Biology subjects

Jungwirth, J.

Publications and source records attributed to Jungwirth, J..

3 recordsLinked to original sources

bioRxiv2024

TACSTD2 expression marks the early transition to colon adenomas

This study aimed to address new molecular events occurring in precancerous stages of colorectal cancer (CRC) by integrated analysis of gene expression data and DNA methylation profiles. Whole-transcriptome sequencing analysis was performed on 16 fresh frozen colorectal adenoma and matched mucosa specimens along with validation of candidates in an independent cohort of 20 fresh frozen paired adenoma and adjacent mucosa tissues as well as eight independent public datasets (335 cases). Genome-wide methylation profiles were determined for 5 adenoma pairs and confirmed by pyrosequencing on 20 tissue pairs used for validation. Functional analysis was performed in vitro and in vivo using the inflammation-associated azoxymethane/dextran sodium sulfate (AOM/DSS) and the sporadic colorectal carcinogenesis (six AOM injections) mouse models as well as ApcMin/+ mice. Candidates were investigated by immunohistochemical staining of human adenomas and early-stage (pT1) CRC tumors. A total of 1,917 differentially expressed genes and 148,191 differentially methylated CpG sites were detected in adenomas compared with adjacent mucosa samples. Based on the transcriptome data and relevance to CRC, TACSTD2, MMP7, MMP1, CLDN2, CLDN1, and ETV4, were selected for further validation. TACSTD2 promoter hypomethylation in adenoma tissues was validated in 20 additional tissue pairs and corresponded with increased TACSTD2 expression. TACSTD2 was also overexpressed in an in vitro transformation model of human colonic epithelial cells. The TACSTD2 protein TROP2 was elevated in human adenomas, pT1 tumors, and in murine adenomas, while it was absent in the unaffected adjacent mucosa. TROP2 overexpression might trigger the development of precancerous lesions and could help identify early transformation foci in colon biopsies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/620817v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f23753org.highwire.dtl.DTLVardef@10e6abeorg.highwire.dtl.DTLVardef@181f500org.highwire.dtl.DTLVardef@fc0d38_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

From Microstates to Macrostates in the Conformational Dynamics of GroEL: a Single-Molecule FRET Study

The chaperonin GroEL is a multi-subunit molecular machine that assists in protein folding in the E. coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. However, a measurement of subunit dynamics and their relation to the allosteric cycle of GroEL has been missing. Here, we report single-molecule FRET measurements that directly probe the conformational transitions of one subunit within GroEL and its single-ring variant under equilibrium conditions. We find that four microstates span the conformational manifold of the protein and interconvert on the submillisecond time scale. A unique set of relative populations of these microstates, termed a macrostate, is obtained by varying solution conditions, e.g., adding different nucleotides or the co-chaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-liganded conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis, confirming the essential role of the observed dynamics in the function of GroEL. Significance StatementGroEL is a large protein-folding machine whose activity is accompanied by considerable conformational motions. Here, we use single-molecule FRET spectroscopy in combination with photon-by-photon statistical analysis to characterize the motions of a single GroEL subunit in real time and in the presence of ADP, ATP, and the co-chaperone GroES. Our results reveal transitions between four conformations on a timescale much faster than the functional cycle. We show that the motions of an individual subunit are directly coupled to the concerted allosteric mechanism of GroEL. This work, therefore, further demonstrates the impact of fast conformational dynamics on the biochemical function of molecular machines.

biophysics↗

bioRxiv2020

The conformational distribution of a major facilitator superfamily peptide transporter is modulated by the membrane composition

While structural biology aims at explaining the biological function of membrane proteins with their structure, it is unclear how these proteins are modulated by the complex lipid composition of membranes. Here, we address this question by mapping the conformational distribution of the bacterial oligopeptide transporter DtpA using single-molecule fluorescence spectroscopy. We show that DtpA populates ensembles of conformers that respond sensitively to the environment. Detergents trap the transporter in an inward-open ensemble in which the substrate binding site faces the cytosol. However, re-constitution in Saposin nanoparticles with different lipid compositions, reveal a plethora of alternative conformations, including a fully inward-open ensemble whose existence had not been anticipated before. The relative abundance of these ensembles depends on the lipid composition of the nanoparticles. Our results therefore demonstrate that membranes sensitively affect the structural distribution of DtpA and we expect this to be a general property of membrane proteins.

biophysics↗