Biology subjects

Taatjes, D. J.

Publications and source records attributed to Taatjes, D. J..

17 recordsLinked to original sources

bioRxiv2025

Porosome reconstitution reverses Alzheimers in human brain organoids

A central challenge in overcoming diseases resulting from defects in the cellular nanoscale multiprotein porosome complexes, is the consequent alteration of other proteins within the complex. By exploiting the distinct porosomes present in different tissues, Porosome Therapeutics, Inc., has developed a clinically relevant approach to incorporate (reconstitute) functional wild type porosomes into the plasma membrane of diseased cells to overcome secretory defects. In a combinatorial strategy of reprogramming the neuronal secretory and metabolic components, we established a dual-target therapeutic framework that repairs synaptic and metabolic defects to counteract neurodegeneration in Alzheimers. Utilizing iPSC derived human brain organoids, as recommended by the NIH and FDA, we demonstrate both the morphological and functional reversal of Alzheimers following porosome-apigenin treatment. Using the UHD-CMOS-MEA System developed jointly by Sony Semiconductor Solutions (Sony), SCREEN Holdings (SCREEN) and VitroVo, we were able to monitor thousands of neuronal firing in the brain organoids, helping in assessing the restoration of neuronal function in Alzheimers following porosome-apigenin therapy.

neuroscience↗

bioRxiv2025

Extracellular Vesicles Derived from Mesenchymal Stromal Cells Reduce Pseudomonas aeruginosa Lung Infection and Inflammation in Mice

The World Health Organization and the U.S. Centers for Disease Control and Prevention have reported that antibiotic resistant infections with Pseudomonas aeruginosa present a significant health risk world-wide. In the genetic disease Cystic Fibrosis (CF), chronic antibiotic resistant Pseudomonas lung infections and persistent inflammation remain the leading causes of mortality. While highly effective modulator therapy (HEMT) dramatically improves lung function in CF, they fail to eradicate chronic infections or eliminate the associated hyperinflammatory state. Thus, there is an urgent need for innovative therapies that can simultaneously eliminate antibiotic resistant P. aeruginosa lung infection and the attendant hyperinflammatory lung environment. Mesenchymal stromal cell-derived extracellular particles (MSC EPs) represent a promising solution, offering potent anti-inflammatory and antimicrobial properties while being safe and non-toxic. This study demonstrates using a CF mouse model of infection that MSC EPs reduce acute P. aeruginosa lung infection and inflammation. As the first investigation of MSC EPs in CF mice, this research underscores the dual effects of MSC EPs; reducing inflammation and bacterial burden. These findings mark an important advancement in antimicrobial therapy, addressing the unmet need for reducing antibiotic resistant infections and hyperinflammation in CF as well as other diseases with chronic, antibiotic resistant P. aeruginosa infections.

microbiology↗

bioRxiv2025

Multi-omics and biochemical reconstitution reveal CDK7-dependent mechanisms controlling RNA polymerase II function at gene 5'- and 3'-ends

CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases precluded CDK7 analysis with nuclear extracts, we completed biochemical assays with purified factors. Reconstitution of RNAPII transcription initiation showed CDK7 inhibition slowed and/or paused RNAPII promoter-proximal transcription, which reduced re-initiation. These CDK7-regulatory functions were Mediator- and TFIID-dependent. Similarly in human cells, CDK7 inhibition reduced transcription by suppressing RNAPII activity at promoters, consistent with reduced initiation and/or re-initiation. Moreover, widespread 3-end readthrough transcription was observed in CDK7-inhibited cells; mechanistically, this occurred through rapid nuclear depletion of RNAPII elongation and termination factors, including high-confidence CDK7 targets. Collectively, these results define how CDK7 governs RNAPII function at gene 5-ends and 3-ends, and reveal that nuclear abundance of elongation and termination factors is kinase-dependent. Because 3-readthrough transcription is commonly induced during stress, our results further suggest regulated suppression of CDK7 activity may enable this RNAPII transcriptional response.

biochemistry↗

bioRxiv2025

Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters

RNA polymerase II (RNAPII) is regulated by sequence-specific transcription factors (TFs) and the pre-initiation complex (PIC): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator. TFs and Mediator contain intrinsically-disordered regions (IDRs) and form phase-separated condensates, but how IDRs control RNAPII function remains poorly understood. Using purified PIC factors, we developed a Real-time In-vitro Fluorescence Transcription assay (RIFT) for second-by-second visualization of RNAPII transcription at hundreds of promoters simultaneously. We show rapid RNAPII activation is IDR-dependent, without condensate formation. For example, the MED1-IDR can functionally replace a native TF, activating RNAPII with similar (not identical) kinetics; however, MED1-IDR squelches transcription as a condensate, but activates as a single-protein. TFs and Mediator cooperatively activate RNAPII bursting and re-initiation and surprisingly, Mediator can drive TF-promoter recruitment, without TF-DNA binding. Collectively, RIFT addressed questions largely intractable with cell-based methods, yielding mechanistic insights about IDRs, condensates, enhancer-promoter communication, and RNAPII bursting that complement live-cell imaging data.

