Biology subjects

Salokas, K.

Publications and source records attributed to Salokas, K..

8 recordsLinked to original sources

bioRxiv2026

A novel method for obtaining epileptic brain tissue for omic analyses using electrodes from a clinical stereoelectroencephalography study

Abstract Purpose: Developing a valid and practical method for obtaining brain tissue samples for transcriptomic and proteomic analyses using tissue adhered to stereoelectroencephalography (SEEG) electrodes, to enable investigation of the molecular mechanisms underlying chronic epilepsy. Method: Brain tissue samples adhered to SEEG electrodes were collected from six patients with drug-resistant epilepsy and preprocessed in a hospital environment after electrode removal. RNA extraction was initiated immediately, and proteomics samples were snap-frozen until subsequent analysis. The samples were categorized into three groups according to their electrophysiological profile: epileptogenic zone, propagation zone, and least-involved zone. The omic findings between these zones and anatomical brain areas were compared. Results: High-quality RNA and protein samples were obtained from tissue adhered to SEEG electrodes. Neuron- and brain-specific gene expression patterns and proteins were identified. Signs of activation of inflammatory mechanisms were most pronounced in the epileptogenic zone. Transcriptomic and proteomic findings demonstrated concordance. Conclusion: SEEG electrodes are a useful source for obtaining brain tissue for molecular characterization of chronic epilepsy. This method will enable the identification of shared and distinct molecular mechanisms in patients with varying etiologies of epilepsy.

neuroscience↗

bioRxiv2026

Efficient NK cell transduction with VSV-G-pseudotyped lentiviral vectors

The need for safe, allogeneic cell therapies for cancer is driving a growing interest in CAR-NK-based therapies, which, unlike CAR-T cell therapies, offer the potential for off-the-shelf administration. Lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) are commonly used for genetic modification of cell therapy products. Their use in NK cells, however, is limited by low transduction efficiency. This study explores the complexities of NK cell transduction using lentiviral vectors pseudotyped with VSV-G. We demonstrate that efficient transduction depends on multiple factors such as NK cell activation, construct design, lentivirus pseudotype selection, and the use of transduction enhancers. By optimizing these elements, we achieved effective transduction, facilitating the use of VSV-G-pseudotyped LVs for therapeutic NK cell production. Our optimized workflow comprises NK cell activation with interleukins, followed by transduction with a NK cell-specific CAR construct using VSV-G-pseudotyped LVs in the presence of BX795 and Retronectin, resulting in excellent transduction efficiency without compromising NK cell phenotype or growth. This allows for the use of a widely used gene transfer vector with an excellent safety record for producing therapeutic NK cell products.

immunology↗

bioRxiv2025

The non-catalytic ϵ DNA polymerase subunit is an NPF motif recognition protein

Short linear motifs (SLiMs) in disordered protein regions direct numerous protein-protein interactions, yet most remain uncharacterized. The Asn-Pro-Phe (NPF) motif is a well-known EH-domain ligand implicated in endocytosis, but here we reveal that the non-catalytic subunit of human DNA polymerase {varepsilon} (POLE2) also serves as a general NPF-motif receptor. Using a quantitative "native holdup" assay, we find that POLE2 selectively binds diverse NPF-containing peptides, including canonical EH-domain ligands (e.g., SYNJ1) and previously uncharacterized motifs. Biochemical measurements and mutational analysis show that NPF motifs interact with a shallow pocket near the POLE2 C-terminus, and AlphaFold predictions confirm key roles for Y513, E520, and S522 in motif coordination. Proteome-scale affinity screens identify NPF-containing nuclear proteins (e.g., WDHD1, DONSON, TTF2) that bind POLE2 with micromolar affinities, and their motif mutations abolish binding in cell extracts. Although POLE2 primarily tethers the catalytic POLE subunit to replication forks, these results indicate that it can also recruit various NPF-bearing partners involved in replication, DNA repair, and transcription regulation. Notably, NPF motifs optimized for EH-domain binding can still associate with POLE2, highlighting the inherent degeneracy of SLiM-mediated networks. Overall, these findings establish POLE2 as a central hub linking replication with other processes via broad NPF-motif recognition.

