Biology subjects

Olender, T.

Publications and source records attributed to Olender, T..

26 records · Page 2Linked to original sources

bioRxiv2021

The Serine Protease HtrA2 mediates radiation-induced senescence in cancer cells

Radiation therapy can induce cellular senescence in cancer cells leading to short-term tumor growth arrest, yet increased long-term recurrence. To better understand the molecular mechanisms involved, we developed a model of radiation-induced senescence in cultured cancer cells, which exhibited a typical senescent phenotype, including upregulation of p53 and its target p21, followed by sustained reduction in cellular proliferation, changes in cell size and cytoskeleton organization, and senescence-associated beta-galactosidase activity. A functional siRNA screen using a cell death-related library identified the mitochondrial Ser protease HtrA2 as necessary for senescence development. Mass spectrometry-based proteomic profiling of the senescent cells indicated downregulation of proteins involved in cell cycle progression and DNA repair, and upregulation of proteins associated with malignancy, while irradiation with HtrA2 inhibition upregulated cell proliferation components. In search of direct HtrA2 substrates following radiation, we determined that HtrA2 cleaves the intermediate filament protein vimentin, affecting its cytoplasmic organization. Ectopic expression of active cytosolic HtrA2 resulted in similar changes to vimentin filament assembly. Thus HtrA2, contributes to several hallmarks of senescence and is involved in the cytoskeletal reorganization that accompanies radiation-induced senescence. SummaryHere the authors identify the Ser protease HtrA2 as a novel mediator of radiation-induced senescence, necessary for sustained proliferation arrest and reorganization of the vimentin filament network.

cancer biology↗

bioRxiv2020

Canonical Wnt signaling exerts bidirectional control on choroid plexus epithelial development

The choroid plexus (ChP) secretes cerebrospinal fluid and is critical for the development and function of the brain. In the telencephalon, the ChP epithelium (ChPe) arises from the Wnt-expressing cortical hem. Embryonic mouse and human ChPe both express nuclear {beta}-CATENIN, a canonical Wnt signaling pathway effector, indicating that this pathway is active during ChPe development. Point mutations in human {beta}-CATENIN result in the constitutive activation of canonical Wnt signaling. In a mouse model that recapitulates this perturbation, we report a loss of ChPe identity and an apparent transformation of the ChPe to a neuronal identity. Aspects of this phenomenon are recapitulated in human embryonic stem cell (hESC)-derived organoids. The ChPe is also disrupted when {beta}-Catenin is conditionally inactivated in the mouse. Together, our results indicate that canonical Wnt signaling is required in a precise and regulated manner for normal ChPe development in the mammalian brain.

developmental biology↗

bioRxiv2020

SMPD3-mediated extracellular vesicle biogenesis inhibits oligodendroglioma growth

Isocitrate dehydrogenase (IDH) mutant gliomas, including oligodendroglioma (IDH-O) and astrocytoma (IDH-A), have signature slow-growth rates that are poorly understood. Here, we reveal that SMPD3, a ceramide-producing sphingomyelinase implicated as a tumor suppressor gene and involved in extracellular vesicle biogenesis, suppresses IDH-mutant tumor growth via autocrine and paracrine actions. In patients with IDH-mutant gliomas, higher SMPD3 expression levels correlate with longer survival, consistent with ceramide acting as an anti-oncometabolite. SMPD3 knock-down in patient-derived IDH-O cells enhances proliferation cell-autonomously in 2D-culture and 3D-human cerebral organoids, and accelerates tumor growth in mouse orthotopic xenografts. Supporting paracrine actions, IDH-O-derived extracellular vesicles, enriched in ribosomal proteins, induce astrocytic death in vitro. Furthermore, non-neoplastic glia in IDH-O tumors proliferate abnormally yet undergo apoptosis, concomitant with the acquisition of a translation-enriched transcriptional signature by tumor-associated oligodendrocytes. SMPD3 thus suppresses IDH-mutant glioma growth cell-autonomously and phenotypically alters normal glia via extracellular vesicle biogenesis and paracrine actions.