biochemistry↗

bioRxiv2024

Porosome reconstitution therapy: A biologic rescue from cystic fibrosis

Cystic fibrosis (CF) is a genetic disorder resulting from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene that codes for a chloride transporting channel at the cell plasma membrane. In CF, highly viscous mucus is secreted in the airways preventing its clearance, leading to lung infections and respiratory failure. A major challenge in treating CF patients has been the presence of more than 2,000 different CFTR mutations or due to the absence of CFTR expression. CFTR is among the 34 major proteins composing the 100 nm porosome secretory machinery in the human airway epithelia, involved in mucin secretion. The airways is coated with a thin film of mucus, composed primarily of mucin MUC5AC and MUC5B. Sputum from patients with CF show a >70% decrease in MUC5B and MUC5AC secretion. Our studies using differentiated 3D cultures of human airway epithelial cell line, also demonstrate loss of both chloride and mucus secretion following exposure to CFTR inhibitors thiazolidinone 172 or the hydrazide GlyH101. Our studies show that human bronchial epithelial (HBE) cells with {Delta}F508 CFTR mutation, affects nearly a dozen porosome proteins including CFTR. Therefore, we hypothesized that the introduction of normal functional porosomes carrying wild type CFTR into the cell plasma membrane of CF cells would rescue from all forms of CF. Air liquid interface (ALI) 3D differentiated HBE WT-CFTR cells and {Delta}F508-CFTR CF HBE cell cultures mimicking normal lung physiology, responding to CFTR inhibitors and CF corrector and modulator drugs Tezacaftor, Ivacaftor and TRIKAFTA, was used in the study. Introduction of functional porosome complexes obtained from WT-CFTR HBE cells into the plasma membrane (PM) of {Delta}F508-CFTR CF cells, was demonstrated by an increase in PM-associated CFTR using Magnify expansion microscopy. Mucin secretion assays demonstrate porosome reconstitution to restore mucin secretion more than twice as effectively as TRIKAFTA. These results are further supported by preliminary nasal potential different studies in {Delta}F508 mice, where treatment of the nasal passage with porosome isolates from WT-CFTR HBE cells, restore chloride secretion in the nasal passage of mice, a further validation of the highly effective porosome reconstitution therapy for CF.

cell biology↗

bioRxiv2024

A transcription factor (TF) inference method that broadly measures TF activity and identifies mechanistically distinct TF networks

TF profiler is a method of inferring transcription factor regulatory activity, i.e. when a TF is present and actively regulating transcription, directly directly from nascent sequencing assays such as PRO-seq and GRO-seq. Transcription factors orchestrate transcription and play a critical role in cellular maintenance, identity and response to external stimuli. While ChIP assays have measured DNA localization, they fall short of identifying when and where transcription factors are actively regulating transcription. Our method, on the other hand, uses RNA polymerase activity to infer TF activity across hundreds of data sets and transcription factors. Based on these classifications we identify three distinct classes of transcription factors: ubiquitous factors that play roles in cellular homeostasis, driving basal gene programs across tissues and cell types, tissue specific factors that act almost exclusively at enhancers and are themselves regulated at transcription, and stimulus responsive TFs which are regulated post-transcriptionally but act predominantly at enhancers. TF profiler is broadly applicable, providing regulatory insights on any PRO-seq sample for any transcription factor with a known binding motif.

genomics↗

bioRxiv2023

Marker-based CRISPR screening reveals a MED12-p63 interaction that activates basal identity in pancreatic ductal adenocarcinoma

The presence of basal lineage characteristics signifies hyper-aggressive human adenocarcinomas of the breast, bladder, and pancreas. However, the biochemical mechanisms that maintain this aberrant cell state are poorly understood. Here we performed marker-based genetic screens in search of factors needed to maintain basal identity in pancreatic ductal adenocarcinoma (PDAC). This approach revealed MED12 as a powerful regulator of the basal cell state in this disease. Using biochemical reconstitution and epigenomics, we show that MED12 carries out this function by bridging the transcription factor p63, a known master regulator of the basal lineage, with the Mediator complex to activate lineage-specific enhancer elements. Consistent with this finding, the growth of basal-like PDAC is hypersensitive to MED12 loss when compared to classical PDAC. Taken together, our comprehensive genetic screens have revealed a biochemical interaction that sustains basal identity in human cancer, which could serve as a target for tumor lineage-directed therapeutics.

