Biology subjects

Saleh, A. M.

Publications and source records attributed to Saleh, A. M..

4 recordsLinked to original sources

bioRxiv2025

A new corrector rescues F508del-CFTR folding through stabilization of the TMD1-NBD1 linker

While Trikafta has turned many CF patients into people that lead relatively healthy lives, additional modulators are still needed. We developed an acylsulfonamide-type C2 corrector X307810 that is potent and shows a distinctive mode of action in rescuing F508del-CFTR from degradation at the endoplasmic reticulum. We tracked the de-novo folding and assembly of each CFTR domain with a radio-active pulse chase - limited proteolysis folding assay and determined when and where X307810 works on the newly synthesized protein. X307810 rescued the Golgi form of F508del-CFTR to 80% of wild-type values. To our knowledge, such a dramatic increase has never been attained with a single corrector. Zooming in on X307810 mode of action revealed that the corrector changed protease resistance of the linker between TMD1 and NBD1 during late stages of CFTR folding. X307810 is the first corrector that added a unique new fragment to the typical limited-proteolysis pattern. Other C2 correctors do not change fragments, but portray an increase in fragment quantity characteristic of domain assembly in wild-type and corrected F508del-CFTR. X307810 instead extended protection of TMD1 from intracellular loop 2 to the unstructured regulatory insertion of NBD1. These results provide new and important insights into the impact of acylsulfonamide correctors on CFTR structure and function. SIGNIFICANCE STATEMENTCFTR is an anion channel, mutations in which cause cystic fibrosis (CF). Corrector compounds allow misfolded missense CFTR mutants to escape from degradation in the endoplasmic reticulum, which is the basis for CF therapy, in combination with a potentiator. How correctors improve folding is incompletely understood. We here report on X307810 having a novel mode of action on post-translational domain assembly of newly synthesized CFTR. Our in-cell folding assay shows that X307810 stabilizes the linker between the first transmembrane domain (TMD1) and the first nucleotide binding domain (NBD1) into the Regulatory Insertion; it strengthens the TMD1-NBD1-ICL4 interface. The action of X307810 boosts egress of F508del-CFTR from the ER to an unprecedented 80% for a single modulator.

biochemistry↗

bioRxiv2021

AN INTEGRATIVE BIOLOGY APPROACH TO QUANTIFY THE BIODISTRIBUTION OF AZIDOHOMOALANINE IN VIVO

Identification and quantitation of newly synthesized proteins (NSPs) are critical to understanding protein dynamics in development and disease. Probing the nascent proteome can be achieved using non-canonical amino acids (ncAAs) to selectively label the NSPs utilizing endogenous translation machinery, which can then be quantitated with mass spectrometry. Since its conception, ncAA labeling has been applied to study many in vitro systems and more recently the in vivo proteomes of complex organisms such as rodents. In vivo labeling is typically achieved by introducing ncAAs into diet, which requires extended labeling times. We have previously demonstrated that labeling the murine proteome is feasible via injection of azidohomoalanine (Aha), a ncAA and methionine (Met) analog, without the need for Met depletion. With the ability to isolate NSPs without applying stress from dietary changes, Aha labeling can address biological questions wherein temporal protein dynamics are significant. However, accessing this temporal resolution requires a more complete understanding of Aha distribution kinetics in tissues. Furthermore, studies of physiological effects of ncAA administration have been limited to gross observation of animal appearance. To address these gaps, we created a deterministic, compartmental model of the biokinetic transport and incorporation of Aha in mice. Parameters were informed from literature and experimentally. Model results demonstrate the ability to predict Aha distribution and labeling under a variety of dosing paradigms and confirms the use of the model as a tool for design of future studies. To establish the suitability of the method for in vivo studies, we investigated the impact of Aha administration on normal physiology by analyzing the plasma metabolome following Aha injection. We show that Aha administration does not significantly perturb cellular functions as reflected by an unchanged plasma metabolome compared to non-injected controls. Author SummaryAs the machinery of life, proteins play a key role in dynamic processes within an organism. As such, the response of the proteome to perturbation is increasingly becoming a critical component of biological and medical studies. Dysregulation of protein mechanisms following exposure to experimental treatment conditions can implicate physiological mechanisms of health and disease, elucidate toxin/drug response, and highlight potential targets for novel therapies. Traditionally, these questions have been probed by studying perturbations in total proteins following an experimental treatment. However, the proteome is expansive and noisy, often an early response can be indiscernible against the background of unperturbed proteins. Here, we apply a technique to selectively label newly synthesized proteins, which enables capturing early changes in protein behavior. We utilize an amino acid analog that naturally incorporates into proteins, and investigate the tissue distribution, protein labeling efficiency, and potential physiological impact of this analog in mice. Our results demonstrate that we can reproducibly predict protein labeling and that the administration of this analog does not significantly alter in vivo physiology over the course of our experimental study. We further present a computational model that can be used to guide future experiments utilizing this technique to study proteomic responses to stimuli.

bioengineering↗

bioRxiv2020

Extracellular matrix protein composition dynamically changes during murine forelimb development

The extracellular matrix (ECM) is an integral part of multicellular organisms, connecting different cell layers and tissue types. During morphogenesis and growth, tissues undergo substantial reorganization involving cellular proliferation, migration, and differentiation. While it is intuitive that the ECM remodels in concert, little is known regarding how matrix composition and organization change during development. We utilized tissue fractionation and mass spectrometry to define ECM protein (matrisome) dynamics during murine forelimb development and resolved significant differences in ECM composition as a function of development, disease and tissue type. Additionally, we used bioorthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized ECM within the developing forelimb. We demonstrate the feasibility of using BONCAT to enrich for newly synthesized matrisome components and identified differences in ECM synthesis between morphogenesis and growth. This resource will guide future research investigating the role of the matrisome during complex tissue development.

developmental biology↗

bioRxiv2019

Distribution and diversity of fish species exposed to artisanal fishery along the Sudanese Red Sea coast.

The semi-enclosed Red Sea harbours one of the longest coral-reef ecosystems on the planet. The {approx} 850 km section of the western shore, comprising the coastline of the Red Sea State of the Republic of Sudan, has however been sparsely studied. Sudans coral reef fishery provides livelihoods to fishers and business opportunities by means of local and regional trade, however, the knowledge level of the state of the natural resources and the impacts of fisheries are poorly known. Here we report the results from the first three comprehensive fisheries research surveys spanning the entire Sudanese coast in 2012-13, representing a new baseline for the western coast fisheries resources. The surveys covered the entire coast from inshore down to about 150 m bottom depth using a combination of baited traps, gillnets and handlines to sample the various reef habitats and fish assemblages. The results demonstrate a uniform latitudinal species distribution with peak catch per unit effort rates in and around the Dungonab Bay area in the north and the outer Suakin archipelago in the south. Functional diversity (Raos Q index) was found to be highest in and around the Dungonab Bay area, thus coming through as a regional hot-spot of biodiversity. The results form a baseline for future research and monitoring, thus representing key input for an ecosystem approach to management of Sudans coastal artisanal fisheries.

ecology↗