Receptor Recycling Research Articles

Role of PCSK9 inhibition during the inflammatory stage of SARS-COV-2: an updated review

The potential role of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition in the management of COVID-19 and other medical conditions has emerged as an intriguing area of research. PCSK9 is primarily known for its impact on cholesterol metabolism, but recent studies have unveiled its involvement in various physiological processes, including inflammation, immune regulation, and thrombosis. In this abstract, the authors review the rationale and potential implications of PCSK9 inhibition during the inflammatory stage of SARS-CoV-2 infection. Severe cases of COVID-19 are characterized by an uncontrolled inflammatory response, often referred to as the cytokine storm, which can lead to widespread tissue damage and organ failure. Preclinical studies suggest that PCSK9 inhibition could dampen this inflammatory cascade by reducing the production of pro-inflammatory cytokines. Additionally, PCSK9 inhibition may protect against acute respiratory distress syndrome (ARDS) through its effects on lung injury and inflammation. COVID-19 has been linked to an increased risk of cardiovascular complications, especially in patients with pre-existing cardiovascular conditions or dyslipidemia. PCSK9 inhibitors are known for their ability to lower low-density lipoprotein (LDL) cholesterol levels by enhancing the recycling of LDL receptors in the liver. By reducing LDL cholesterol, PCSK9 inhibition might protect blood vessels from further damage and lower the risk of atherosclerotic plaque formation. Moreover, PCSK9 inhibitors have shown potential antithrombotic effects in preclinical studies, making them a potential avenue to mitigate the increased risk of coagulation disorders and thrombotic events observed in COVID-19. While the potential implications of PCSK9 inhibition are promising, safety considerations and possible risks need careful evaluation. Hypocholesterolemia, drug interactions, and long-term safety are some of the key concerns that should be addressed. Clinical trials are needed to establish the efficacy and safety of PCSK9 inhibitors in COVID-19 patients and to determine the optimal timing and dosing for treatment. Future research opportunities encompass investigating the immune response, evaluating long-term safety, exploring combination therapy possibilities, and advancing personalized medicine approaches. Collaborative efforts from researchers, clinicians, and policymakers are essential to fully harness the therapeutic potential of PCSK9 inhibition and translate these findings into meaningful clinical outcomes.

Plant signalling: The case of the recycled receptor

RsbQ from bacteria and KAI2 from plants are highly related α/β-hydrolase proteins with unknown ligands. In a new study, Melville, Kamran etal. attempt to understand the ligand binding of RsbQ using knowledge from studies of KAI2, with surprising results.

Prodigiosin Inhibits Transforming Growth Factor β Signaling by Interfering Receptor Recycling and Subcellular Translocation in Epithelial Cells.

Prodigiosin (PG) is a naturally occurring polypyrrole red pigment produced by numerous microorganisms including some Serratia and Streptomyces strains. PG has exhibited promising anticancer activity; however, the molecular mechanisms of action of PG on malignant cells remain ambiguous. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that governs a wide array of cellular processes in development and tissue homeostasis. Malfunctions of TGF-β signaling are associated with numerous human cancers. Emerging evidence underscores the significance of internalized TGF-β receptors and their intracellular trafficking in initiating signaling cascades. In this study, we identified PG as a potent inhibitor of the TGF-β pathway. PG blocked TGF-β signaling by targeting multiple sites of this pathway, including facilitating the sequestering of TGF-β receptors in the cytoplasm by impeding the recycling of type II TGF-β receptors to the cell surface. Additionally, PG prompts a reduction in the abundance of receptors on the cell surface through the disruption of the receptor glycosylation. In human Caucasian lung carcinoma cells and human hepatocellular cancer cell line cells, nanomolar concentrations of PG substantially diminish TGF-β-triggered phosphorylation of Smad2 protein. This attenuation is further reflected in the suppression of downstream target gene expression, including those encoding fibronectin, plasminogen activator inhibitor-1, and N-cadherin. SIGNIFICANCE STATEMENT: Prodigiosin (PG) emerges from this study as a potent TGF-β pathway inhibitor, disrupting receptor trafficking and glycosylation and reducing TGF-β signaling and downstream gene expression. These findings not only shed light on PG's potential therapeutic role but also present a captivating avenue towards future anti-TGF-β strategies.

Somatostatin receptors and the associated intracellular machinery: the two sides of the coin.

