Biology subjects

Nitz, M.

Publications and source records attributed to Nitz, M..

9 recordsLinked to original sources

bioRxiv2026

A Metabolic Labeling Strategy for Tracking Protein Synthesis in Complex Biological Systems

Protein synthesis supports most biological processes. In the brain in particular, protein synthesis plays a critical role in physiological and pathological states. Here, we describe Tellurophene-Alkyne Cycloaddition-mediated Amino acid Tagging (TeACAT), a versatile strategy for fast, facile, and flexible tagging of newly synthesized proteins in mice. TeACAT is based on metabolic incorporation of the non-canonical amino acid TePhe into proteins by the endogenous protein synthesis machinery. Due to their high similarity, TePhe can efficiently replace canonical Phe without dietary or genetic manipulation. The subsequent bio-orthogonal reaction of TePhe with either fluorescent dyes or affinity handles enables both visualization and affinity enrichment of proteins synthesized during TePhe exposure. TeACAT is compatible with immunofluorescence for cell-type specific visualization of protein synthesis with subcellular resolution and can be used in conjunction with routine proteomics to identify and quantify newly synthesized proteins. Robust incorporation into the mouse proteome was observed on the scale of hours to days, allowing the interrogation of various biological processes. In summary, TeACAT enables the visualization and quantification of protein synthesis with minimal perturbation for biological discoveries.

molecular biology↗

bioRxiv2025

Single recipient cell tracking of tellurium-labeled extracellular vesicle proteomes (TeLEV) identifies EV-driven immunomodulation

Extracellular vesicles (EVs) mediate tumor-immune cell communication by carrying protein cargo that can immediately modulate signaling and antigen presentation. Yet mapping the uptake of primary EV proteomes by human immune cells at single-cell resolution has been constrained by a lack of labeling strategies. We show here that TeLEV, a tellurium-based metabolic mass tagging approach that incorporates L-2-tellurienylalanine (TePhe) into EV proteomes, can produce a biologically rare monoisotopic signal, which is detectable by mass cytometry, imaging mass cytometry, and nanoscale SIMS, without perturbing EV morphology, yield, or proteome composition. We applied TeLEV to label primary malignant B-cell-derived EVs (MBC-EVs) from chronic lymphocytic leukemia (CLL) patients and could follow EV uptake by distinct cell populations of healthy donor peripheral blood mononuclear cells. MBC-EV uptake occurred predominantly in cells of myeloid lineages. In direct control experiments with matched secreted proteins, a machine learning approach identified CD123, CD127, and CD25 as key markers distinguishing primary MBC-EV recipients from matched secreted protein recipient cells. Nanoscale imaging enabled localization of EV-delivered proteins within heterochromatin, whereas Te-labeled secreted proteins accumulated in the cytoplasm of recipient cells. We then generated a pan-immune EV uptake atlas by tracing the uptake of primary and cell-line EVs from nine cell lines and six donors with chronic lymphocytic leukemia into 2,977,094 recipient cells across 43 cell types and subpopulations. We found that the uptake of MBC-EVs by myeloid recipients induced monocyte-derived dendritic-cell polarization characterized by the co-expression of the interleukin-receptor triad (IL-RT: CD123, CD127, CD25) identified above. Time-resolved EV uptake analysis showed a rapid, transient expression of CD123/CD127 followed by CD25, both tightly coupled to MBC-EV uptake by myeloid cells. The intensity of IL-RT expression correlated with that of PD-L1 and BCL-2. Using different STAT degraders to bidirectionally modify the EV-induced STAT5 signal, we observed that MBC-EV uptake and IL-RT, PD-L1, and BCL-2 expression increased with STAT3 degradation and decreased with STAT5 degradation. To investigate the functional consequences of the MBC-EV-induced changes, we showed that MBC-EVs in the presence of IL-2 induced a high-CD25 immune state with low cytotoxic and high B cell proliferation. Taken together, TeLEV represents a novel tool for single-cell tracking of EV proteomes, revealing STAT5-dependent immune remodeling of recipient cells.