biochemistry↗

bioRxiv2024

Human skeletal muscle possesses both reversible proteomic signatures and a retained proteomic memory after repeated resistance training

Investigating repeated resistance training separated by a training break enables exploration of the potential for a proteomic memory of resistance training (RT)-induced skeletal muscle growth. Our aim was to examine skeletal muscle proteome response to 10-week RT (RT1) followed by 10-week training cessation (i.e. detraining, DT), and finally, 10-week retraining (RT2). Thirty healthy, untrained participants conducted either periodic RT (RT1-DT-RT2, n=17) or a 10-week no-training control period (n=13) followed by 20 weeks of RT (n=11). RT included twice-weekly supervised whole-body RT sessions, and resting vastus lateralis biopsies were obtained every ten weeks for proteomics analysis using high-end DIA-PASEFs mass spectrometry. The first RT period altered 150 proteins (93% increased) involved in e.g. energy metabolism and protein processing compared with minor changes during the no-training control period. The proteome adaptations were similar after the second RT compared to baseline demonstrating reproducibility in proteome adaptations to RT. Many of the proteins induced by RT1 were reversed towards baseline after detraining and increased again after retraining. These reversible proteins were especially involved in aerobic energy metabolism. Interestingly, several proteins increased after RT1 remain elevated after detraining, including carbonyl reductase 1 (CBR1) and proteins involved in muscle contraction, cytoskeleton and calcium-binding. Amongst the latter, calcium-activated protease calpain-2 (CAPN2) has been recently identified as an epigenetic muscle memory gene. We show that resistance training evokes retained protein levels even after 2.5 months of no training. This is the first study to demonstrate a potential proteomic memory of resistance training-induced muscle growth in human skeletal muscle. Key pointsO_LIRepeated resistance training in humans separated by a training break (i.e. detraining) enables the identification of temporal protein signatures over the training, detraining, and retraining periods as well as studying reproducibility of protein changes to resistance training. C_LIO_LIMuscle proteome adaptations were similar after a second period of resistance training when compared to baseline, demonstrating reproducibility in proteome adaptations to earlier resistance training. C_LIO_LIMany of the proteins induced by resistance training were reversed towards baseline after detraining and increased again after retraining. These reversible proteins were especially involved in aerobic energy metabolism. C_LIO_LISeveral proteins increased after resistance training remain elevated after detraining, including carbonyl reductase 1 (CBR1) and calcium-binding proteins such as calpain-2 (CAPN2), a recently identified epigenetic muscle memory gene. C_LIO_LIHuman skeletal muscle experiences retained protein changes following resistance training persisting over two months demonstrating a potential proteomic memory of resistance training-induced muscle growth. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/624068v3_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1f0ae2corg.highwire.dtl.DTLVardef@3af3b4org.highwire.dtl.DTLVardef@55d064org.highwire.dtl.DTLVardef@1e46bf9_HPS_FORMAT_FIGEXP M_FIG Human skeletal muscle proteome response was investigated after 10-week resistance training (RT1) followed by 10-week training cessation (i.e. detraining, DT), and finally, 10-week retraining (RT2). Many of the proteins were reversed towards baseline after DT and increased again after RT2. These reversible proteins were especially involved in aerobic energy metabolism. However, several RT-induced proteins remain elevated after DT, including carbonyl reductase 1 (CBR1) and many proteins involved in muscle contraction or cytoskeleton and calcium-binding. Amongst the latter, calcium-activated protease calpain-2 (CAPN2) is a recently identified epigenetic muscle memory gene. This study shows that resistance training evokes retained protein levels even after 2.5 months of no training and demonstrates a potential proteomic memory of RT-induced muscle growth in human skeletal muscle. Created in BioRender.com. C_FIG

