Biology subjects

Mikl, M.

Publications and source records attributed to Mikl, M..

6 recordsLinked to original sources

bioRxiv2024

Activity-dependent COX-2 proteolysis generates a catalytically inactive fragment that affects aerobic respiration

Cyclooxygenase-2 (COX-2) catalyzes arachidonic acid (AA) into PGH2, the single source of all prostaglandins (PGs), ligands that activate multiple inflammatory pathways. AA catalysis quickly results in suicide inactivation, rendering the enzyme catalytically inactive. We show that the catalytic activity also leads to controlled cleavage of COX-2, an event that is differentially regulated by fatty acids, and blocked by COX inhibitors. We also observe COX-2 cleavage in human colon tumors. Using mass spectrometry, we identify two adjacent cleavage points within the catalytic domain, which give rise to COX-2 fragments that are catalytically inactive and localize to different cellular compartments. One of these fragments significantly alters the expression of mitochondrial electron transport genes and functional assays show that it leads to reduced mitochondrial function, increased lactate production, and enhanced proliferation. We propose that in addition to its role in generating PGs, COX-2 has subsequent PG-independent cellular functions that may account for the complex role of COX-2 in proliferative diseases and chronic inflammation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/588559v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@15d589corg.highwire.dtl.DTLVardef@1648e5eorg.highwire.dtl.DTLVardef@bededcorg.highwire.dtl.DTLVardef@1195684_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Polyclonal lymphoid expansion drives paraneoplastic autoimmunity in neuroblastoma

Neuroblastoma is a lethal childhood solid tumor of developing peripheral nerves. Two percent of children with neuroblastoma develop Opsoclonus Myoclonus Ataxia Syndrome (OMAS), a paraneoplastic disease characterized by cerebellar and brainstem-directed autoimmunity, but typically with outstanding cancer-related outcomes. We compared tumor transcriptomes and tumor infiltrating T- and B-cell repertoires from 38 OMAS subjects with neuroblastoma to 26 non- OMAS associated neuroblastomas. We found greater B- and T-cell infiltration in OMAS- associated tumors compared to controls, but unexpectedly showed that both were polyclonal expansions. Tertiary lymphoid structures (TLS) were enriched in OMAS-associated tumors. We identified significant enrichment of the MHC Class II allele HLA-DOB*01:01 in OMAS patients. OMAS severity scores were associated with the expression of several candidate autoimmune genes. We propose a model in which polyclonal autoreactive B lymphocytes act as antigen presenting cells and drive TLS formation, thereby crucially supporting both sustained polyclonal T-cell-mediated anti-tumor immunity and paraneoplastic OMAS neuropathology.

cancer biology↗

bioRxiv2021

Biological Factors and Self-Perception of Stress Predict Human Freeze-Like Responses in the Context of Self-Defence Training and Personal Experience with Violence

Many animals react to threatening stimuli such as a predator attacks by freezing. However, little experimental research investigated freeze response in humans. Here, we have employed practices commonly used in self-defence training to create two unique scenarios simulating armed physical attacks. Sixty healthy men volunteers divided into three groups of twenty (untrained, trained but unexperienced, trained and experienced) underwent these scenarios accompanied by measurement of biochemical, physiological, and psychological markers of stress. All participants also underwent an fMRI session during which they observed neutral and negative images from the International Affective Picture System (IAPS). Our results show that scenarios simulating physical attacks can induce a freeze-like response in men. In addition, we demonstrate that while electrodermal activity (EDA), subjective stress perception, and brain activity in fMRI predict freeze-like response in men, their effect on freeze-like response is entirely dependent on the level of training and experience of a given individual.

neuroscience↗

bioRxiv2021

A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons

Asymmetric subcellular localization of mRNA is a common cellular phenomenon that is thought to contribute to spatial gene regulation. In highly polar neurons, subcellular transcript localization and translation are thought to enhance cellular efficiency and timely responses to external cues. Although mRNA localization has been observed in many tissues and numerous examples of the functional importance of this process exist, we still lack a systematic understanding of how the transcript sorting machinery works in a sequence-specific manner. Here, we addressed these gaps by combining subcellular transcriptomics and rationally designed sequence libraries. We developed a massively parallel reporter assay (MPRA) for mRNA localization and tested ~50,000 sequences for their ability to drive RNA localization to neurites of neuronal cell lines. By scanning the 3UTR of >300 genes we identified many previously unknown localization regions and mapped the localization potential of endogenous sequences. Our data suggest two ways the localization potential can be encoded in the 3UTR: focused localization motifs and broadly encoded localization potential based on small contributions. We identified sequence motifs enriched in dendritically localized transcripts and tested the potential of these motifs to affect the localization behavior of an mRNA. This assay revealed sequence elements with the ability to bias localization towards neurite as well as soma. Depletion of RNA binding proteins predicted or experimentally shown to bind these motifs abolished the effect on localization, suggesting that these motifs act by recruiting specific RNA-binding proteins. Based on our dataset we developed machine learning models that accurately predict the localization behavior of novel sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native transcripts. Applying similar systematic high-throughput approaches to other cell types will open the door for a comparative perspective on RNA localization across tissues and reveal the commonalities and differences of this crucial regulatory mechanism.

genomics↗

bioRxiv2018

Extensive programmed ribosomal frameshifting in human as revealed by a massively parallel reporter assay

Programmed ribosomal frameshifting is the controlled slippage of the translating ribosome to an alternative frame. This tightly regulated process is widely employed by human viruses such as HIV and SARS coronavirus and is critical for their life cycle and virulence. It is also utilized from yeast to human to implement a feedback control mechanism to regulate polyamine levels. Here, we developed a high-throughput, fluorescence-based approach to assess the frameshifting potential of a sequence. We designed and tested >12,000 sequences based on 15 viral and human frameshifting events, allowing us to elucidate the rules governing ribosomal frameshifting in a systematic way and to discover novel regulatory inputs based on amino acid properties and tRNA availability. We assessed the natural variation in HIV gag-pol frameshifting rates by testing >500 clinical isolates and identified subtype-specific differences as well as associations between viral load in patients and the optimality of gag-pol frameshifting rates. We further devised computational models that accurately predict frameshifting potential (up to auROC=0.93) and frameshifting rates (up to Pearson r=0.81) of novel variants, including subtle differences between HIV clinical isolates (r=0.60). Taken together, this systematic approach can contribute to the development of antiviral agents acting on programmed ribosomal frameshifting.

genomics↗

bioRxiv2018

Dissecting splicing decisions and cell-to-cell variability with designed sequence libraries

Most human genes are alternatively spliced, allowing for a large expansion of the proteome.The multitude of regulatory inputs to splicing limits the potential to infer general principles from investigating native sequences. Here, we created a rationally designed library of >32,000 splicing events to dissect the complexity of splicing regulation through systematicsequence alterations. Measuring RNA and protein splice isoforms allowed us to investigate bothcause and effect of splicing decisions, quantify diverse regulatory inputs and accurately predict (R2=0.75-0.85) isoform ratios from sequence and secondary structure. By profiling individual cells, we measure the cell-to-cell variability of splicing decisions and show that it can be encoded in the DNA and influenced by regulatory inputs, opening the door for a novel,single-cell perspective on splicing regulation.

genomics↗