cancer biology↗

bioRxiv2024

Structural and functional analysis of Pseudomonas aeruginosa PelA provides insight into the modification of the Pel exopolysaccharide

A major biofilm matrix determinant of Pseudomonas aeruginosa is the partially deacetylated -1,4 linked N-acetylgalactosamine polymer, Pel. After synthesis and transport of the GalNAc polysaccharide across the inner membrane, PelA partially deacetylates and hydrolyzes Pel before its export out of the cell via PelB. While the Pel modification and export proteins are known to interact in the periplasm, it is unclear how the interaction of PelA and PelB coordinates these processes. To determine how PelA modifies the polymer, we determined its structure to 2.1 [A] and found a unique arrangement of four distinct domains. We have shown previously that the hydrolase domain exhibits endo--1,4-N-acetylgalactosaminidase activity. Characterization of the deacetylase domain revealed that PelA is the founding member of a new carbohydrate esterase family, CE#. Further, we found that the PelAB interaction enhances the deacetylation of N-acetylgalactosamine oligosaccharides. Using the PelA structure in conjunction with AlphaFold2 modelling of the PelAB complex, we propose a model wherein PelB guides Pel to the deacetylase domain of PelA and subsequently to the porin domain of PelB for export. Perturbation or loss of the PelAB interaction would result in less efficient deacetylation and potentially result in increased Pel hydrolysis. In PelA homologues across many phyla, the predicted structure and active sites are conserved, suggesting that there is a common modification mechanism in Gram-negative bacterial species that contain a functional pel operon.

microbiology↗

bioRxiv2023

The Metabolic Usage and Glycan Destinations of GlcNAz in E. coli

Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars such as N-acetylglucosamine (GlcNAc) being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAc-containing glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway, and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc, N-azidoacetylglucosamine (GlcNAz), for metabolic labelling in E. coli. The cytosolic production of UDP-GlcNAz was confirmed using fluorescence assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated, was interrogated by analyzing potential sites including: peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-{beta}-1,6-N-acetylglucosamine (PNAG), lipopolysaccharide (LPS) and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) towards UDP-GlcNAz in vitro and lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo confirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/553294v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1c4a9c9org.highwire.dtl.DTLVardef@66f71corg.highwire.dtl.DTLVardef@1e981bcorg.highwire.dtl.DTLVardef@14489b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

A new strategy for identifying polysialylated proteins reveals they are secreted from cancer cells as soluble proteins and as part of extracellular vesicles

Polysialic acid (polySia) is a long homopolymer consisting of 2,8-linked sialic acid with tightly regulated expression in humans. In healthy adults, it occurs on cell surface glycoproteins in neuronal, reproductive, and immune tissues; however, it is aberrantly present in many cancers and its overexpression correlates with significantly increased metastasis and poor prognosis. Prompted by the observation that the MCF-7 breast cancer cell line contains only intracellular polySia, we investigated the secretion of polySia from MCF-7 cells. PolySia was found predominantly on soluble proteins in MCF-7 conditioned media, but also on extracellular vesicles (EVs), secreted from the cells. Since MCF-7 cells do not express known polysialylated proteins, we developed a robust method for purifying polysialylated proteins that uses a metabolic labelling strategy to introduce a bioorthogonal functionality into polySia. Using this method we identified three previously unknown polysialylated proteins, and found that two of these proteins - AGR2 and QSOX2 - were secreted from MCF-7 cells. We confirmed that QSOX2 found in EV-depleted MCF-7 cell conditioned media was polysialylated. Herein we report the secretion of polysialic acid on both soluble and EV-associated proteins from MCF-7 cancer cells and introduce a new method to efficiently identify polysialylated proteins. These findings have exciting implications for understanding the roles of polySia in cancer progression and metastasis and for identifying new cancer biomarkers.

