Deficiency of Nuclear Factor-κB c-Rel Accelerates the Development of Autoimmune Diabetes in NOD Mice

The nuclear factor-κB protein c-Rel plays a critical role in controlling autoimmunity. c-Rel–deficient mice are resistant to streptozotocin-induced diabetes, a drug-induced model of autoimmune diabetes. We generated c-Rel–deficient NOD mice to examine the role of c-Rel in the development of spontaneous autoimmune diabetes. We found that both CD4^+ and CD8^+ T cells from c-Rel–deficient NOD mice showed significantly decreased T-cell receptor–induced IL-2, IFN-γ, and GM-CSF expression. Despite compromised T-cell function, c-Rel deficiency dramatically accelerated insulitis and hyperglycemia in NOD mice along with a substantial reduction in T-regulatory (Treg) cell numbers. Supplementation of isogenic c-Rel–competent Treg cells from prediabetic NOD mice reversed the accelerated diabetes development in c-Rel–deficient NOD mice. The results suggest that c-Rel–dependent Treg cell function is critical in suppressing early-onset autoimmune diabetogenesis in NOD mice. This study provides a novel natural system to study autoimmune diabetes pathogenesis and reveals a previously unknown c-Rel–dependent mechanistic difference between chemically induced and spontaneous diabetogenesis. The study also reveals a unique protective role of c-Rel in autoimmune diabetes, which is distinct from other T-cell–dependent autoimmune diseases such as arthritis and experimental autoimmune encephalomyelitis, where c-Rel promotes autoimmunity

Deficiency of Nuclear Factor-κB c-Rel Accelerates the Development of Autoimmune Diabetes in NOD Mice

The nuclear factor-κB protein c-Rel plays a critical role in controlling autoimmunity. c-Rel–deficient mice are resistant to streptozotocin-induced diabetes, a drug-induced model of autoimmune diabetes. We generated c-Rel–deficient NOD mice to examine the role of c-Rel in the development of spontaneous autoimmune diabetes. We found that both CD4^+ and CD8^+ T cells from c-Rel–deficient NOD mice showed significantly decreased T-cell receptor–induced IL-2, IFN-γ, and GM-CSF expression. Despite compromised T-cell function, c-Rel deficiency dramatically accelerated insulitis and hyperglycemia in NOD mice along with a substantial reduction in T-regulatory (Treg) cell numbers. Supplementation of isogenic c-Rel–competent Treg cells from prediabetic NOD mice reversed the accelerated diabetes development in c-Rel–deficient NOD mice. The results suggest that c-Rel–dependent Treg cell function is critical in suppressing early-onset autoimmune diabetogenesis in NOD mice. This study provides a novel natural system to study autoimmune diabetes pathogenesis and reveals a previously unknown c-Rel–dependent mechanistic difference between chemically induced and spontaneous diabetogenesis. The study also reveals a unique protective role of c-Rel in autoimmune diabetes, which is distinct from other T-cell–dependent autoimmune diseases such as arthritis and experimental autoimmune encephalomyelitis, where c-Rel promotes autoimmunity

Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome

Purpose Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort. Methods We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. Results Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. Conclusion Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome

Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome

Purpose: Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort. Methods: We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. Results: Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. Conclusion: Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome

Intrinsic and extrinsic determinants of protein folding, aggregation and abundance.

Misfolding and aggregation are major challenges in proteopathies, including Alzheimer's and Parkinson's diseases, and in biochemical and therapeutic industries. Aggregation is caused by misfolded or unfolded proteins that escape the protein quality control machinery of the cell, which employs a large network of molecules and primarily consists of molecular chaperones and proteases which continuously monitor the cell and ensure protein homeostasis. Molecular chaperones help to fold proteins, which are otherwise unable to fold themselves as per "Anfinsen's rule", into native conformation or disaggregate those that are aggregated. Proteases degrade and clear out the misfolded and aggregated proteins which cannot be rescued by chaperones. At molecular level, certain mutations destabilize proteins and thereby expose hydrophobic segments which form the core in the protein's native conformation. Such hydrophobic segments called "aggregation prone regions (APRs)" can also be exposed during translation, domain swapping and protein trafficking. Their exposure contributes to aggregation. Thus, exposed APRs in misfolded and unfolded proteins are the leading cause of aggregation. Similarly, any disturbance of native structure due to physical or chemical factors such as change in optimum temperature, pressure, oxidation status and pH for growth and protein's structural and sequence features -for instance presence of disordered segments and moreover ageing and accompanying weakness in quality control mechanism are all responsible for contributing to the production of non-functional proteins via misfolding and aggregation. Finally, protein abundance exceeding the saturation limit of its solubility can itself cause aggregation. In this thesis, the interdependence between different factors that contribute to folding and aggregation of proteins centred around the three Escherichia coli cytosolic chaperones, namely, DnaKJ, GroELS and TF were analyzed. Focusing on Escherichia coli provided the advantage of a well-documented list of chaperone dependent proteins and other cellular, sequence and structural parameters than any other model organisms. Moreover, being a prokaryote, lack of cellular compartmentalization made it less complicated to analyze and compare protein features. Chaperones being a vital part of a cell, and the subject of extensive experimental and computational studies, the striking feature by which they differentiate healthy and folded proteins from un/misfolded and aggregated proteins remains an unresolved question. The generally accepted view is: high-abundant proteins are soluble and less aggregation prone; aggregation prone proteins are expressed at low level in the cells and require folding assistance from chaperones. Considering the inconsistencies between and limitations of previous approaches; in this work, a new in-depth but wide and simplistic meta-analysis approach was developed and implemented to readdress the contributing factors of misfolding and aggregation, focusing mainly on chaperones that control folding, aggregation and abundance of proteins. Firstly, we could successfully describe that external changes play a critical role in modulating the biogenesis of a functional proteome in vivo. Both high-abundant and low-abundant proteins are aggregation prone in different contexts. Proteins with low expression levels that depend on chaperones are highly and inherently prone to misfold; they require DnaKJ /GroELS or both to remain soluble and to maintain their native state. On the other hand, high abundant and big proteins aggregate mainly in the absence of TF and DnaKJ, indicating they require co-translational interaction with either TF/DnaKJ or both to remain soluble and some of them need to interact with GroELS to attain the functional state. Secondly, through a detailed analysis at the transcript level, we could predict the contribution of translation kinetics as a determinant of in vivo chaperone dependency. Repeating the analysis through cross-validation strategies on a limited but more reliable set of proteins and on highly debated TIM barrel proteins in the chaperone biology, we produced more evidence in supporting translation kinetics as a modulator of folding efficiency and thus is a determinant of chaperone dependency of proteins. This in silico observation was well substantiated experimentally by designing GFP variants where fast translated variants showed more dependency to DnaKJ and slow variants towards GroELS, despite GFP being a natural GroELS dependent protein. Finally, the proteostatic data collected and processed for the analysis were made available to the use of general public via PHDB database (http://phdb.switchlab.org/#/home). The tools implemented along with the database provide statistical comparison of proteins for all the proteostatic parameters. As an application, the database and the pipeline established for proteostatic data analysis, were later used successfully to differentiate cytoplasmic and secretory protein. The findings of our research have considerable implications on understanding chaperone mediated protein folding and interaction in detail, in vivo proteins folding in general and solving proteins misfolding, aggregation and solubility related issues. Notably, the PHDB database is a valuable resource not only for chaperone related and proteostatic studies but helps in quick analysis and comparison of any Escherichia coli protein(s).status: publishe

