Biology subjects

Otterlei, M.

Publications and source records attributed to Otterlei, M..

4 recordsLinked to original sources

bioRxiv2024

Responses to ligand binding in the bacterial DNA sliding clamp beta-clamp manifest in dynamic allosteric effects

The homo-dimeric, ring-shaped bacterial DNA sliding clamp, {beta}-clamp, is a central hub in DNA replication and repair. It interacts with a plethora of proteins via short linear motifs, which bind to the same hydrophobic binding pocket on {beta}-clamp. Although the structure, functions and interactions of {beta}-clamp have been amply studied, less focus has been on understanding its dynamics and how this is influenced by ligand binding. In this work, we have made a backbone nuclear magnetic resonance (NMR) assignment of the 83 kDa dimeric {beta}-clamp and used NMR in combination with hydrogen-deuterium exchange mass spectrometry to scrutinize the dynamics of {beta}-clamp and how different ligands affect this. We found that binding of a small peptide from the polymerase III subunit affects the dynamics and stability of {beta}-clamp. This effect not only appears locally around the binding pocket, but also globally through dynamic allosteric connections to distant regions of the protein, including the dimer interface. This dissipated dynamic effect is likely a consequence of the binding pocket architecture and may reflect a common mechanism of structural plasticity, where different ligands impose differential responses in the structure and dynamics of {beta}-clamp. HighlightsO_LINMR spectroscopy of {beta}-clamp revealed its inherent dynamics C_LIO_LIA peptide ligand from polymerase III stabilizes {beta}-clamp from global unfolding C_LIO_LIPeptide binding causes allosteric effects that manifest in changes in dynamics C_LIO_LIDynamic changes occur distant to the binding pocket in the dimer interface C_LIO_LIAllostery may be a mechanism for differential responses to interaction partners C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/600769v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1a2fa22org.highwire.dtl.DTLVardef@d40d98org.highwire.dtl.DTLVardef@1c87aaforg.highwire.dtl.DTLVardef@ee8462_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Activation of the Cpx-envelope stress response system promotes tolerance to antibacterials delivered by arginine-rich peptides and aminoglycosides in Escherichia coli

Cell penetrating peptides (CPP) are increasingly used for cellular drug delivery in both pro- and eukaryotic cells, and oligoarginines have attracted special attention. However; their mechanism of action, particularly for prokaryotes is still unknown. Arginine-rich CPPs (R-CPP) efficiently delivers the antimicrobial peptide nucleic acid (PNA) into bacteria. Here, we show that resistance to an R-CPP PNA conjugate in Escherichia coli requires multiple genetic modifications and is specific to R-CPP and not to the PNA-part. An integral part of the resistance was the constitutively activated Cpx-envelope stress response system (cpx*), which decreased the cytoplasmic membrane potential and thereby indicates an indirectly energy dependent uptake mechanism. Interestingly, cpx* mutants also showed increased tolerance to aminoglycosides and R-CPP conjugated to a peptide targeting the DNA sliding clamp; i.e., similar uptake in E. coli for these antimicrobial compounds. We speculate that the cpx* phenotype could create an evolutionary opportunity to adapt and evolve in the presence of either compounds. Author summaryThe emergence of multidrug resistant bacteria is raising the need for new classes of antibiotics. Peptide nucleic acids (PNAs) may fill this requirement by their ability to block translation of essential mRNAs and hence inhibit growth. PNA needs conjugation to a delivery peptide (cell penetrating peptide; CPP) to enter the bacteria. Arginine-rich CPPs (CPPR) are receiving a lot of attention for use as delivery vessels. Here, we show, for the first time, CPPR-PNA resistance in Escherichia coli directed towards the delivery peptide. Consequently, resistance also applies to other antimicrobial compounds delivered by the same carrier. An integral part of CPPR resistance is due to a constitutive active Cpx-response system, which leads to a decreased electric potential ({Delta}{Psi}) across the inner membrane. The decreased {Delta}{Psi} is a result of down-regulation of two aerobic respiratory operons, namely NADH:ubiquinone oxidoreductase complex I and cytochrome bo3 ubiquinol oxidase. The decreased {Delta}{Psi} also led to increased tolerance to aminoglycosides. This shows that a (large) negative {Delta}{Psi} is important for providing sufficient free energy for membrane translocation of both CPPR and that the inner membrane is the main barrier for entry of both arginine-rich delivery peptides and aminoglycosides.

microbiology↗

bioRxiv2020

PCNA has specific functions in regulation of metabolism in haematological cells

PCNAs essential roles in DNA replication and repair are well established, while its recently discovered cytosolic roles are less explored. Here we show that impairing PCNAs cytosolic scaffold functions led to massive changes in cellular signaling, and most strikingly, a strong reduction in glycolytic metabolite and nucleoside phosphate pools in haematological cancer cells. This was not detected in cells from solid tissues nor in primary monocytes from healthy donors. However, lipopolysaccharide stimulated monocytes responded to targeting PCNAs scaffold function similarly to haematological cancer cells, suggesting that cellular stress is an important factor for the observed response. Integrated transcriptome, proteome and metabolome analysis revealed that pathways involved in protein stability/folding and immune responses were affected in haematopoietic cancer cells. Altogether, our data suggests that PCNA has an important role in regulation of cytoplasmic stress via regulation of central carbon metabolism in haematological cells, which potentially can be targeted in cancer treatment. HighlightA strong reduction in glycolytic metabolites and the nucleoside phosphate pools was detected in haematological cancer cells upon inhibiting PCNA scaffold functions. These metabolic changes were not found in cancer cells from other tissues, indicating a special role of PCNA in regulation of metabolism in cells of haematological origin.

cell biology↗

bioRxiv2019

Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression

Base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG; aka MPG) is essential for removal of aberrantly methylated DNA bases. Genome instability and accumulation of aberrant bases accompany multiple diseases including cancer and neurological disorders. While BER is well studied on naked DNA, it remains unclear how BER efficiently operates on chromatin. Here we show that AAG binds to chromatin and forms complex with active RNA polymerase (pol) II. This occurs through direct interaction with Elongator and results in transcriptional co-regulation. Importantly, at co-regulated genes aberrantly methylated bases accumulate towards 3end, in regions enriched for BER enzymes AAG and APE1, Elongator and active RNA pol II. Active transcription and functional Elongator are further crucial to ensure efficient BER, by promoting AAG and APE1 chromatin recruitment. Our findings provide novel insights to maintaining genome stability in actively transcribing chromatin, and reveal roles of aberrantly methylated bases in regulation of gene expression.

molecular biology↗