biochemistry↗

bioRxiv2021

Allosteric modulation of the adenosine A2A receptor by cholesterol

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A2AR, as seen through enhanced basal signaling and a small decrease in agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy suggests that this enhancement arises from an increase in the receptors active state populations and stronger G protein coupling. 19F NMR of fluorinated cholesterol analogs reveals transient and non-specific interactions with A2AR, indicating a lack of high-affinity binding sites or direct allosteric modulation. This is confirmed by computational analysis which suggests that cholesterol contacts confer a weak and possibly negative allosteric effect. The combined results suggest that the observed cholesterol allostery in A2AR is likely a result of indirect membrane effects through cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

biophysics↗

bioRxiv2020

PelX is a UDP-N-acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation

Pel is an N-acetylgalactosamine rich polysaccharide that contributes to the structure and function of Pseudomonas aeruginosa biofilms. The pelABCDEFG operon is highly conserved among diverse bacterial species, and thus Pel may be a widespread biofilm determinant. Previous annotation of pel gene clusters led us to identify an additional gene, pelX, that is found adjacent to pelABCDEFG in over 100 different bacterial species. The pelX gene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily of enzymes, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have used Pseudomonas protegens Pf-5 as a model to understand PelX function as P. aeruginosa lacks a pelX homologue in its pel gene cluster. We find that P. protegens forms Pel-dependent biofilms, however, despite expression of pelX under these conditions, biofilm formation was unaffected in a {Delta}pelX strain. This observation led to our identification of the pelX paralogue, PFL_5533, which we designate pgnE, that appears to be functionally redundant to pelX. In line with this, a {Delta}pelX {Delta}pgnE double mutant was substantially impaired in its ability to form Pel-dependent biofilms. To understand the molecular basis for this observation, we determined the structure of PelX to 2.1[A] resolution. The structure revealed that PelX resembles UDP-N-acetylglucosamine (UDP-GlcNAc) C4-epimerases and, using 1H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Taken together, our results demonstrate that Pel-dependent biofilm formation requires a UDP-GlcNAc C4-epimerase that generates the UDP-GalNAc precursors required by the Pel synthase machinery for polymer production.

microbiology↗

bioRxiv2019

Single-Cell Signalling Analysis of Heterocellular Organoids

Organoids are powerful biomimetic tissue models. Despite their widespread adoption, methods to analyse cell-type specific post-translational modification (PTM) signalling networks in organoids are absent. Here we report multivariate single-cell analysis of cell-type specific signalling networks in organoids and organoid co-cultures. Simultaneous measurement of 28 PTMs in >1 million single small intestinal organoid cells by mass cytometry reveals cell-type and cell-state specific signalling networks in stem, Paneth, enteroendocrine, tuft, goblet cells, and enterocytes. Integrating single-cell PTM analysis with Thiol-reactive Organoid Barcoding in situ (TOBis) enables high-throughput comparison of signalling networks between organoid cultures. Multivariate cell-type specific PTM analysis of colorectal cancer tumour microenvironment organoids reveals that shApc, KrasG12D, and Trp53R172H cell-autonomously mimic signalling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type specific signalling analysis of healthy and cancerous organoids.

cell biology↗

bioRxiv2019

Methods for Analyzing Imaging Mass Cytometry Data with Tellurium Probes

Imaging mass cytometry (IMC) is a technique allowing visualization and quantification of over 40 biological parameters in a single experiment with subcellular spatial resolution, however most IMC experiments are limited to endpoint analysis with antibodies and DNA stains. Small molecules containing tellurium are promising probes for IMC due to their cell permeability, synthetic versatility, and most importantly their application to sequential labelling with isotopologous probes (SLIP) experiments. SLIP experiments with tellurium-containing probes allow quantification of intracellular biology at multiple timepoints with IMC. Despite the promise of tellurium in IMC, there are unique challenges in image processing associated with tellurium IMC data. Here, we address some of these issues by demonstrating the removal of xenon background signal, combining channels to improve signal-to-noise ratio, and calculating isotope transmission efficiency biases. These developments add accuracy to the unique temporal resolution afforded by tellurium IMC probes.

bioinformatics↗