cancer biology↗

bioRxiv2020

Identification and enrichment of SECReTE cis-acting RNA elements in the Coronaviridae and other (+) single-strand RNA viruses

cis-acting RNA motifs play a major role in regulating many aspects of RNA biology including posttranscriptional processing, nuclear export, RNA localization, translation and degradation. Here we analyzed the genomes of SARS-CoV-2 and other single-strand RNA (ssRNA) viruses for the presence of a unique cis RNA element called SECReTE. This motif consists of 10 or more consecutive triplet nucleotide repeats where a pyrimidine nucleotide (C or U) in present every third base, and which we identified in mRNAs encoding secreted proteins in bacteria, yeast, and humans. This motif facilitates mRNA localization to the endoplasmic reticulum (ER), along with the enhanced translation and secretion of translated protein. We now examined for SECReTE presence in Group IV and V RNA viruses, the former including the Coronaviridae, like SARS-CoV-2 and other positive (+)ssRNA viruses, and the latter consisting of negative (-) ssRNA viruses. Interestingly, the SARS-CoV-2 genome contains 40 SECReTE motifs at an abundance of ~1.3 SECReTEs/kilobase (kb). Moreover, all ssRNA viruses we examined contain multiple copies of this motif and appears in (+)ssRNA viruses as non-random in occurrence and independent of genome length. Importantly, (+)ssRNA viruses (e.g. Coronaviruses and Hepaciviruses), which utilize ER membranes to create double membrane vesicles to serve as viral replication centers (VRCs), contain more SECReTE motifs per kb as compared to (-)ssRNA viruses (e.g. Rabies, Mumps, and Influenza), that replicate in the nucleus or the cytoplasm, or other (+)ssRNA viruses (e.g. Enteroviruses and Flaviviruses) which employ different organellar membranes. As predicted by our earlier work, SECReTE sequences are mostly found in membranal or ER-associated/secreted proteins. Thus, we propose that SECReTE motifs could be important for the efficient translation and secretion of secreted viral proteins, as well as for VRC formation. Future studies of SECReTE function and identification of SECReTE-binding proteins could provide new drug targets to treat COVID-19 and other (+)ssRNA related diseases.

bioinformatics↗

bioRxiv2020

Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors

The connection between different tissues is vital for the development and function of any organs and systems. In the musculoskeletal system, the attachment of elastic tendons to stiff bones poses a mechanical challenge that is solved by the formation of a transitional tissue, which allows the transfer of muscle forces to the skeleton without tearing. Here, we show that tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, which is regulated by sharing regulatory elements with these cells and by Kruppel-like factors transcription factors (KLF). To uncover the molecular identity of attachment cells, we first applied high-throughput RNA sequencing to murine humeral attachment cells. The results, which were validated by in situ hybridization and single-molecule in situ hybridization, reveal that attachment cells express hundreds of chondrogenic and tenogenic genes. In search for the underlying mechanism allowing these cells to express these genes, we performed ATAC sequencing and found that attachment cells share a significant fraction of accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis further revealed transcriptional enhancer signatures for the majority of these regions. We then examined a subset of these regions using transgenic mouse enhancer reporter. Results verified the shared activity of some of these enhancers, supporting the possibility that the transcriptome of attachment cells is regulated by enhancers with shared activities in tenocytes or chondrocytes. Finally, integrative chromatin and motif analyses, as well as the transcriptome data, indicated that KLFs are regulators of attachment cells. Indeed, blocking the expression of Klf2 and Klf4 in the developing limb mesenchyme led to abnormal differentiation of attachment cells, establishing these factors as key regulators of the fate of these cells. In summary, our findings show how the molecular identity of bi-fated attachment cells enables the formation of the unique transitional tissue that connect tendon to bone. More broadly, we show how mixing the transcriptomes of two cell types through shared enhancers and a dedicated set of transcription factors can lead to the formation of a new cell fate that connects them.

developmental biology↗

bioRxiv2020

Spatio-temporal Proteomic Analysis of Stress Granule disassembly using APEX Reveals Regulation by SUMOylation and links to ALS pathogenesis