Somatostatin receptors (SSTs) are widely expressed in pituitary tumors and neuroendocrine neoplasms (NENs) of different origins, i.e. the gastrointestinal tract and the thorax (lungs and thymus), thus representing a well-established target for medical treatment with SST ligands (SRLs). However, the response to SRLs is highly heterogeneous between tumors. Two main factors can contribute to this variability: (i) the differential SST expression among tumor types and (ii) the differential expression/modulation of the SST-related intracellular machinery. In this literature review, we provide an overview of available data on the variable expression of SSTs in pituitary tumors and NENs, together with the resulting clinical implications. Moreover, we aim to describe the complex intracellular machinery involved in SST signaling and trafficking. Particularly, we will focus on β-arrestins and describe their role in receptor internalization and recycling, as well as the various functions of these scaffold molecules in tumor pathogenesis and progression. This review highlights the interplay between membrane receptors and intracellular machinery, together with its role in determining the clinical behavior of the tumor and the response to treatment in patients with pituitary tumors or NENs.

Myosin Va: Capturing cAMP for synaptic plasticity.

The plus-end directed actin-dependent motor protein, myosin Va, is of particular relevance for outward vesicular protein trafficking and for restraining specific cargo vesicles within the actin cortex. The latter is a preferred site of cAMP production, and the specificity of cAMP signaling is largely mediated through the formation of microdomains that spatially couple localized metabotropic receptor activity and cAMP production to selected effectors and downstream targets. This review summarizes the core literature on the role of myosin Va for the creation of such a cAMP microdomain at the mammalian nerve-muscle synapse that serves the activity-dependent recycling of nicotinic acetylcholine receptors (nAChRs)-a principal ligand-gated ion channel which is imperative for voluntary muscle contraction. It is discussed that i) the nerve-muscle synapse is a site with a unique actin-dependent microstructure, ii) myosin Va and protein kinase A regulatory subunit Iα as well as nAChR and its constitutive binding partner, rapsyn, colocalize in endocytic/recycling vesicles near the postsynaptic membrane, and iii) impairment of myosin Va or displacement of protein kinase A regulatory subunit Iα leads to the loss of nAChR stability. Regulation of this signaling process and underlying basic pieces of machinery were covered in previous articles, to which the present review refers.

Partial loss of Sorting Nexin 27 resembles age- and Down syndrome-associated T cell dysfunctions

BackgroundSorting Nexin 27 (SNX27)-retromer complex facilitates cargo recycling from endosomes to the plasma membrane. SNX27 downregulation in neurons, as the result of Trisomy 21 (T21), has been linked with cognitive deficits due to impairment of AMPA and NMDA receptor recycling. Studies in human T cell lines likewise demonstrated that SNX27 regulates the correct delivery of cargoes to the immune synapse limiting the activation of pro-inflammatory pathways. Nevertheless, the physiological consequences of partial SNX27 loss in T cell homeostasis are still unclear.ResultsIn this study, we have explored the consequences of T cell specific partial SNX27 downregulation in mice. T cells with partial SNX27 deficiency show a marked deficit in the CD4+ T cell pool, a hallmark of aging in mice and humans, and a well-characterized comorbidity of individuals with Down syndrome (DS). When analyzed ex vivo, CD4+ T cells with partial SNX27 deletion demonstrate enhanced proliferation but diminished IL-2 production. In contrast, the CD8+ population show enhanced expression of pro-inflammatory cytokines and lytic enzymes.ConclusionsThis mouse model supports the relevance of SNX27 in the organization of the immune synapse, previously described in cell lines, as well as in the control of T cell homeostasis. Individuals with DS experiment an acceleration of the aging process, which particularly affects the immune and central nervous systems. Thus, we hypothesize that reduced SNX27 expression in DS could contribute to the dysregulation of these systems and further research in SNX27 will shed light on the molecular factors underlying the phenotypes observed in people with DS and its contribution to aging.

Research advances in huntingtin-associated protein 1 and its application prospects in diseases.

Huntingtin-associated protein 1 (HAP1) was the first protein discovered to interact with huntingtin. Besides brain, HAP1 is also expressed in the spinal cord, dorsal root ganglion, endocrine, and digestive systems. HAP1 has diverse functions involving in vesicular transport, receptor recycling, gene transcription, and signal transduction. HAP1 is strongly linked to several neurological diseases, including Huntington's disease, Alzheimer's disease, epilepsy, ischemic stroke, and depression. In addition, HAP1 has been proved to participate in cancers and diabetes mellitus. This article provides an overview of HAP1 regarding the tissue distribution, cell localization, functions, and offers fresh perspectives to investigate its role in diseases.

