Biology subjects

Maldosevic, E.

Publications and source records attributed to Maldosevic, E..

4 recordsLinked to original sources

bioRxiv2025

Mechanism of Ribosome Stalling by the AMD1 C-terminal Tail Arrest Peptide

AMD1 encodes Adenosylmethionine decarboxylase 1 (AMD1), a key enzyme required for polyamine biosynthesis. A subset of ribosomes translating the AMD1 coding sequence (CDS) read through the stop codon and pause at the next in-frame stop codon 384 nucleotides downstream. The resulting C-terminal extension (C-tail) is universally conserved across all vertebrates, implying that its molecular function is critical to their fitness. Despite growing evidence that such cis-acting elements regulate translation of their genes, the molecular mechanism by which the C-tail mediates ribosome stalling remains unclear. Here, we determined the structure of the ribosome nascent chain complex paused by the AMD1 C-tail which traps eukaryotic release factor 1 (eRF1) with the ATP-binding cassette sub-family E member 1 (ABCE1). The nascent chain forms a molecular clamp that positions an arginine finger in the peptidyl-transferase center, occluding the accommodation of the eRF1 GGQ motif thereby hampering translation termination. Analysis of aggregated ribosome profiling data revealed several genes with a pattern of stop codon readthrough followed by ribosome stalling at a specific location, suggesting that regulatory readthrough-stall mechanisms may not be limited to AMD1.

biophysics↗

bioRxiv2025

A molecular switch in NAC prevents mitochondrial protein mistargeting by SRP

The nascent polypeptide-associated complex (NAC) co-translationally screens all nascent proteins and regulates their access to the signal recognition particle (SRP) to ensure the fidelity of protein targeting to the endoplasmic reticulum (ER). However, the mechanism by which NAC prevents the mistargeting of nascent mitochondrial proteins remains unclear. Here, we identified a molecular switch in NAC that allows its central barrel domain to adopt a stabilized conformation on ribosomes exposing a mitochondrial targeting sequence (MTS). Mutations of the MTS on the nascent chain or in the NAC switch region increases NAC barrel dynamics and reduces its binding to the ribosome. This leads to an impaired ability of NAC to prevent mistargeting by SRP and causes ER stress in human cells. Our work reveals how NAC detects nascent mitochondrial proteins early in translation and prevents their promiscuous access to SRP, elucidating the structural basis that underlies this role and providing novel insights into protein targeting fidelity with broader implications for cellular proteostasis.

biophysics↗

bioRxiv2025

Structural basis and pathological implications of the dimeric OS9-SEL1L-HRD1 ERAD Core Complex

The SEL1L-HRD1 complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD), a critical pathway that clears misfolded proteins to maintain ER proteostasis. However, the molecular organization and pathogenic mechanisms of mammalian ERAD have remained elusive. Here, we report the first cryo-EM structure of the core mammalian ERAD complex, comprising the ER lectin OS9, SEL1L, and the E3 ubiquitin ligase HRD1. The structure, validated by mutagenesis and crosslinking assays, reveals a dimeric assembly of the core complex in which SEL1L and OS9 form a claw-like configuration in the ER lumen that mediates substrate engagement, while HRD1 dimerizes within the membrane to facilitate substrate translocation. Pathogenic SEL1L mutations at the SEL1L-OS9 (Gly585Asp) and SEL1L-HRD1 (Ser658Pro) interfaces disrupt complex formation and impair ERAD activity. A newly identified disease-associated HRD1 variant (Ala91Asp), located in transmembrane helix 3, impairs HRD1 dimerization and substrate processing, underscoring the functional necessity of this interface and HRD1 dimerization. Finally, the structure also reveals two methionine-rich crevices flanking the HRD1 dimer, suggestive of substrate-conducting channels analogous to those in the ER membrane protein complex (EMC). These findings establish a structural framework for mammalian ERAD and elucidate how mutations destabilizing this machinery contribute to human disease. SUMMARYThe dimeric structure of the human SEL1L-HRD1 ERAD core complex reveals key architectural and functional principles underlying the recognition and processing of misfolded proteins linked to human disease.

molecular biology↗

bioRxiv2021

Progressive sub-MIC Exposure of Klebsiella pneumoniae 43816 to Cephalothin Induces the Evolution of beta-lactam Resistance without Acquisition of beta-lactamase Genes

Bacterial exposure to antibiotic concentrations below the minimum inhibitory concentration (MIC) may result in a selection window allowing for the rapid evolution of resistance. These sub-MIC concentrations are commonly found in the greater environment. This study aimed to evaluate the adaptive genetic changes in Klebsiella pneumoniae 43816 after prolonged but increasing sub-MIC levels of the common antibiotic cephalothin over a fourteen-day period. Over the course of the experiment, antibiotic concentrations increased from 0.5 g/mL to 7.5 g/mL. At the end of this extended exposure, the final adapted bacterial culture exhibited clinical resistance to both cephalothin and tetracycline, altered cellular and colony morphology, and a highly mucoid phenotype. Cephalothin resistance exceeded 125 g/mL without the acquisition of beta-lactamase genes. Whole genome sequencing identified a series of genetic changes that could be mapped over the fourteen-day exposure period to the onset of antibiotic resistance. Specifically, mutations in the rpoB subunit of RNA Polymerase, the tetR/acrR regulator, and the wcaJ sugar transferase each fix at specific timepoints in the exposure regimen where the MIC susceptibility dramatically increases. These mutations indicate that alterations in the secretion of colanic acid and attachment of colonic acid to LPS, may contribute to the resistant phenotype. These data demonstrate that very low, sub-MIC concentrations of antibiotics can have dramatic impacts on the bacterial evolution of resistance. Additionally, this study demonstrates that beta-lactam resistance can be achieved through sequential accumulation of specific mutations without the acquisition of a beta-lactamase gene. ImportanceBacteria are constantly exposed to low levels of antibiotics in the environment. The impact of this low-level exposure on bacterial evolution is not well understood. In this work, we developed a model to expose Klebsiella pneumoniae to progressive, low doses of the antibiotic cephalothin. After a fourteen-day exposure regimen, our culture exhibited full clinical resistance to this antibiotic without the traditional acquisition of inactivating genes. This culture also exhibited resistance to tetracycline, had a highly mucoid appearance, and exhibited altered, elongated cellular morphology. Whole genome sequencing identified a collection of mutations to the bacterial genome that could be mapped to the emergence of the resistant phenotype. This study demonstrates that antibiotic resistance can be achieved in response to low level antibiotic exposure and without the traditional acquisition of resistance genes. Further, this study identifies new genes that may play a role in the evolution of antibiotic resistant bacteria.

microbiology↗