Biology subjects

Konvalinka, J.

Publications and source records attributed to Konvalinka, J..

8 recordsLinked to original sources

bioRxiv2026

Xeno amino acid alphabets form peptides with familiar secondary structure

Summary paragraphThe central dogma of molecular biology describes how genetic information stored in nucleic acids guides the formation of structured proteins from a single molecular alphabet of twenty amino acids (C20)1,2. The extent to which C20 is uniquely capable of forming structural polymers remains a fundamental open question. Here we demonstrate that peptides built from other, "xeno" amino acid alphabets can adopt protein-like secondary structural motifs. Having designed two different xeno alphabets, with them we constructed both combinatorial random sequence libraries and specific, designed 25-mer sequences. We report sequence-dependent structural motifs that result according to circular dichroism, infrared spectroscopy and nuclear magnetic resonance interpreted by molecular dynamics simulations, supported by extensive quantum mechanical calculations. This evidence, including a solution-phase NMR-resolved helical motif, demonstrates that the potential for amino acids to form structure bearing sequences is not unique to lifes alphabet, and thereby reveals a previously unexplored sequence-structure space. Results inform the search for extraterrestrial life, lay foundations for incorporating novel synthetic functional groups and heteroatoms into structure-bearing alphabets, and provide the first truly independent data with which to test and improve all that has been learned about protein folding from the study of lifes 20 side chains.

biochemistry↗

bioRxiv2025

Prebiotically Plausible Peptides can Self-assemble into β-rich Nanostructures

Peptides can self-assemble into diverse morphologies in a programmable manner and hence are privileged building blocks used widely in nanotechnology. Most reported peptide nanostructures consist of one (or a few) defined sequence(s), as self-similarity is presumed to be essential for promoting assembly. While oligomerisation is seen as an important feature of the earliest functional polymers during the origin of life, prebiotic peptides were likely short, statistical, and non-templated - traits that seem incommensurate with robust self-assembly. Here we show that random 25-mer peptides can efficiently and spontaneously form highly thermostable, soluble assemblies rich with beta-sheets. Notably, these nanostructures only emerge when random peptides are constructed with an early alphabet, consisting of the 10 canonical amino acids that were also prebiotically abundant - but not other alphabets tested. Hence, our findings show that peptide self-assembly does not require purity, and in fact compositional complexity is adaptive for preventing formation of insoluble structures. Altogether, this study showcases that unevolved sequences of prebiotically-abundant amino acids can readily produce foldable self-assembling polymers, thereby providing a potential steppingstone toward the first proteins, prior to the onset of purifying selection.

biochemistry↗

bioRxiv2025

A novel screening assay for insulin receptor

Insulin is a key hormone in glucose homeostasis. Its lack causes severe health complications and has to be compensated by regular administration of insulin. Despite intense long-lasting research, a more stable and efficient substitute has yet to be discovered to alleviate patients issues. Here we report the development of a new assay for screening potential insulin receptor ligands based on the DNA-linked inhibitor antibody assay (DIANA). Our assay meets the need for a fast, sensitive, non-radioactive method as an alternative to the commonly used radioligand receptor binding assay.

biochemistry↗

bioRxiv2024

Sensitive quantification of fibroblast activation protein and high-throughput screening for inhibition by FDA-approved compounds

