Biology subjects

Kropp, E. M.

Publications and source records attributed to Kropp, E. M..

4 recordsLinked to original sources

bioRxiv2025

Identification of the role of SEL1L in platelet function through a multi-species genetic investigation

SEL1L is a well-known protein in the endoplasmic reticulum associated degradation (ERAD) pathway. While it is known to be expressed in platelets, SEL1L has never been shown to play an active role. Here we find evidence that SEL1L regulates platelet function. We first identified SEL1L through the study of Atypical Equine Thrombasthenia (AET), an autosomal recessive platelet disorder found in Thoroughbred horses. A missense variant in SEL1L (c.1810A>G p.Ile604Val) was found in AET-affected horses, which we show is associated with decreased protein expression. SEL1L is intracellular in equine platelets and localizes to the surface upon activation with thrombin. Platelets from homozygous horses exhibit significant decreases in spreading on immobilized collagen. Human megakaryocytes were found to have two SEL1L protein isoforms that increase in expression during megakaryopoiesis, although only one is delivered to mature platelets. Studies using inducible mouse and constitutive zebrafish knockouts demonstrate that SEL1L is necessary for efficient platelet or thrombocyte (fish equivalent) adhesion to sites of endothelial injury. These data reveal a previously undescribed and conserved role for the ERAD pathway in the etiology of AET and platelet function, which may play a role in human platelet disorders as well. Brief SummaryUsing a multi-species approach, SEL1L was determined to have a role in platelet function, specifically in helping platelets properly adhere to sites of injury,

genetics↗

bioRxiv2025

Identification of Omaveloxolone as An Endoplasmic Reticulum Associated Degradation Inhibitor That Induces Early Apoptotic Signaling in Multiple Myeloma

Endoplasmic reticulum-associated degradation (ERAD) is essential for maintaining protein homeostasis, yet its regulatory mechanisms remain poorly understood. A major challenge in studying ERAD is the lack of specific inhibitors targeting the ERAD complex. To address this, we conducted a cell-based high-throughput screen using the FDA-repurposing library and identified omaveloxolone (RTA408) as a potent ERAD inhibitor that selectively impairs the degradation of ER luminal and membrane substrates. Beyond its utility in identifying ERAD substrates, RTA408 exhibits strong cytotoxic effects in multiple myeloma (MM), an incurable plasma cell malignancy. RTA408 inhibits ERAD activity and rapidly induces apoptotic signaling via caspase 8 and the death-inducing signaling complex (DISC). Notably, RTA408 is cytotoxic to malignant plasma cells, including those resistant to proteasome inhibitors, and demonstrates in vivo anti-myeloma activity. Our findings establish ERAD inhibitors as valuable tools for dissecting ERAD regulation while also highlighting their potential as therapeutic agents for MM. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/646787v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@96254aorg.highwire.dtl.DTLVardef@16a3f6dorg.highwire.dtl.DTLVardef@6a1503org.highwire.dtl.DTLVardef@897c45_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIWe developed a cell-based screening approach to identify novel modulators of endoplasmic reticulum associated degradation (ERAD), with implications in studying ERAD biology and targeting plasma cell neoplasms. C_LIO_LIScreening of the FDA-repurposing library identified Omaveloxolone (RTA408) as an inhibitor of luminal and membrane ERAD substrate degradation, which can be leveraged to identify ERAD substrates. C_LIO_LImaveloxolone treatment rapidly induces the unfolded protein response and apoptosis that is dependent on caspase 8 and death-inducing signaling complex (DISC) in multiple myeloma cells. C_LIO_LImaveloxolone exhibits cytotoxic effects against multiple myeloma cells in vitro and in vivo and induces apoptosis in primary plasma cells from patients with relapsed/refractory myeloma C_LI

cell biology↗

bioRxiv2024

PERK Signaling Maintains Hematopoietic Stem Cell Pool Integrity under Endoplasmic Reticulum Stress by Promoting Proliferation

The integrity of the hematopoietic stem cell (HSC) pool relies on efficient long-term self-renewal and the timely removal of damaged or differentiation-prone HSCs. Previous studies have demonstrated the PERK branch of the unfolded protein response (UPR) drives specific programmed cell death programs to maintain HSC pool integrity in response to ER stress. However, the role of PERK in regulating HSC fate in vivo remains unclear. Here, we demonstrate that PERK is dispensable for normal hematopoiesis and HSC self-renewal under steady-state conditions. In contrast, PERK is activated to promote HSC proliferation and depletion in response to ER stress induced by the inactivation of ER-associated degradation (ERAD), via the knockout of key components of ERAD Sel1L or Hrd1. Inhibition of PERK, either through genetic knockout or knock-in of a point mutation that eliminates PERK kinase activity, significantly restores the HSC defects induced by Sel1L or Hrd1 knockout. Mechanistic studies reveal that ERAD deficiency does not lead to HSC death or ROS accumulation. Instead, PERK promotes the activation of mTOR signaling and drives abnormal proliferation of HSCs, impairing their self-renewal potential. This process removes stressed HSCs, thereby maintaining HSC pool integrity. Our study uncovers a PERK-centered strategy employed by HSCs to preserve their pool integrity independently of apoptosis. Key pointsO_LIPERK is not required for steady-state hematopoiesis but preserves hematopoietic stem cell pool integrity in response to increased ER stress. C_LIO_LIUnder ER stress induced by ERAD deficiency, PERK is activated to promote mTOR signaling and HSC hyper-proliferation, depleting damaged HSCs. C_LI

cell biology↗

bioRxiv2018

Are these cardiomyocytes? Protocol development reveals impact of sample preparation on the accuracy of identifying cardiomyocytes by flow cytometry

Modern differentiation protocols enable efficient, yet imperfect, differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM). As the number of laboratories and studies implementing this technology expands, the accurate assessment of cell identity in differentiation cultures is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity in hPSC-CM cultures has not yet been established. To address this gap, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in hPSC-CM and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges of interpreting data generated by published methods and informed the development of a robust protocol for routine assessment of hPSC-CM. Overall, the new data, workflow for evaluating fit-for-purpose use of antibodies, and standardized protocol described here should benefit investigators new to this field as well as those with expertise in hPSC-CM differentiation.

developmental biology↗