Biology subjects

Jankevics, A.

Publications and source records attributed to Jankevics, A..

8 recordsLinked to original sources

bioRxiv2025

Glutamine antagonism suppresses tumor growth in adrenocortical carcinoma through inhibition of de novo nucleotide biosynthesis

Dysregulation of cellular metabolism is a hallmark of cancer, which remains poorly understood in adrenocortical carcinoma (ACC). Here, we dissected ACC metabolism by integrating transcriptional profiling from human and mouse ACC, targeted tissue metabolomics from a mouse ACC model, and untargeted serum metabolomics from a large patient cohort, providing cross-species validation of metabolic rewiring in ACC. This study revealed global metabolic dysregulation, involving glutamine-dependent pathways such as non-essential amino-acid and hexosamine biosynthesis, nucleotide metabolism, and glutathione biosynthesis, suggesting glutamine catabolism is a critical metabolic vulnerability in ACC. Treatment with glutamine antagonists 6-Diazo-5-Oxo-L-Norleucine (DON) and JHU-083 elicited robust anti-tumor responses. Mechanistic studies revealed DONs anti-tumor effect was primarily driven by selective inhibition of glutamine-fueled de novo nucleotide biosynthesis. Additionally, DON led to DNA damage, which yielded potent synergism with inhibition of the DNA damage response pathway. Collectively, this work highlights glutamine metabolism as a central metabolic dependency and therapeutic target in ACC. HighlightsO_LIMouse and human ACC share conserved transcriptional-metabolic programs, revealing Gln metabolism as a central, targetable vulnerability. C_LIO_LITargeted tissue metabolomic analysis in a mouse model of ACC validates dysregulation in Gln-dependent metabolic pathways. C_LIO_LITargeting of Gln metabolism with JHU-083 (6-diazo-5-oxo-L-norleucine (DON) pro-drug) achieves marked inhibition of tumor growth in vivo. C_LIO_LIHigh expression of Gln-metabolizing genes mediating de novo nucleotide biosynthesis is associated with poor prognosis in ACC. C_LIO_LIDON drives nucleotide depletion and DNA damage, leading to potent synergy with inhibition of the DNA damage response. C_LIO_LIUntargeted serum metabolomic analysis in a large cohort of patients with adrenal tumors demonstrates dysregulation of Gln and nucleotide metabolism in ACC. C_LI

cancer biology↗

bioRxiv2025

An optimized SP3 sample processing workflow for in-depth and re-producible phosphoproteomics

Protein phosphorylation is a ubiquitous post-translational modification (PTM) found across the kingdoms of life, and is critical for the regulation of protein function in health & disease. Advances in high-throughput mass spectrometry have transformed our ability to interrogate the phosphoproteome. However, sample preparation methodologies optimized for phosphoproteomics have not kept pace, compromising the ability to fully exploit these technological advances. In this study, we present an optimized phosphoproteomics workflow using carboxylated SP3 magnetic beads which have simplified proteomics sample preparation. By employing a washing step with 8 M urea and omitting the conventional C18 SPE clean-up, we demonstrate a significant improvement in phosphopeptide identifications, with application of this refined protocol to HEK-293T cell extracts increasing the number nearly 2-fold compared to standard SP3 techniques (7908 cf. 4129). We also observed a substantial improvement in the detection of multiply phosphorylated peptides. Our findings suggest that the complexity of PTM crosstalk using current peptide-based proteomics workflows is currently under-represented and underscores the necessity of methodological innovations to better capture the intricacies of the phosphoproteome landscape.

biochemistry↗

bioRxiv2023

Fast and accurate disulfide bridge detection

Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multi-billion-dollar industry. Essential here is quality control assessment of critical attributes such as sequence fidelity, proper folding, and post-translational modifications (PTMs). Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two Cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis (MAAH), the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer (XlinkX/PD) to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within one hour, allowing for high-throughput, high-accuracy disulfide mapping.

biochemistry↗

bioRxiv2022

MRPS36 provides a missing link in the eukaryotic 2-oxoglutarate dehydrogenase complex for recruitment of E3 to the E2 core

The tricarboxylic acid (TCA) cycle, or Krebs cycle, is the central pathway of energy production in eukaryotic cells and plays a key part in aerobic respiration throughout all kingdoms of life. The enzymes involved in this cycle generate the reducing equivalents NADH and FADH2 by a series of enzymatic reactions, which are utilized by the electron transport chain to produce ATP. One of the pivotal enzymes in this cycle is 2-oxoglutarate dehydrogenase complex (OGDHC), which generates NADH by oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. OGDHC is a megadalton protein complex originally thought to be assembled just from three catalytically active subunits (E1o, E2o, E3). In fungi and animals, however, the protein MRPS36 has more recently been proposed as a putative additional component. Based on extensive XL-MS data obtained from measurements in mice and bovine heart mitochondria, supported by phylogenetic analyses, we provide evidence that MRPS36 is an essential member of OGDHC, albeit only in eukaryotes. Comparative sequence analysis and computational structure predictions reveal that in eukaryotic OGDHC, E2o does not contain the peripheral subunit-binding domain (PSBD), present in bacterial and archaeal E2os. We propose that in eukaryotes MRPS36 evolved as an E3 adaptor protein, functionally replacing the PSBD. We further provide a refined structural model of the complete eukaryotic OGDHC containing 16 E1o, 12 E3, and 6 subunits of MRPS36 accommodated around the OGDHC core composed of 24 E2o subunits (3.45 MDa). The model provides new insights into the OGDH complex topology and stipulates putative mechanistic implications.