Fibroblast activation protein (FAP) has been extensively studied as a cancer biomarker for decades. Recently, small-molecule FAP inhibitors have been widely adopted as a targeting moiety of experimental theranostic radiotracers. Here we present a fast qPCR-based analytical method allowing FAP inhibition screening in a high-throughput regime. In order to identify clinically relevant compounds that might interfere with FAP-targeted approaches, we focused on the library of FDA-approved drugs. Using the DNA-linked Inhibitor Antibody Assay (DIANA), we tested a library of 2,667 compounds within just few hours and identified numerous FDA-approved drugs as novel FAP inhibitors. Notably, prodrugs of cephalosporin antibiotics, reverse-transcriptase inhibitors, and one elastase inhibitor were the most potent FAP inhibitors in our dataset. In addition, by employing FAP DIANA in quantification mode, we were able to determine FAP concentrations in human plasma samples. Together, our work expands the repertoire of FAP inhibitors, underscores the potential interference of co-administered drugs with FAP-targeting strategies, and presents a sensitive and low-consumption ELISA alternative for FAP quantification with a detection limit of 50 pg/ml. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/600598v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1c19d55org.highwire.dtl.DTLVardef@405b3aorg.highwire.dtl.DTLVardef@1255281org.highwire.dtl.DTLVardef@14bc80e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Polymer-tethered quenched fluorescent probes for enhanced imaging of tumor associated proteases

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

bioengineering↗

bioRxiv2020

DDI2 protease activity controls embryonic development and inflammation via TCF11/NRF1

DDI2 is an aspartic protease that cleaves polyubiquitinated substrates. Upon proteotoxic stress, DDI2 activates the ER-bound transcription factor TCF11/NRF1 (NFE2L1), a master regulator of proteostasis maintenance in mammalian cells, and ensures the expression of rescue factors including proteasome subunits. Here we describe the consequences of DDI2 ablation both in vivo and in cells. Knock-out of DDI2 in mice resulted in embryonic lethality at E12.5 with severe developmental failure. Molecular characterization of the embryos and surrogate DDI2 knock-out cell lines showed insufficient proteasome expression with proteotoxic stress, accumulation of high molecular weight ubiquitin conjugates, and induction of the unfolded protein and integrated stress responses. We also show that DDI2 KO-induced proteotoxic stress causes the cell-autonomous innate immune system to induce a type I interferon signature. These results indicate an important role for DDI2 in the proteostasis network of cells and tissues and in the maintenance of a balanced immune response. HighlightsO_LIDDI2-deficiency in mice causes severe developmental failure and embryonic lethality at mid-late gestation C_LIO_LIDDI2-deficiency causes severe proteotoxic stress and proteasome impairment C_LIO_LIDDI2-deficiency induces the UPR and ISR signaling pathways C_LIO_LIDDI2-deficient cells survive via STAT3-dependent interferon signaling C_LI

cell biology↗

bioRxiv2019

MCC950/CRID3 potently targets the NACHT domain of wildtype NLRP3 but not disease-associated mutants for inflammasome inhibition

The NLRP3 inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndromes (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight in the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome. Here, we show that the NACHT domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labelling of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance, MCC950/CRID3 failed to inhibit NLRP3- driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1{beta} and IL-18 in LPS-challenged wildtype mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wildtype NLRP3 as the molecular target of MCC950/CRID3, and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wildtype NLRP3, but not CAPS-associated mutants.

immunology↗

bioRxiv2019

The yeast proteases Ddi1 and Wss1 are both involved in the DNA replication stress response.

Genome integrity and cell survival are dependent on proper replication stress response. Multiple repair pathways addressing obstacles generated by replication stress arose during evolution, and a detailed understanding of these processes is crucial for treatment of numerous human diseases. Here, we investigated the strong negative genetic interaction between two proteases involved in the DNA replication stress response, yeast Wss1 and Ddi1. While Wss1 proteolytically acts on DNA-protein crosslinks, mammalian DDI1 and DDI2 proteins remove RTF2 from stalled forks via a proposed proteasome shuttle hypothesis. We show that the double-deleted {Delta}ddi1, {Delta}wss1 yeast strain is hypersensitive to the replication drug hydroxyurea and that this phenotype can be complemented only by catalytically competent Ddi1 protease. Furthermore, our data show the key involvement of the helical domain preceding the Ddi1 protease domain in response to replication stress caused by hydroxyurea, offering the first suggestion of this domains biological function. Overall, our study provides a basis for a novel dual protease-based mechanism enabling yeast cells to counteract DNA replication stress.

molecular biology↗