Biology subjects

Gorna, M. W.

Publications and source records attributed to Gorna, M. W..

6 recordsLinked to original sources

bioRxiv2026

Evaluation of degron motifs in Escherichia coli using a fluorescent reporter

Fluorescent reporters provide a useful tool for studying degron motifs. Fusing a degron of interest to a fluorescent protein allows to accurately track protein levels overtime to characterise the degradation kinetics of studied degrons. Here we describe a rapid and simple method to study degron peptides in Escherichia coli using plasmid-encoded eGFP-degron fusion constructs. The described methods provide an accessible workflow to evaluate degrons. We provide protocols for generation of pBAD plasmids encoding the studied constructs and two different methods for evaluating degrons - an end-point fluorescence measurement on agar plates and a kinetic measurement in liquid cultures in a 96-well format for high-throughput degron studies.

microbiology↗

bioRxiv2024

Human FASTK preferentially binds single-stranded and G-rich RNA

Fas-activated serine/threonine kinase (FASTK) is the founding member of the FASTKD protein family, which was shown to regulate the fate of mRNA molecules on multiple levels. The mitochondrial variant of FASTK co-localizes with mitochondrial RNA granules and regulates degradation of mitochondrial mRNAs, whereas the cytoplasmic and nuclear forms of FAST are involved in regulation of alternative splicing, cytoplasmic RNA granule formation and mRNA translation. Despite these multiple roles of FASTK in mRNA biology, the exact rules of RNA recognition by this protein remained undetermined. Here, we demonstrate direct RNA binding by purified human FASTK and show its preference for single-stranded G-rich sites and RNA G-quadruplexes. Addition of FASTK alone was sufficient to achieve protection of mitochondrial mRNAs from degradation by the degradosome. Structural characterization by SAXS showed that FASTK in solution is a monomer with an extended conformation. Point mutagenesis studies supported the structural predictions of an exposed RNA-binding interface in the central helical region, preceded by a smaller, flexibly attached, helical N-terminal domain. We provide the first such extensive in vitro characterization of the RNA binding properties for a representative of the FASTKD protein family, and suggest how these intrinsic properties may underly FASTK function in mRNA metabolism.

molecular biology↗

bioRxiv2024

Depletion of essential GroEL protein in Escherichia coli using Clp-Interacting Peptidic Protein Erasers (CLIPPERs)

New, universal tools for targeted protein degradation in bacteria can help to accelerate protein function studies and antimicrobial research. We have developed a new method for degrading bacterial proteins using plasmid-encoded degrader peptides which deliver target proteins for degradation by a highly conserved ClpXP protease. We demonstrated the mode of action of the degraders on a challenging essential target, GroEL. The studies in bacteria were complemented by in vitro binding and structural studies. Expression of degrader peptides resulted in a temperature-dependent growth inhibition and depletion of GroEL levels over time. The reduction of GroEL levels was accompanied by dramatic proteome alterations. The presented method offers a new alternative approach for regulating protein levels in bacteria without genomic modifications or tag fusions. Our studies demonstrate that ClpXP is an attractive protease for the future use in bacterial targeted protein degradation.

molecular biology↗

bioRxiv2022

MultiOMICs landscape of SARS-CoV-2-induced host responses in human lung epithelial cells

Despite the availability of vaccines and approved therapeutics, the COVID-19 pandemic continues to rise owing to the emergence of newer variants. Several multi-omics studies have made available extensive evidence on host-pathogen interactions and potential therapeutic targets. Nonetheless, an increased understanding of host signaling networks regulated by post-translational modifications and their ensuing effect on the biochemical and cellular dynamics is critical to expanding the current knowledge on the host response to SARS-CoV-2 infections. Here, employing unbiased global transcriptomics, proteomics, acetylomics, phosphoproteomics, and exometabolome analysis of a lung-derived human cell line, we show that SARS-CoV-2 Norway/Trondheim-S15 strain induces time-dependent alterations in the induction of type I IFN response, activation of DNA damage response, dysregulated Hippo signaling, among others. We provide evidence for the interplay of phosphorylation and acetylation dynamics on host proteins and its effect on the altered release of metabolites, especially organic acids and ketone bodies. Together, our findings serve as a resource of potential targets that can aid in designing novel host-directed therapeutic strategies.

systems biology↗

bioRxiv2022

The Repeating, Modular Architecture of the HtrA Proteases

A conserved, 26 residue sequence [AA(X2)[A/G][G/L](X2)GDV[I/L](X2)[V/L]NGE(X1)V(X6)] and corresponding structure repeating module was identified within the HtrA protease family using a non-redundant set (N=20) of publically available structures. While the repeats themselves were far from sequence perfect they had notable conservation to a statistically significant level with three or more repetitions identified within one protein at a level that would be expected to randomly occur only once per 1031 residues. This sequence repeat was associated with a six stranded antiparallel {beta}-barrel module, two of which are present in the core of the structures of the PA clan of serine proteases, while a modified version of this module could be identified in the PDZ-like domains. Automated structural alignment methods had difficulties in superimposing these {beta}-barrels but use of a target human HtrA2 structure showed that these modules had an average RMSD across the set of structures of less than 2 [A] (mean and median). Our findings support Dayhoffs hypothesis that complex proteins arose through duplication of simpler peptide motifs and domains.

bioinformatics↗

bioRxiv2021

A uniform benchmark for testing ssrA-derived degrons in the Escherichia coli ClpXP pathway

The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder developments of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches. SUMMARYThis work lays standards for assays used to compare engagement of SspB and ClpXP by a set of ssrA-derived degrons that can be used to fine-tune tools for protein stability control.

biochemistry↗