Martin Hubálek

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

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Jiří Stulík

University of Defence

Lenka Hernychová

Masaryk Memorial Cancer Institute

Libor Krásný

Czech Academy of Sciences

HŠ

Hana Šanderová

Czech Academy of Sciences

Josef Cvačka

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

Jiří Pospíšil

Czech Academy of Sciences

MŠ

Michaela Šiková

Czech Academy of Sciences, Institute of Microbiology

Alena Křenková

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

Juraj Lenčo

University of Hradec Králové

Oldřích Benada

Czech Academy of Sciences, Institute of Microbiology

Cooperative Institutions

Czech Academy of Sciences

186

Charles University

156

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

141

Czech Academy of Sciences, Institute of Microbiology

University of Chemistry and Technology, Prague

Czech Academy of Sciences, Institute of Molecular Genetics

Masaryk University

Czech Academy of Sciences, Institute of Biotechnology

University of Defence

Centre National de la Recherche Scientifique

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PRMT5: A novel regulator of Hepatitis B virus replication and an arginine methylase of HBV core

PLoS ONE (2017)

Barbora Lubyová Jan Hodek Aleš Zábranský Hana Prouzova Martin Hubálek

In mammals, protein arginine methyltransferase 5, PRMT5, is the main type II enzyme responsible for the majority of symmetric dimethylarginine formation in polypeptides. Recent study reported that PRMT5 restricts Hepatitis B virus (HBV) replication through epigenetic repression of HBV DNA transcription and interference with encapsidation of pregenomic RNA. Here we demonstrate that PRMT5 interacts with the HBV core (HBc) protein and dimethylates arginine residues within the arginine-rich domain (ARD) of the carboxyl-terminus. ARD consists of four arginine rich subdomains, ARDI, ARDII, ARDIII and ARDIV. Mutation analysis of ARDs revealed that arginine methylation of HBc required the wild-type status of both ARDI and ARDII. Mass spectrometry analysis of HBc identified multiple potential ubiquitination, methylation and phosphorylation sites, out of which lysine K7 and arginines R150 (within ARDI) and R156 (outside ARDs) were shown to be modified by ubiquitination and methylation, respectively. The HBc symmetric dimethylation appeared to be linked to serine phosphorylation and nuclear import of HBc protein. Conversely, the monomethylated HBc retained in the cytoplasm. Thus, overexpression of PRMT5 led to increased nuclear accumulation of HBc, and vice versa, down-regulation of PRMT5 resulted in reduced levels of HBc in nuclei of transfected cells. In summary, we identified PRMT5 as a potent controller of HBc cell trafficking and function and described two novel types of HBc post-translational modifications (PTMs), arginine methylation and ubiquitination.

cccDNA

10.1371/journal.pone.0186982

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Citations (46)

Data and statistical analysis for: Bacterial nanotubes are a manifestation of cell death

Zenodo (CERN European Organization for Nuclear Research) (2020)

Jiří Pospíšil Dragana Vítkovská Olga Kofroňová Katarı́na Muchová Hana Šanderová

Contains all data and code to reproduce the statistical analysis in the Supplementary file 2 for the paper "Bacterial nanotubes are a manifestation of cell death" to be published in Nature Communications. Contents: statistical_analysis.Rmd is the main document written in R Markdown statistical_analysis.html is a compiled version of statistical_analysis.Rmd showing all the computed results. The .xlsx files contain raw data used for the analysis - their names indiciate the figure they refer to. See the main file for code that can read the data. The code can also be accessed at https://github.com/cas-bioinf/nanotubes-death

Statistical Analysis

10.5281/zenodo.3988112

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Glyoxal‐Linked Nucleotides and DNA for Bioconjugations and Crosslinking with Arginine‐Containing Peptides and Proteins

Chemistry - A European Journal (2022)

Denise‐Liu' Leone Radek Pohl Martin Hubálek Marta Kadeřábková Matouš Krömer

Glyoxal-linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of 4-azidophenylglyoxal or a Sonogashira reaction of 4-bromophenylglyoxal with 5-ethynyl-dUMP or -dUTP. The triphosphates were used as substrates for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The glyoxal-linked nucleotides reacted with arginine-containing peptides to form stable imizadolone-linked conjugates. This reactive glyoxal modification in DNA was used for efficient bioconjugations and crosslinking with Arg-containing peptides or proteins (e. g., histones) and was found to be more reactive than previously reported 1,3-diketone-linked DNA probes.

Glyoxal

Deoxyribonucleosides

Sonogashira coupling

10.1002/chem.202104208

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Citations (13)

MoaB2, a newly identified transcription factor, binds to σ ^A in Mycobacterium smegmatis

Journal of Bacteriology (2024)

Barbora Brezovská Subhash Narasimhan Michaela Šiková Hana Šanderová T. Kovaĺ

ABSTRACT In mycobacteria, σ A is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σ A . The search revealed a number of proteins; prominent among them was MoaB2. The σ A -MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors ( e.g. , closely related σ B ) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σ A was identified to play a role in the σ A -MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σ A -dependent (but not σ B -dependent) transcription and may increase the stability of σ A in the cell. We propose that MoaB2, by sequestering σ A , has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σ A , paving the way for future investigation of this phenomenon. IMPORTANCE Mycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σ A , the primary sigma factor, and characterize its effects on transcription and σ A stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.