Cytoplasmic stress granules (SG) form in response to a variety of cellular stresses by phase-separation of proteins associated with non-translating mRNAs. SG provide insight into the biology of neurodegeneration, including amyotrophic lateral sclerosis (ALS) because they approximate some of the molecular conditions for nucleation of insoluble aggregates in neuropathological inclusions. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SG undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of SG temporal disassembly sequence, using multi-bait APEX proximity-proteomics. We discover 109 novel SG-proteins and characterize at proteomic resolution two biophysically distinct SG substructures. We further demonstrate that dozens of additional proteins are recruited to SG specifically during disassembly, indicating that it is a highly regulated process. The involved proteins link SG disassembly, to mitochondrial biology and the cytoskeleton. Parallel analysis with C9ORF72-associated dipeptides, which are found in patients with ALS and frontotemporal dementia, demonstrated compositional changes in SG during the course of disassembly and focused our attention on the roles SUMOylation in SG disassembly. We demonstrate that broad SUMOylation of SG-proteins is required for SG disassembly and is impaired by C9ORF72-associated dipeptides, representing an unexplored potential molecular mechanism of neurodegeneration. Altogether, out study fundamentally increases the knowledge about SG composition in human cells by dissecting the SG spatio-temporal proteomic landscape, provides an in-depth resource for future work on SG function and reveals basic and disease-relevant mechanisms of SG disassembly. HighlightsO_LIMulti bait APEX proximity labelling reveals 109 novel SG proteins and two distinct SG substructures. C_LIO_LIProteomic dissection of SG temporal disassembly under basal conditions and with a model of neurodegeneration. C_LIO_LIDisassembly-engaged proteins (DEPs) include SUMO ligases that are recruited during normal disassembly and dysregulated in ALS-like conditions. C_LIO_LIPervasive SG protein SUMOylation during SG disassembly is impaired by ALS-like conditions. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/830133v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@15899daorg.highwire.dtl.DTLVardef@1dcd399org.highwire.dtl.DTLVardef@c7ef12org.highwire.dtl.DTLVardef@20c096_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

A novel calcium-concentrating compartment drives biofilm formation and persistent infections

In nature, bacteria reside in biofilms - multicellular differentiated communities held together by extracellular matrix. In this work, we identified a novel subpopulation essential for biofilm formation - mineral-forming cells. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community. The molecular mechanisms promoting calcite scaffold formation were conserved between three distant phyla - the Gram-positive Bacillus subtilis, Gram-negative Pseudomonas aeruginosa and the actinobacterium Mycobacterium abscessus. Biofilm development of all three species was similarly impaired by inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization both significantly reduced the attachment of P. aeruginosa to the lung, as well as the subsequent damage inflicted by biofilms to lung tissues, and restored their sensitivity to antibiotics. The evolutionary conserved cellular pathway controlling the fundamental feature of biofilm development uncovered in this work offers novel druggable targets for antibiotics to combat otherwise untreatable biofilm infections.

microbiology↗

bioRxiv2018

Identification of a secretion-enhancing cis regulatory targeting element (SECReTE) involved in mRNA localization and protein synthesis

Earlier dogma states that mRNAs encoding secreted and membrane protein (mSMPs) reach the ER in a translation-dependent manner through the signal recognition particle (SRP) pathway. In this pathway, the signal sequence of the translation product is recognized by SRP and the mRNA-ribosome-nascent-chain-SRP complex is recruited to the ER via the interaction with an endoplasmic reticulum (ER)-localized SRP receptor. This model suggests that the translation product dictates the delivery of mRNAs to the ER and that the mRNA is a passive passenger. However, new evidence challenges this model and implies the existence of both translation - and SRP-independent mRNA localization to the ER, raising the possibility that mRNAs have an active role in determining their localization to the ER.\n\nBesides serving as a template for protein translation, mRNAs carry information required for other regulatory processes such as mRNA processing, translation and transcription efficiency, degradation and localization. In yeast, mRNA localization governed by cis-acting sequence elements has been characterized for asymmetrically (e.g. bud) localized mRNAs that localize to, and are transported with, cortical ER. Now, we identify a cis motif in mSMPs that targets mRNAs mainly to the nuclear ER in yeast and increases both protein synthesis and secretion. Termed SECReTE, for secretion-enhancing cis regulatory targeting element, this motif was identified by computational analysis of genes encoding secretome proteins. SECReTE consists of [≥]10 repetitive triplets enriched with pyrimidines (i.e. Cs and Us) every third base (i.e. NNY, N - any nucleotide, Y - pyrimidine), and is found particularly in mRNAs coding for cell wall proteins. To study the physiological relevance of SECReTE, we introduced synonymous mutations that either elevate or decrease its overall score in genes coding for secreted proteins, without changing the protein sequence, and examined the physiological effects in yeast. An increase in the SECReTE score elevated the synthesis and secretion of endogenous proteins while, in contrast, a reduction led to less secretion and physiological defects. Importantly, the addition of SECReTE to the 3UTR of an exogenous protein (e.g. SS-GFP) led to its increased secretion from yeast. SECReTE is present all through evolution and, thus, constitutes a novel RNA targeting motif found in both prokaryotes and eukaryotes.

cell biology↗