biochemistry↗

bioRxiv2022

Oxonium Ion-Guided Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro

Spatial separation of ions in the gas-phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro series combines a trapped ion mobility device with a quadrupole, collision-cell and a time-of-flight analyser to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from non-modified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared to non-modified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and non-modified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. This new approach was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation, combined with the focussing of the mass spectrometer, allows for improved extraction of information from the samples, even at shorter LC gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known make-up, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

biochemistry↗

bioRxiv2022

Xlink Mapping and AnalySis (XMAS) - Smooth Integrative Modeling in ChimeraX

Crosslinking mass spectrometry (XL-MS) adds tremendous value to structural biology investigations. Its main strength lies in uncovering structural information in the form of distance constraints between neighboring amino acids of proteins, protein regions and complex samples, which are difficult to assess by other analytical techniques. However, although several approaches have been proposed, interpreting XL-MS data in a structural context has been cumbersome. ChimeraX has gained momentum as a flexible and widely used software package for the visualization of structural data, but is currently lacking functionalities for integration of experimental XL-MS data. Here, we introduce XMAS, a bundle that allows users to load results from several XL-MS search engines directly into ChimeraX and map the information onto protein structures. Besides automatically locating distance constraints on protein structures, XMAS offers the possibility to work with replicate experiments and/or different crosslinkers, and filter this data based on the number of replicates for which a given distance constraint was detected, thereby increasing the data quality. Additionally, we introduce the concept of self-links, which allows easy modeling of homo-dimeric interactions. Its core functionality is extended by the implementation of seamless connections to the HADDOCK suite to streamline otherwise time-consuming tasks in structural modeling pipelines. We demonstrate these key elements of the XMAS bundle by modeling crosslinking data obtained from human fibrin clots. The software is freely available from the ChimeraX toolshed, with an extensive user manual and example datasets.

bioinformatics↗

bioRxiv2020

DGAT1 is a lipid metabolism oncoprotein that enables cancer cells to accumulate fatty acid while avoiding lipotoxicity

ABSTRACTDysregulated cellular metabolism is a hallmark of cancer. As yet, few druggable oncoproteins directly responsible for this hallmark have been identified. Increased fatty acid acquisition allows cancer cells to meet their membrane biogenesis, ATP, and signaling needs. Excess fatty acids suppress growth factor signaling and cause oxidative stress in non-transformed cells, but surprisingly not in cancer cells. Molecules underlying this cancer adaptation may provide new drug targets. Here, we identify Diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, as a frequently up-regulated oncoprotein allowing cancer cells to tolerate excess fatty acids. DGAT1 over-expression alone induced melanoma in zebrafish melanocytes, and co-operated with oncogenic BRAF or NRAS for more rapid melanoma formation. Mechanistically, DGAT1 stimulated melanoma cell growth through sustaining mTOR kinase–S6 kinase signaling and suppressed cell death by tempering fatty acid oxidation, thereby preventing accumulation of reactive oxygen species including lipid peroxides.SIGNIFICANCE We show that DGAT1 is a bona fide oncoprotein capable of inducing melanoma formation and co-operating with other known drivers of melanoma. DGAT1 facilitates enhanced fatty acid acquisition by melanoma cells through suppressing lipototoxicity. DGAT1 is also critical for maintaining S6K activity required for melanoma cell growth.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2020

mzMLb: a future-proof raw mass spectrometry data format based on standards-compliant mzML and optimized for speed and storage requirements

With ever-increasing amounts of data produced by mass spectrometry (MS) proteomics and metabolomics, and the sheer volume of samples now analyzed, the need for a common open format possessing both file size efficiency and faster read/write speeds has become paramount to drive the next generation of data analysis pipelines. The Proteomics Standards Initiative (PSI) has established a clear and precise XML representation for data interchange, mzML, receiving substantial uptake; nevertheless, storage and file access efficiency has not been the main focus. We propose an HDF5 file format mzMLb that is optimised for both read/write speed and storage of the raw mass spectrometry data. We provide extensive validation of write speed, random read speed and storage size, demonstrating a flexible format that with or without compression is faster than all existing approaches in virtually all cases, while with compression, is comparable in size to proprietary vendor file formats. Since our approach uniquely preserves the XML encoding of the metadata, the format implicitly supports future versions of mzML and is straightforward to implement: mzMLbs design adheres to both HDF5 and NetCDF4 standard implementations, which allows it to be easily utilised by third parties due to their widespread programming language support. A reference implementation within the established ProteoWizard toolkit is provided.

bioinformatics↗