Biology subjects

Krssakova, G.

Publications and source records attributed to Krssakova, G..

9 recordsLinked to original sources

bioRxiv2026

Emergence of histone-based chromatin complexity in Asgard archaea

The emergence of the eukaryotes coincided with the diversification of histone proteins and their post-translational modifications by enzymes that constitute the core of eukaryotic chromatin. Yet the evolutionary origins of this regulatory machinery are unknown. Here, we show that the key molecular components of histone-based chromatin regulation are present in the Asgard archaea, the closest prokaryotic relatives of eukaryotes. Asgard histones are abundant and have extended N-terminal tails rich in lysine residues that can be post-translationally modified, all of which are features shared with eukaryotic histones. In line with these findings, we identify enzymes from Asgard archaea that deposit or remove lysine acetylation on histone tails in vitro. Moreover, Asgard sirtuin deacetylases (SIR2 proteins) restore chromatin silencing in yeast, demonstrating the functional compatibility of Asgard enzymes with eukaryotic histone substrates. Our findings establish that the foundations of histone-based chromatin predate eukaryogenesis and place Asgard archaea as an evolutionary intermediate in the emergence of eukaryotic chromatin.

evolutionary biology↗

bioRxiv2026

The SPARK complex forms the molecular basis of vertebrate fertilization

Fertilization requires gamete recognition and membrane fusion, yet the molecular basis of this process in vertebrates remains unknown. Here we identify SPARK (sperm protein assembly and receptor-binding key), a conserved multi-protein complex that integrates all known sperm fertilization factors, including TMEM81-IZUMO1-SPACA6 and DCST1/2, together with two newly identified components, TMDD1 and FAM187A. SPARK subunits are mutually dependent for stability in mature sperm, and disruption of any single component causes male sterility in zebrafish and mice. Incubating zebrafish sperm with soluble egg receptor Bouncer partially rescues fertilization of Bouncer-deficient eggs in a SPARK-dependent manner, consistent with egg receptor binding priming the complex for fusion. Thus, we propose SPARK as a con-served molecular machine that couples gamete recognition to membrane fusion.

developmental biology↗

bioRxiv2025

Hierarchical assembly and functional resilience of the mammalian RNA exosome

Most eukaryotic proteins assemble into multisubunit complexes that coordinate essential cellular functions, yet the principles governing their assembly and proteostatic control remain largely undefined. Here, we systematically dissect the cellular assembly and functional organization of the RNA exosome, an essential ribonucleolytic complex, using an inducible dual-guide CRISPR/Cas9 system in mouse embryonic stem cells. We reveal a sequential assembly pathway where Exosc2, Exosc4, and Exosc7 initiate complex formation, facilitating the incorporation of barrel and cap subunits in a defined hierarchy. Unlike other structural subunits, the terminally incorporated cap subunit Exosc1 is dispensable for cell viability, revealing a modular, functionally resilient architecture. We demonstrate that orphan subunits are selectively degraded via the ubiquitin-proteasome system, enforcing stringent quality control over RNA exosome biogenesis. These findings establish a framework for decoding the assembly logic of essential macromolecular machines and uncover previously unrecognized plasticity in the composition and function of the RNA exosome.

molecular biology↗

bioRxiv2024

Urinary multi-omics reveal non-invasive diagnostic biomarkers in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the kidney malignancy with the highest incidence and mortality rates. Despite the high patient burden, there are no biomarkers for rapid diagnosis and public health surveillance. Urine would be an ideal source of ccRCC biomarkers due to the low invasiveness, easy accessibility, and the kidneys intrinsic role in filtering urine. In the present work, by combining proteomics, lipidomics and metabolomics, we detected urogenital metabolic dysregulation in ccRCC patients with increased lipid metabolism, altered mitochondrial respiration signatures and increased urinary lipid content. Importantly, we identify three early-stage diagnostic biomarkers for ccRCC in urine samples: Serum amyloid A1 (SAA1), Haptoglobin (HP) and Lipocalin 15 (LCN15). We further implemented a parallel reaction monitoring mass spectrometry protocol for rapid and sensitive detection of SAA1, HP and LCN15 and combined all three proteins into a diagnostic UrineScore. In our discovery cohort, this score had a performance accuracy of 96% in receiver operating characteristic curve (ROC) analysis for classification of ccRCC versus control cases. Our data identifies tractable and highly efficacious urinary biomarkers for ccRCC diagnosis and serve as a first step towards the development of more rapid and accessible urinary diagnostic platforms.

cancer biology↗

bioRxiv2023

A conserved fertilization complex of Izumo1, Spaca6, and Tmem81 mediates sperm-egg interaction in vertebrates

Fertilization, the fusion of sperm and egg, is essential for sexual reproduction. While several proteins have been demonstrated to be essential for the binding and fusion of gametes in vertebrates, the molecular mechanisms driving this key process are poorly understood. Here, we performed a protein interaction screen using AlphaFold-Multimer to uncover protein-protein interactions in fertilization. This screen resulted in the prediction of a trimeric complex composed of the essential fertilization factors Izumo1 and Spaca6, and Tmem81, a protein previously not implicated in fertilization. We show that Tmem81 is a conserved, testis-expressed transmembrane protein that is evolutionarily related to Izumo1 and Spaca6 and is essential for male fertility in fish and mice. Consistent with trimer formation in vivo, zebrafish izumo1-/-, spaca6-/-, and tmem81-/- mutants exhibit the same sperm-egg binding defect and show co-depletion of all three proteins in sperm. Moreover, we provide experimental evidence that Izumo1, Spaca6, and Tmem81 interact in zebrafish sperm. Strikingly, the Izumo1-Spaca6 interaction is predicted to form a cleft that serves as a binding site for Bouncer, the only identified egg protein essential for fertilization in zebrafish. Together, these results provide compelling evidence for a conserved sperm factor complex in vertebrates that forms a specific interface for the sperm-egg interaction required for successful fertilization.

