Biology subjects

Grzemska, M.

Publications and source records attributed to Grzemska, M..

6 recordsLinked to original sources

bioRxiv2026

PPa1 insufficiency drives lysosomal storage disease and inflammatory macrophage expansion in the bone marrow.

Inorganic pyrophosphatase-1 (PPa1) is an essential enzyme proposed to limit accumulation of the ubiquitous metabolic byproduct inorganic pyrophosphate, yet its role beyond a general housekeeping function remains poorly understood. We generated viable hypomorphic alleles of PPa1 through random mutagenesis in mice and unexpectedly found that PPa1 insufficiency causes a lysosomal storage disease of the bone marrow. Mutant mice developed impaired hematopoiesis and defective skeletal mineralization in a hematopoietic-intrinsic manner. The bone marrow was infiltrated by glycolipid-laden macrophages that were marked by high Spp1 and CD14 expression, blunted lysosomal acidification, metabolic stress, and a distinct inflammatory transcriptional program. Consistent with a lysosomal storage defect, PPa1-deficient bone marrow accumulated elevated levels of long-chain glucosyl-sphingolipids and sphingosine. These findings identify PPa1 as a previously unrecognized regulator of lysosomal function in myeloid cells that restrains inflammatory macrophage expansion and prevents bone marrow lysosomal storage pathology.

immunology↗

bioRxiv2025

The Structural Basis of Pacs1-Wdr37 Complex Assembly and Stability

Phosphofurin acidic cluster sorting protein 1 (Pacs1) is a multidomain adaptor proposed to bind transmembrane cargo proteins to facilitate their intracellular trafficking. Pacs1 also forms a complex with WD-repeat protein 37 (Wdr37), which is essential for lymphocyte homeostasis. Despite numerous proposed binding partners, a validated structure for Pacs1-containing protein complexes is lacking. Here, we present the cryo-electron microscopy structure of the Pacs1-Wdr37 complex. Pacs1 binds Wdr37 through a conserved interface within its furin-binding region (FBR), the domain previously linked to cargo recognition. This interaction stabilizes Wdr37 and is critical for the expression of both proteins. A gain-of-function mutation in PACS1 (R203W) causes a highly penetrant neurodevelopmental syndrome. This pathogenic mutation lies on a solvent-exposed surface of the FBR and does not disrupt complex formation. Instead, Pacs1-R203W remains dependent on Wdr37 for stability and its levels can be reduced through targeted Wdr37 proteolytic degradation. Structural homology of the FBR to synaptotagmin C2 domains reveals a previously unrecognized ability of Pacs1 to bind negatively charged phospholipids through a unique positively charged cleft. Together these findings define the structural basis for Pacs1-Wdr37 complex assembly and stability, present potential strategies for Pacs1-mediated neurodevelopmental disease, and suggest novel Pacs1 functions in membrane association.

biochemistry↗

bioRxiv2024

Predictive and Experimental Motif Interaction Analysis Identifies Functions of the WNK-OSR1/SPAK Pathway

The WNK-OSR1/SPAK protein kinase signaling pathway regulates ion homeostasis and cell volume, but its other functions are poorly understood. To uncover undefined signaling functions of the pathway we analyzed the binding specificity of the conserved C-terminal (CCT) domains of OSR1 and SPAK to find all possible interaction motifs in human proteins. These kinases bind the core consensus sequences R-F-x-V/I and R-x-F-x-V/I. Motifs were ranked based on sequence, conservation, cellular localization, and solvent accessibility. Out of nearly 3,700 motifs identified, 90% of previously published motifs were within the top 2% of those predicted. Selected candidates (TSC22D1, CAVIN1, ATG9A, NOS3, ARHGEF5) were tested. Upstream kinases WNKs 1-4 and their close relatives, the pseudokinases NRBP1/2, contain CCT-like domains as well. We identified additional distinct motif variants lacking the conserved arginine previously thought to be required, and found that the NRBP1 CCT-like domain binds TSC22D1 via the same motif as OSR1 and SPAK. Our results further highlight the rich and diverse functionality of CCT and CCT-like domains in connecting WNK signaling to cellular processes.

biochemistry↗

bioRxiv2021

WNK1 Enhances Migration and Invasion in Breast Cancer Models

Metastasis is the major cause of mortality in breast cancer patients. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase WNK1 (with no lysine (K) 1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.

cancer biology↗

bioRxiv2019

Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex

The nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the eleven (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a sub-complex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N-terminus of DRC4 which is now shown to contribute to the core scaffold of the N-DRC and C-terminus of DRC5. Our results reveal the overall architecture of N-DRC, with the three subunits, DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the \"functional subunits\" associate, namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.\n\nSignificance StatementCilia and flagella are small hair-like appendages in eukaryotic cells that play essential roles in cell sensing, signaling, and motility. The highly conserved nexin-dynein regulatory complex (N-DRC) is one of the key regulators for ciliary motility. At least 11 proteins (DRC1-11) have been assigned to the N-DRC, but their precise arrangement within the large N-DRC structure is not yet known. Here, using cryo-electron tomography combined with genetic approaches, we have localized DRC7, the sub-complex DRC8/DRC11, the N-terminus of DRC4, and the C-terminus of DRC5. Our results provide insights into the N-DRC structure, its function in the regulation of dynein activity, and the mechanism by which n-drc mutations can lead to defects in ciliary motility that cause disease.

cell biology↗

bioRxiv2018

Chromomycin A2 potently inhibits glucose-stimulated insulin secretion from pancreatic beta cells.

Enhancers or inhibitors of insulin secretion could become therapeutics as well as lead to the identification of requisite {beta}-cell regulatory pathways and increase our understanding of pancreatic islet function. Toward this goal, we previously used an insulin-linked luciferase that is co-secreted with insulin in MIN6 {beta}-cells to perform a high-throughput natural product screen for chronic effects on glucose-stimulated insulin secretion. Using multiple phenotypic analyses, we identified that one of the top natural product hits, chromomycin A2 (CMA2), potently inhibited insulin secretion through at least three mechanisms: disruption of Wnt signaling, interfering with {beta}-cell gene expression, and suppression of triggering calcium (Ca2+) influx. Chronic treatment with CMA2 largely ablated glucose-stimulated insulin secretion even post-washout, but did not inhibit glucose-stimulated generation of ATP or Ca2+ influx. However, by using the KATP channel-opener diazoxide, we uncovered defects in depolarization-induced Ca2+ influx which may contribute to the suppressed secretory response. Glucose-responsive ERK1/2 and S6 phosphorylation were also disrupted by chronic CMA2 treatment. The FUSION bioinformatic database indicated that the phenotypic effects of CMA2 clustered with a number of Wnt/GSK3 pathway-related genes. Consistently, CMA2 decreased GSK3 phosphorylation and suppressed activation of a {beta}-catenin activity reporter. CMA2 and a related compound mithramycin are described to have DNA-interaction properties, possibly abrogating transcription factor binding to critical {beta}-cell gene promoters. We observed that CMA2, but not mithramycin, suppressed expression of PDX1 and UCN3. However, neither expression of INSI/II nor insulin content was affected by chronic CMA2. The mechanisms of CMA2-induced insulin secretion defects may involve components both proximal and distal to Ca2+ influx. Therefore, CMA2 is an example of a chemical that can simultaneously disrupt {beta}-cell function through both non-cytotoxic and cytotoxic mechanisms. Future applications of CMA2 and similar aureolic acid analogs for disease therapies should consider the potential impacts on pancreatic islet function.

cell biology↗