Assessment of Non-canonical Functions of the Autophagy Proteins in LC3-Associated Phagocytosis and LC3-Associated Endocytosis.

The identification and characterization of noncanonical functions within the autophagy pathway have unveiled intricate cellular processes, including LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO). These phenomena play pivotal roles in the conjugation of ATG8 with single-membrane phagosomes and endosomes, shedding light on the dynamic interplay between autophagy and cellular homeostasis. Here, we present detailed protocols for both qualitative and quantitative assessment of LAP, including immunofluorescence, flow cytometry, and Western blotting of isolated LAPosomes. Additionally, the protocol for the evaluation of LANDO through immunofluorescent detection of receptor recycling is outlined. The methodologies presented herein serve as a practical guide for researchers seeking to unravel the intricacies of LAP and LANDO. By providing step-by-step instructions, accompanied by insights into potential challenges and optimization strategies, this chapter aims to empower investigators in the exploration of these noncanonical functions of autophagy proteins.

Extracellular vesicle-mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin-kexin type 9 (Pcsk9) in primary mouse hepatocytes.

The loss-of-function of the proprotein convertase subtilisin-kexin type 9 (Pcsk9) gene has been associated with significant reductions in plasma serum low-density lipoprotein cholesterol (LDL-C) levels. Both CRISPR/Cas9 and CRISPR-based editor-mediated Pcsk9 inactivation have successfully lowered plasma LDL-C and PCSK9 levels in preclinical models. Despite the promising preclinical results, these studies did not report how vehicle-mediated CRISPR delivery inactivating Pcsk9 affected low-density lipoprotein receptor recycling in vitro or ex vivo. Extracellular vesicles (EVs) have shown promise as a biocompatible delivery vehicle, and CRISPR/Cas9 ribonucleoprotein (RNP) has been demonstrated to mediate safe genome editing. Therefore, we investigated EV-mediated RNP targeting of the Pcsk9 gene ex vivo in primary mouse hepatocytes. We engineered EVs with the rapamycin-interacting heterodimer FK506-binding protein (FKBP12) to contain its binding partner, the T82L mutant FKBP12-rapamycin binding (FRB) domain, fused to the Cas9 protein. By integrating the vesicular stomatitis virus glycoprotein on the EV membrane, the engineered Cas9 EVs were used for intracellular CRISPR/Cas9 RNPdelivery, achieving genome editing with an efficacy of ±28.1% in Cas9 stoplight reporter cells. Administration of Cas9 EVs in mouse hepatocytes successfully inactivated the Pcsk9 gene, leading to a reduction in Pcsk9 mRNA and increased uptake of the low-density lipoprotein receptor and LDL-C. These readouts can be used in future experiments to assess the efficacy of vehicle-mediated delivery of genome editing technologies targeting Pcsk9. The ex vivo data could be a step towards reducing animal testing and serve as a precursor to future in vivo studies for EV-mediated CRISPR/Cas9 RNP delivery targeting Pcsk9.

Probing the pH-dependency of DC-SIGN/R multivalent lectin-glycan interactions using polyvalent glycan-gold nanoparticles.

The dendritic cell tetrameric lectin, DC-SIGN, and its closely related endothelial cell lectin, DC-SIGNR (collectively abbreviated as DC-SIGN/R) play a key role in the binding and transmission of deadly viruses, including Ebola, HIV, HCV, and SARS-CoV-2. Their virus binding/release processes involve a gradually acidifying environment following the natural intracellular trafficking pathways. Therefore, understanding DC-SIGN/R's pH-dependent binding properties with glycan ligands is of great importance. We have recently developed densely glycosylated gold nanoparticles (glycan-GNPs) as a powerful new tool for probing DC-SIGN/R multivalent lectin-glycan interaction (MLGI) mechanisms. They can provide not only quantitative MLGI affinities but also important structural information, such as binding site orientation and binding modes. Herein, we further employ the glycan-GNP probes to investigate the pH dependency of DC-SIGN/R MLGI properties. We find that DC-SIGN/R MLGIs exhibit distinct pH dependence over the normal physiological (7.4) to lysosomal (∼4.6) pH range. DC-SIGN binds glycan-GNPs strongly and stably from pH 7.4 to ∼5.8, but the binding is weakened significantly as pH decreases to ≤5.4 and may be fully dissociated at pH 4.6. This behaviour is fully consistent with DC-SIGN's role as an endocytic recycling receptor. In contrast, DC-SIGNR's affinity with glycan-GNPs is enhanced with the decreasing pH from 7.4 to 5.4, peaking at pH 5.4, and then reduced as pH is further lowered. Interestingly, both DC-SIGN/R binding with glycan-GNPs are found to be partially reversible in a pH-dependent manner.