developmental biology↗

bioRxiv2022

Wide Window Acquisition and AI-based data analysis to reach deep proteome coverage for a wide sample range, including single cell proteomic inputs

A comprehensive proteome map is essential to elucidate molecular pathways and protein functions. Although great improvements in sample preparation, instrumentation and data analysis already yielded impressive results, current studies suffer from a limited proteomic depth and dynamic range therefore lacking low abundant or highly hydrophobic proteins. Here, we combine and benchmark advanced micro pillar array columns ({micro}PAC) operated at nanoflow with Wide Window Acquisition (WWA) and the AI-based CHIMERYS search engine for data analysis to maximize chromatographic separation power, sensitivity and proteome coverage. Our data shows that {micro}PAC columns clearly outperform classical packed bed columns boosting peptide IDs by up to 140%. Already at classical narrow isolation widths CHIMERYS boosted ID rates by a factor of 2.6 compared to the conventional search engine MS Amanda 2.0. By combining CHIMERYS with WWA, even a 4.6-fold increase in ID rates could be achieved. Using our optimized workflow, we were further able to identify more than 10,000 proteins from a single 2 h gradient shotgun analysis. We further investigated the applicability of WWA for single cell inputs and found that the choice of the optimal isolation window width depends on sample input and complexity. Using a short 5.5 cm column and very high flow rates during loading and column equilibration we improved sample throughput to [~]100 samples per day while maintaining high protein ID numbers. We believe that this is especially important for the single cell field where throughput is one of the most limiting factors. Finally, we applied our optimized workflow on immunoprecipitations of Smarca5/SNF2H and found 32 additional interaction partners compared to the original workflow utilizing a packed bed column. These additional interaction partners include previously described interaction partners of Smarca5 like Baz2b as well as undescribed interactors including Arid1a, which is also involved in chromatin remodeling and has been described as key player in neurodevelopmental and malignant disorders.

biochemistry↗

bioRxiv2021

Quantitative accuracy and precision in multiplexed single-cell proteomics.

Single-cell proteomics workflows have considerably improved in sensitivity and reproducibility to characterize yet unknown biological phenomena. With the emergence of multiplexed single-cell proteomics, studies increasingly present single-cell measurements in conjunction with an abundant congruent carrier to improve precursor selection and enhance identifications. While these extreme carrier spikes are often >100-times more abundant than the investigated samples, undoubtedly the total ion current increases, but quantitative accuracy possibly is affected. We here focus on narrowly titrated carrier spikes (i.e., <20x) and assess their elimination for comparable sensitivity at superior accuracy. We find that subtle changes in the carrier ratio can severely impact measurement variability and describe alternative multiplexing strategies to evaluate data quality. Lastly, we demonstrate elevated replicate overlap while preserving acquisition throughput at improved quantitative accuracy with DIA-TMT and discuss optimized experimental designs for multiplexed proteomics of trace samples. This comprehensive benchmarking gives an overview of currently available techniques and guides conceptualizing the optimal single-cell proteomics experiment.

biochemistry↗

bioRxiv2021

Comparative proteome signatures of ultra-low input samples by TMT multiplexed Data-Independent Acquisition

Single cell transcriptomics has revolutionized our understanding of basic biology and disease. Since transcript levels often do not correlate with protein expression, it is crucial to complement transcriptomics approaches with proteome analyses at single cell resolution. Despite continuous technological improvements in sensitivity, mass spectrometry-based single cell proteomics ultimately faces the challenge of reproducibly comparing the protein expression profiles of thousands of individual cells. Here, we combine two hitherto opposing analytical strategies, DIA and Tandem-Mass-Tag (TMT)-multiplexing, to generate highly reproducible, quantitative proteome signatures from ultra-low input samples. While conventional, data-dependent shotgun proteomics (DDA) of ultra-low input samples critically suffers from the accumulation of missing values with increasing sample-cohort size, data-independent acquisition (DIA) strategies do usually not take full advantage of isotope-encoded sample multiplexing. We developed a novel, identification-independent proteomics data-analysis pipeline that allows to quantitatively compare DIA-TMT proteome signatures across hundreds of samples independent of their biological origin, and to identify cell types and single protein knockouts. We validate our approach using integrative data analysis of different human cell lines and standard database searches for knockouts of defined proteins. These data establish a novel and reproducible approach to markedly expand the numbers of proteins one detects from ultra-low input samples, such as single cells.

biochemistry↗

bioRxiv2019

Improved Sensitivity in Low-Input Proteomics using Micro-Pillar Array-based Chromatography

Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nano-LC-ESI-MS/MS is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples.\n\nHowever, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nano-flow HPLC, current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology.\n\nWith the recent introduction of the {micro}PAC system, which provides perfectly ordered micro-pillar array based chromatographic support materials, completely new chromatographic concepts for optimization towards the needs of ultra-sensitive proteomics become available.\n\nHere we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micro-pillar array column to a widely used nano-flow HPLC column for the proteomics analysis of 10 ng tryptic HeLa cell digest.\n\nComparative analysis of LC-MS/MS-data corroborated that micro-pillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, increase sensitivity in the analysis of low-input proteomics samples, and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional nano-flow HPLC columns.

biochemistry↗