physiology↗

bioRxiv2024

FINCA disease mouse model exhibits altered behaviour and immune response

Fibrosis, neurodegeneration and cerebral angiomatosis (FINCA) is a childhood-onset multi-organ neurodevelopmental disorder associated with multi-organ manifestations and recurrent infections. The disease is caused by variants in NHLRC2 initiating a cascade of unknown pathological events. Previously, we have demonstrated that despite the significant decrease at the molecular level, the compound heterozygosity of knock out and p.Asp148Tyr alleles in NHLRC2 does not lead to a severe phenotype in mice. Here, we analysed the behavioural and immunological phenotype of the FINCA mice and studied the molecular pathways affected by p.Asp148Tyr in NHLRC2 using mouse and human-derived cell culture models. The FINCA mice displayed a mild hyperactivity and deficient early immune response when challenged with LPS leading to altered cytokine responses, including IFN{gamma}, IL-12, and TNF. By comparing gene expression and putative interaction partners affected by p.Asp148Tyr, we identified Rho GTPase signalling as the common pathway. Altogether, these results establish a multi-dimensional impact of the p.Asp148Tyr variant in NHLRC2. Knowledge of the molecular pathways affected by NHLRC2 and the natural course of FINCA disease progression are instrumental for the development of effective therapeutics. Summary statementFINCA is a paediatric neurodevelopmental and multi-organ disorder caused by variants in NHLRC2. Here, mild hyperactivity in connection with altered early immune response is described in the FINCA mouse model.

neuroscience↗

bioRxiv2021

Physical and functional interactome atlas of human receptor tyrosine kinases

Much cell-to-cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we used three complementary methods to map the molecular context and substrate profiles of RTKs. We used affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK-protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also used kinase-deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well-studied RTKs, offers insights into the functions of less well-studied RTKs, and highlights RTK-RTK interactions and shared signaling pathways.

systems biology↗

bioRxiv2020

Human Transcription Factor and Protein Kinase Gene Fusions in Human Cancer

Oncogenic gene fusions are estimated to account for up-to 20 % of cancer morbidity. Originally, oncofusions were identified in blood cancer, but recently multiple sequence-level studies of cancer genomes have established oncofusions throughout all tissue types. However, the functional implications of the identified oncofusions have often not been investigated. In this study, the identified oncofusions from a fusion detection approach (DEEPEST) were analyzed in more detail. In total, DEEPEST contains 28863 unique fusions. From sequence analysis, we found that almost 30% of them (8225) are expected to produce functional fusion proteins with features from both parent genes. Kinases and transcription factors were found to be the main gene families of the protein producing fusions. Considering their role as initiators, actors, and termination points of cellular signaling pathways, we focused our in-depth analyses on them. The domain architecture of the fusions, as well as of their expected interactors, suggests that abnormal molecular context of intact protein domains brought about by fusion events may unlock the oncogenic potential of the wild type counterparts of the fusion proteins. To understand overall effects of oncofusions on cellular signaling, we performed differential expression analysis using TCGA cancer project samples. Results indicated oncofusion-specific alterations in expression levels of individual genes, and overall lowering of the expression levels of key cellular pathways, such as signal transduction, proteolysis, microtubule cytoskeleton organization, and in particular regulation of transcription. The sum of our results suggests that kinase and transcription factor oncofusions globally deregulate cellular signaling, possibly via acquiring novel functions.

bioinformatics↗

bioRxiv2018

An integrated approach to comprehensively map the molecular context of proteins

Protein-protein interactions underlie almost all cellular functions. The comprehensive mapping of these complex cellular networks of stable and transient associations has been made available by affinity purification mass spectrometry (AP-MS) and more recently by proximity based labelling methods such as BioID. Due the advancements in both methods and MS instrumentation, an in-depth analysis of the whole human proteome is at grasps. In order to facilitate this, we designed and optimized an integrated approach utilizing MAC-tag combining both AP-MS and BioID in a single construct. We systematically applied this approach to 18 subcellular localization markers and generated a molecular context database, which can be used to define molecular locations for any protein of interest. In addition, we show that by combining the AP-MS and BioID results we can also obtain interaction distances within a complex. Taken together, our combined strategy offers comprehensive approach for mapping physical and functional protein interactions.

systems biology↗