Differential proteostatic regulation of insoluble and abundant proteins

MOTIVATION: Despite intense effort, it has been difficult to explain chaperone dependencies of proteins from sequence or structural properties. RESULTS: We constructed a database collecting all publicly available data of experimental chaperone interaction and dependency data for the Escherichia coli proteome, and enriched it with an extensive set of protein-specific as well as cell-context-dependent proteostatic parameters. Employing this new resource, we performed a comprehensive meta-analysis of the key determinants of chaperone interaction. Our study confirms that GroEL client proteins are biased toward insoluble proteins of low abundance, but for client proteins of the Trigger Factor/DnaK axis, we instead find that cellular parameters such as high protein abundance, translational efficiency and mRNA turnover are key determinants. We experimentally confirmed the finding that chaperone dependence is a function of translation rate and not protein-intrinsic parameters by tuning chaperone dependence of Green Fluorescent Protein (GFP) in E.coli by synonymous mutations only. The juxtaposition of both protein-intrinsic and cell-contextual chaperone triage mechanisms explains how the E.coli proteome achieves combining reliable production of abundant and conserved proteins, while also enabling the evolution of diverging metabolic functions. AVAILABILITY AND IMPLEMENTATION: The database will be made available via http://phdb.switchlab.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.status: publishe

Protein Homeostasis Database: protein quality control in E.coli

MOTIVATION: In vivo protein folding is governed by molecular chaperones, that escort proteins from their translational birth to their proteolytic degradation. In E.coli the main classes of chaperones that interact with the nascent chain are trigger factor, DnaK/J and GroEL/ES and several authors have performed whole-genome experiments to construct exhaustive client lists for each of these. RESULTS: We constructed a database collecting all publicly available data of experimental chaperone-interaction and -dependency data for the E.coli proteome, and enriched it with an extensive set of protein-specific as well as cell context-dependent proteostatic parameters. We made this publicly accessible via a web interface that allows to search for proteins or chaperone client lists, but also to profile user-specified datasets against all the collected parameters. We hope this will accelerate research in this field by quickly identifying differentiating features in datasets. AVAILABILITY AND IMPLEMENTATION: The Protein Homeostasis Database is freely available without any registration requirement at http://PHDB.switchlab.org/.status: publishe

Structural Basis of the Subcellular Topology Landscape of Escherichia coli

Cellular proteomes are distributed in multiple compartments: on DNA, ribosomes, on and inside membranes, or they become secreted. Structural properties that allow polypeptides to occupy subcellular niches, particularly to after crossing membranes, remain unclear. We compared intrinsic and extrinsic features in cytoplasmic and secreted polypeptides of the Escherichia coli K-12 proteome. Structural features between the cytoplasmome and secretome are sharply distinct, such that a signal peptide-agnostic machine learning tool distinguishes cytoplasmic from secreted proteins with 95.5% success. Cytoplasmic polypeptides are enriched in aliphatic, aromatic, charged and hydrophobic residues, unique folds and higher early folding propensities. Secretory polypeptides are enriched in polar/small amino acids, β folds, have higher backbone dynamics, higher disorder and contact order and are more often intrinsically disordered. These non-random distributions and experimental evidence imply that evolutionary pressure selected enhanced secretome flexibility, slow folding and looser structures, placing the secretome in a distinct protein class. These adaptations protect the secretome from premature folding during its cytoplasmic transit, optimize its lipid bilayer crossing and allowed it to acquire cell envelope specific chemistries. The latter may favor promiscuous multi-ligand binding, sensing of stress and cell envelope structure changes. In conclusion, enhanced flexibility, slow folding, looser structures and unique folds differentiate the secretome from the cytoplasmome. These findings have wide implications on the structural diversity and evolution of modern proteomes and the protein folding problem.status: publishe