cancer biology↗

bioRxiv2023

Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome

Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS over a 75min - 24h timeframe. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A (CA), via rapid suppression of IFN-responsive transcription factor (TF) activity. We also discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. To further probe Mediator kinase function, we completed cytokine screens and metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways in cell type-specific ways, and broad up-regulation of anti-inflammatory lipid mediators occurred specifically in kinase-inhibited cells during hyperactive IFN{gamma} signaling. A subset of these lipids (e.g. oleamide, desmosterol) serve as ligands for nuclear receptors PPAR and LXR, and activation of these receptors occurred specifically during hyperactive IFN signaling in CA-treated cells, revealing mechanistic links between Mediator kinases, lipid metabolism, and nuclear receptor function. Collectively, our results establish CDK8/CDK19 as context-specific metabolic regulators, and reveal that these kinases control gene expression not only via TFs, but also through metabolic changes and splicing. Moreover, we establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.

biochemistry↗

bioRxiv2022

Mediator Kinase Inhibition Impedes Transcriptional Plasticity and Prevents Resistance to ERK/MAPK-Targeted Therapy in KRAS-Mutant Cancers

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this novel class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and tumor-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved population-level process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, tumor-specific resistance programs.

cancer biology↗

bioRxiv2022

A global atlas of substrate specificities for the human serine/threonine kinome

Protein phosphorylation is one of the most widespread post-translational modifications in biology. With the advent of mass spectrometry-based phosphoproteomics, more than 200,000 sites of serine and threonine phosphorylation have been reported, of which several thousand have been associated with human diseases and biological processes. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein Ser/Thr kinases encoded in the human genome is responsible. Here, we utilize synthetic peptide libraries to profile the substrate sequence specificity of nearly every functional human Ser/Thr kinase. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. Our kinome-wide dataset was used to computationally annotate and identify the most likely protein kinases for every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites where the protein kinases involved have been previously identified, our predictions were in excellent agreement. When this approach was applied to examine the signaling response of tissues and cell lines to hormones, growth factors, targeted inhibitors, and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the full extent of substrate specificity of the human Ser/Thr kinome, illuminate cellular signaling responses, and provide a rich resource to link unannotated phosphorylation events to biological pathways.

systems biology↗

bioRxiv2022

Possible Mechanism of Porosome-Mediated Exosome Release

Supramolecular cup-shaped lipoprotein structures called porosomes embedded in the cell plasma membrane mediate fractional release of intra-vesicular contents from cells during secretion. The presence of porosomes, have been documented in many cell types including neurons, acinar cells of the exocrine pancreas, growth hormone secreting cells of the pituitary, and mouse insulin-secreting beta cell insulinomas called Min6. Electron micrographs of the nerve terminal in rat brain and of Min6 cells, suggest that besides the docking, fusion and content release of neurotransmitter containing synaptic vesicles and insulin containing granules, multi-vesicular bodies and exosome-like vesicles are released at the porosome complex. To test this hypothesis, the porosome-associated calcium transporting ATPase 1 [SPCA1] encoded by the ATP2C1 gene, was knocked-out in Min6 cells using CRISPR, to study glucose-stimulated insulin and exosome release. In agreement with electron micrographs, results from the study demonstrate a loss of both glucose-stimulated insulin and exosome release in the ATP2C1 knockout Min6 cells. These results further confirm the role of the porosome complex in insulin secretion and establishes a new paradigm in porosome-mediated exosome release in beta cells of the endocrine pancreas.

cell biology↗

bioRxiv2020

The role of XPB/Ssl2 double-stranded DNA translocase processivity in transcription start-site scanning.

The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates the C-terminal domain to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning.

biochemistry↗

bioRxiv2020

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibition results in mucus accumulation in human airway epithelia Calu-3 cells: Experimental and Machine Learning Studies

Porosomes are cup-shaped lipoprotein structures at the cell plasma membrane involved in fractional release of intra-vesicular contents during secretion. At the base of the porosome facing the cell cytoplasm, secretory vesicles dock, fuse and swell, to release intra-vesicular content during secretion. Earlier studies demonstrate the cystic fibrosis trans-membrane conductance regulator (CFTR) associated with the porosome in human airways epithelial Calu-3 mucous-secreting cells, suggesting its possible involvement in porosome-mediated mucus secretion. The current study was undertaken to test this hypothesis. Electron microscopy followed by morphometric analysis using manual and computational machine learning approaches were used to assess changes in secretory vesicle size and content, following stimulation of secretion in the absence and presence of CFTR inhibitors. Results from the study demonstrate that stimulated Calu-3 cells pre-exposed to CFTR inhibitors, demonstrate attenuation of secretory vesicle swelling and the release of mucin. Consequently, accumulation of intracellular mucin is observed in cells exposed to CFTR inhibitors. These results further suggest that mucin secretion from Calu-3 cells involve CFTR both at the secretory vesicle membrane to regulate vesicle volume and hydration, and at the porosome to facilitate mucin hydration and secretion. These new findings progress our understanding of the involvement of CFTR on mucus hydration and secretion, providing critical insights into the etiology of CF disease.