Mycobacterium smegmatis

Sigma factor

Transcription

General transcription factor

Bacterial transcription

RNA polymerase II

10.1128/jb.00066-24

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A myristoyl switch at the plasma membrane triggers cleavage and oligomerization of Mason-Pfizer monkey virus matrix protein

eLife (2024)

Markéta Častorálová Jakub Sýs Jan Prchal Anna Pavlů Lucie Prokopová

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

Cleavage (geology)

Budding

C-terminus

10.7554/elife.93489

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Citations (3)

Analysis ofFrancisella TularensisAcetonitrile Extracts

Lenka Hernychová Martin Hubálek Jana Udržalová

This chapter contains sections titled: Introduction Material and Methods Results Conclusions Acknowledgments References

Tularemia

10.1002/9783527638192.ch8

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Bacterial nanotubes as a manifestation of cell death

Nature Communications (2020)

Jiří Pospíšil Dragana Vítovská Olga Kofroňová Katarı́na Muchová Hana Šanderová

Abstract Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis . We show that nanotube formation is associated with stress conditions, and is highly sensitive to the cells’ genetic background, growth phase, and sample preparation methods. Remarkably, nanotubes appear to be extruded exclusively from dying cells, likely as a result of biophysical forces. Their emergence is extremely fast, occurring within seconds by cannibalizing the cell membrane. Subsequent experiments reveal that cell-to-cell transfer of non-conjugative plasmids depends strictly on the competence system of the cell, and not on nanotube formation. Our study thus supports the notion that bacterial nanotubes are a post mortem phenomenon involved in cell disintegration, and are unlikely to be involved in cytoplasmic content exchange between live cells.

Cell envelope

10.1038/s41467-020-18800-2

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Citations (41)

1,3‐Diketone‐Modified Nucleotides and DNA for Cross‐Linking with Arginine‐Containing Peptides and Proteins

Angewandte Chemie (2021)

Denise‐Liu' Leone Martin Hubálek Radek Pohl Veronika Sýkorová Michal Hocek

Abstract Linear or branched 1,3‐diketone‐linked thymidine 5′‐ O ‐mono‐ and triphosphate were synthesized through CuAAC click reaction of diketone‐alkynes with 5‐azidomethyl‐dUMP or ‐dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5‐dioxohexyl group (HDO) efficiently reacted with arginine‐containing peptides to form stable pyrimidine‐linked conjugates, whereas the branched 2‐acetyl‐3‐oxo‐butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross‐linking with Arg‐containing peptides or proteins (e.g. histones).

Protecting group

Primer (cosmetics)

Thymidine

Thymine

10.1002/ange.202105126

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Citations (5)

Francisella tularensis live vaccine strain: Proteomic analysis of membrane proteins enriched fraction

PROTEOMICS (2005)

Ivona Pávková Martin Hubálek Jana Zechovská Juraj Lenčo Jiří Stulík

Abstract Proteome analysis of Gram‐negative facultative intracellular pathogen Francisella tularensis ( F. tularensis ) live vaccine strain has been performed only on whole‐cell extracts so far. This is the first study dealing with the analysis of the membrane subproteome of this microorganism. A fraction enriched in membrane proteins obtained by carbonate extraction was separated using two‐dimensional electrophoresis and all visualized spots were identified by mass spectrometry. The reference map is the basis for further comparative analyses of virulent and non‐virulent F. tularensis strains.

Proteome

Francisella

Strain (injury)

10.1002/pmic.200401213

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Citations (31)

The matrix‐assisted laser desorption/ionisation in‐source decay of peptides using ion mobility enabled quadrupole‐time‐of‐flight mass spectrometry

Rapid Communications in Mass Spectrometry (2018)

Vladimír Vrkoslav Alexander Muck Jeffery M. Brown Martin Hubálek Josef Cvačka

Rationale In‐source decay (ISD) matrix‐assisted laser desorption/ionisation (MALDI) mass spectrometry with a 1,5‐diaminonaphthalene (1,5‐DAN) matrix is used for the structural characterisation of peptides. However, MALDI spectra are intrinsically complicated by the presence of matrix ions, which interfere with the peptide fragments. This may cause false‐positive results or reduced sequence coverage. This paper reports investigations of ISD processes in an intermediate pressure MALDI ion source and a protocol for the removal of interfering ions using ion mobility separation (IMS). Methods An intermediate pressure MALDI source of a Q‐IMS‐Q‐TOF instrument (Synapt G2) has been employed for the ISD of selected peptides using a 1,5‐DAN matrix. Results Successful coupling of the MALDI source tuned for ISD experiments using IMS is demonstrated. The IMS made it possible to remove interfering matrix ions effectively from the spectra and thus to increase the confidence of spectral interpretation. Extensive fragment series corresponding to N–C α bond cleavages were observed under optimised conditions; on the other hand, weaker series of ions caused by peptide bond cleavages were prevalent for default conditions and/or the α‐hydroxycinnamic acid matrix. Conclusions Ion mobility has been used for the elimination of matrix ions. The technique has been applied to top‐down sequencing of non‐tryptic peptides, such as the human palmitoylated analogue of prolactin‐releasing peptide used in recent obesity studies, and human and insect antimicrobial peptides.

Matrix (chemical analysis)

Ion-mobility spectrometry

MALDI imaging

10.1002/rcm.8284

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Citations (3)