Biology subjects

Fleischmann, J.

Publications and source records attributed to Fleischmann, J..

4 recordsLinked to original sources

bioRxiv2024

Kinases in motion: impact of protein and small molecule interactions on kinase conformations

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the Kinase Conformation (KinCon)-reporter system. We [fi]rst track BRAF-kinase activity conformation changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1-kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP-kinases in response to TNF-pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

biochemistry↗

bioRxiv2023

A comprehensive study of SARS-CoV-2 main protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system

Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available PIs (nirmatrelvir and ensitrelvir) with cell-based and biochemical assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease-inhibitor-resistance mechanisms and show the relevance of specific mutations in the clinic, thereby informing treatment decisions. TeaserUnderstanding how SARS-CoV-2 could counter the antiviral drug nirmatrelvir and what it means for the future of COVID-19 treatment.

microbiology↗

bioRxiv2021

RNAP II produces capped 18S and 25S ribosomal RNAs resistant to 5-monophosphate dependent processive 5-3 exonuclease in polymerase switched Saccharomyces cerevisiae

We have previously found in the pathogenic yeast Candida albicans, 18S and 25S ribosomal RNA components, containing more than one phosphate on their 5-end, resistant to 5-monophosphate requiring 5[->]3" exonuclease. Several lines of evidence pointed to RNAP II as the enzyme producing them. We now show in Saccharomyces cerevisiae, permanently switched to RNAP II, due to deletion part of RNAP I upstream activator alone or in combination with deletion of one component of RNAP I itself, the production of such 18S and 25S rRNAs. They contain multiple phosphates at their 5-end and an anti-cap specific antibody binds to them indicating capping of these molecules. These molecules are found in RNA isolated from nuclei, therefore are unlikely to be capped in the cytoplasm. This would be unlike recapping of decapped mRNAs which occurs in the cytoplasm. Our data confirm the existence of such molecules and firmly establish RNA II playing a role in their production. The fact that we see these molecules in wild type Saccharomyces cerevisiae indicates that they are not only a result of mutations but are part of the cells physiology. This adds another way RNAP II is involved in ribosome production in addition to their role in the production of ribosome associated proteins.

molecular biology↗

bioRxiv2019

RNA Polymerase II is involved in 18S and 25S ribosomal RNA transcription, in Candida albicans

Introduction Introduction Results Discussion Methods Reference While in eukaryotes three RNA polymerases are involved in ribosome production [1] under usual growth conditions, the 18S and 25S ribosomal RNA (rRNA) components are thought to be exclusively the products of transcription by RNA polymerase I (Pol I) followed by processing [2]. We have observed recently, in Candida albicans during nutritional depletion and with TOR inhibition, the appearance of 18S and 25S rRNA molecules, resisting digestion by a 5'-phosphate-dependent exonuclease, indicating that they were different from the usual processed rRNA transcripts [3]. Candida albicans, a eukaryotic yeast, is a major cause of invasive fungal disease especially in immune compromised patients [4]. ...

molecular biology↗