cell biology↗

bioRxiv2020

Transcription factor enrichment analysis (TFEA): Quantifying the activity of hundreds of transcription factors from a single experiment

1Detecting differential activation of transcription factors (TFs) in response to perturbation provides insight into cellular processes. Transcription Factor Enrichment Analysis (TFEA) is a robust and reliable computational method that detects differential activity of hundreds of TFs given any set of perturbation data. TFEA draws inspiration from GSEA and detects positional motif enrichment within a list of ranked regions of interest (ROIs). As ROIs are typically inferred from the data, we also introduce muMerge, a statistically principled method of generating a consensus list of ROIs from multiple replicates and conditions. TFEA is broadly applicable to data that informs on transcriptional regulation including nascent (eg. PRO-Seq), CAGE, ChIP-Seq, and accessibility (e.g. ATAC-Seq). TFEA not only identifies the key regulators responding to a perturbation, but also temporally unravels regulatory networks with time series data. Consequently, TFEA serves as a hypothesis-generating tool that provides an easy, rigorous, and cost-effective means to broadly assess TF activity yielding new biological insights.

bioinformatics↗

bioRxiv2019

Suppression of p53 response by targeting p53-Mediator binding with a stapled peptide

DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator; we sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterized a stapled peptide, with functional mimics of both p53 activation domains, that selectively inhibited p53- and Mediator-dependent transcription in vitro. This \"bivalent peptide\" also suppressed p53 transcriptional response in human cancer cells. Our strategy circumvents the TF and instead targets the TF-Mediator interface, with desired transcriptional outcomes. Different TFs target Mediator through different subunits, suggesting this strategy could be broadly applied.

molecular biology↗

bioRxiv2019

Differential expansion microscopy

Expansion microscopy (ExM) involves the use of hydration-competent polymers to physically expand biological specimens approximately 4-fold linear increase to achieve 70 nanometer resolution using an ordinary diffraction limited optical microscope. Optimal conditions however for antigen retention during the expansion process and the relative expansion between organelles within cells has remained unclear. It is reported that different tissues expand to different extents, suggesting that although isotropic expansion is believed to occur, different subcellular compartments with different composition would undergo anisotropic or differential expansion (DiEx). Consequently, there would be distortion of the native shape and size of subcellular compartments upon expansion, parameters which are critical in assessing cellular states in health and disease. Here we report optimal fixation and expansion conditions that retain structural integrity of cells while exhibiting up to 8-fold linear and therefore 512-fold volumetric expansion. Anisotropic expansion is observed not just between tissues, but between different subcellular compartments and even within subcellular compartments. Combining image analysis and machine learning, we provide an approach for the rapid and precise measurement of cellular and subcellular structures in expanded tissue. Using both manual and computation assessment of morphometric parameters, we demonstrate expansion to be anisotropic and name this method differential expansion microscopy (DiExM).

cell biology↗

bioRxiv2018

The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair

Loss of function mutations in the protein kinase DYRK1A lead to a syndromic form of autism spectrum disorder and intellectual disability. Conversely, increased DYRK1A dosage is implicated in atypical brain development and neurocognitive deficits in trisomy 21. DYRK1A regulates a diverse array of cellular processes through kinase dependent and independent interactions with substrates and binding partners. Recent evidence implicates DYRK1A in direct regulation of the transcriptional machinery, but many of the molecular details are not yet known. Furthermore, the landscape of DYRK1A interactions in the nucleus is incomplete, impeding progress toward understanding its function in transcription. Here, we used immunoaffinity purification and mass spectrometry to identify nuclear interaction partners of endogenous DYRK1A. These were enriched in DNA damage repair factors, transcriptional elongation factors and E3 ubiquitin ligases. We validated an interaction with RNF169, a factor that promotes homology directed repair upon DNA damage. We further show that knockout of DYRK1A or treatment with DYRK1A inhibitors in HeLa cells impaired efficient recruitment of 53BP1 to DNA double strand breaks induced by ionizing radiation. This nuclear interactome thus reveals a new role for DYRK1A in DNA damage repair and provides a resource for exploring new functions of DYRK1A in the nucleus.

systems biology↗