territories have declared an epidemic of black fungus due to its unprecedented emergence, which has adversely affected the already debilitated health system of the country. The advent of the new fungal epidemic in the country is due to the overdosage, panic and injudicious use of corticosteroids among COVID-19 patients, as well as their pre-existing medical history of diabetes, given that India is the diabetes capital of the world. Thus, there is an urgent need to address this public health concern by having nationwide surveillance, diagnostic and management system of the disease, along with public awareness and education to combat the syndemic of COVID-19 and mucormycosis in the country.

Defining the Caprin-1 Interactome in Unstressed and Stressed Conditions.

Cytoplasmic stress granules (SGs) are dynamic foci containing translationally arrested mRNA and RNA-binding proteins (RBPs) that form in response to a variety of cellular stressors. It has been debated that SGs may evolve into cytoplasmic inclusions observed in many neurodegenerative diseases. Recent studies have examined the SG proteome by interrogating the interactome of G3BP1. However, it is widely accepted that multiple baits are required to capture the full SG proteome. To gain further insight into the SG proteome, we employed immunoprecipitation coupled with mass spectrometry of endogenous Caprin-1, an RBP implicated in mRNP granules. Overall, we identified 1543 proteins that interact with Caprin-1. Interactors under stressed conditions were primarily annotated to the ribosome, spliceosome, and RNA transport pathways. We validated four Caprin-1 interactors that localized to arsenite-induced SGs: ANKHD1, TALIN-1, GEMIN5, and SNRNP200. We also validated these stress-induced interactions in SH-SY5Y cells and further determined that SNRNP200 also associated with osmotic- and thermal-induced SGs. Finally, we identified SNRNP200 in cytoplasmic aggregates in amyotrophic lateral sclerosis (ALS) spinal cord and motor cortex. Collectively, our findings provide the first description of the Caprin-1 protein interactome, identify novel cytoplasmic SG components, and reveal a SG protein in cytoplasmic aggregates in ALS patient neurons. Proteomic data collected in this study are available via ProteomeXchange with identifier PXD023271.

GROEL/ES Buffers Entropic Traps in Folding Pathway during Evolution of a Model Substrate.

The folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and entropic traps in the folding pathway of the substrate protein.

Cellular responses to proteostasis perturbations reveal non-optimal feedback in chaperone networks.

The proteostasis network (PN) comprises a plethora of proteins that are dedicated to aid in protein folding and maintenance; some with overlapping functions. Despite this, there are multiple pathophysiological states associated with depletion of chaperones. This is counter-intuitive, assuming cells have the ability to re-program transcriptional outputs in accordance with its proteostasic limitations. Here, we have used S. cerevisiae to understand how cells respond to different types of proteostasis impairments. We monitored the proteostasis status and transcriptome of single deletions of fourteen different Protein Quality Control (PQC) genes. In most cases, cellular response did not activate proteostasis components or pathways that could either complement the function of the missing PQC gene or restore proteostasis. Over-expression of alternate machineries could restore part of the proteostasis defect in two representative PQC gene deletion strains. We posit that S. cerevisiae inherently lacks the ability to sense and respond optimally to defects in proteostasis caused due to deletion of specific PQC components.

Specific Cholesterol Binding Drives Drastic Structural Alterations in Apolipoprotein A1.

Proteins typically adopt a multitude of flexible and rapidly interconverting conformers, many of which are governed by specific protein interaction domains. Whereas disc-shaped oligomeric HDL and its major protein component ApoA1 have been the focus of several investigations, the structural properties of monomeric ApoA1 remain poorly understood. Using tens of independent molecular simulations (>50 µs), we reveal that ApoA1 adopts a compact conformation. Upon the addition of a physiological concentration of cholesterol to ApoA1, the monomeric protein spontaneously formed a circular conformation. Remarkably, these drastic structural perturbations are driven by a specific cholesterol binding site at the C-terminal and a novel cholesterol binding site at the N-terminal. We propose a mechanism whereby ApoA1 opens in a stagewise manner and mutating the N-terminal binding site destroys the open "belt-shaped" topology. Complementary experiments confirm that the structural changes are induced by specific association of cholesterol with ApoA1, not by the nonspecific hydrophobic effect.

Oxidative Homeostasis Regulates the Response to Reductive Endoplasmic Reticulum Stress through Translation Control.

Reductive stress leads to the loss of disulfide bond formation and induces the unfolded protein response of the endoplasmic reticulum (UPR(ER)), necessary to regain proteostasis in the compartment. Here we show that peroxide accumulation during reductive stress attenuates UPR(ER) amplitude by altering translation without any discernible effect on transcription. Through a comprehensive genetic screen in Saccharomyces cerevisiae, we identify modulators of reductive stress-induced UPR(ER) and demonstrate that oxidative quality control (OQC) genes modulate this cellular response in the presence of chronic but not acute reductive stress. Using a combination of microarray and relative quantitative proteomics, we uncover a non-canonical translation attenuation mechanism that acts in a bipartite manner to selectively downregulate highly expressed proteins, decoupling the cell's transcriptional and translational response during reductive ER stress. Finally, we demonstrate that PERK, a canonical translation attenuator in higher eukaryotes, helps in bypassing a ROS-dependent, non-canonical mode of translation attenuation.

Cross-compartment proteostasis regulation during redox imbalance induced ER stress.

Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle-specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ-based LC-MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross-talk between ER- and mitochondrial proteostasis exemplified by an Ire1p-dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross-talk during stress.