The structure and function of cell membrane receptor

Cell membrane receptors play a key role in regulating cell communication and maintaining cell homeostasis. This paper explores the complex relationship between the structure and function of cell membrane receptors, and elucidates their multiple roles in signal transduction, cellular response, and disease pathways. Different receptor types, including as G protein-coupled receptors (GPCRs), ligand-gated ion channels, receptor tyrosine kinases (RTKs), and cytokine receptors, have varied structural properties that serve different biological purposes and are necessary for cell division, proliferation, and metabolism. The function of membrane receptors that translate extracellular signals into intracellular responses is diverse. The organization of lipid rafts and other receptors within membrane microdomains can influence signal transduction efficiency and specificity. There are inseparable interactions among receptor internalization, recycling and degradation, and regulating the duration and intensity of receptor signaling is closely related to the treatment of diseases. In summary, the current research status of cell membrane receptor structure and function is reviewed in this paper. It contributes to a broader understanding of how receptors regulate important cellular processes at the molecular level. Understanding the nuances of receptor structure-function relationships holds great promise for developing new therapeutic strategies and advancing drug discovery for a variety of diseases.

Rab11 suppresses head and neck carcinoma by regulating EGFR and EpCAM exosome secretion

ObjectivesRab11(Rab11a and Rab11b) localizes primarily along recycling endosomes in cells and is involved in various intracellular trafficking processes, including membrane receptor recycling and secretion of exosomes or small extracellular vesicles (EVs). Although Rab11 is closely associated with the progression and metastasis of various cancer types, little is known about Rab11’ role in head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the roles of Rab11a and Rab11b in HNSCC. MethodsThe clinical significance of Rab11 expression in HNSCC was investigated using a public database and tissue microarray analysis. Stable cell lines with loss and gain of Rab11a or Rab11b were originally established to investigate their roles in the proliferative, migratory, and invasive capabilities of HNSCC cells. ResultsDatabase analysis revealed a significant association between Rab11b mRNA expression and a favorable patient survival rate in HNSCC. Tissue microarray analysis revealed that Rab11b expression was the highest in normal tissues and gradually decreased across the stages of HNSCC progression. Overexpression of Rab11a or Rab11b resulted in a decrease in epidermal growth factor receptor (EGFR), Epithelial cell adhesion molecule (EpCAM) exosome secretion, and the migratory and invasive potential of HNSCC cells. The knockdown of Rab11a or Rab11b increased EpCAM/CD9 exosome secretion in addition to the migratory and invasive potential of HNSCC cells. ConclusionsRab11 suppresses HNSCC by regulating EGFR recycling and EpCAM exosome secretion in HNSCC cells. Our results indicate that Rab11b is a superior prognostic indicator of HNSCC and holds promise for developing novel therapeutic strategies.

SORLA‐retromer complex formation rely on receptor dimerization within the endosome

AbstractBackgroundSORLA acts as an endosome recycling receptor together with the retromer complex to escort cargo through the endo‐lysosomal tubular network. SORLA is encoded by the SORL1 gene, which is the gene most frequently mutated in patients with Alzheimer’s disease, although it is still not clear which of the many variants are (the most) pathogenic. Here, we studied the biochemistry and cell biology of a SORL1 variant that is found in several European families.MethodsWe used transfected cells to study SORLA maturation and shedding of the soluble sSORLA by Western blotting. Expression of non‐mutated and mutated receptor at the cell surface was determined by flow cytometry, and the intracellular localization and overlap with markers of Endoplasmic reticulum and endosome compartments investigated by immunocytochemistry and confocal microscopy. The cellular distribution of receptor dimerization was visualized using bimolecular fluorescence complementation and proximity ligation assays, and quantified using the GFP‐trap method.ResultsWe showed that non‐mutated SORLA forms a dimer in retromer‐coated endosome structures, while the pathogenic SORLA protein is unable to form dimers, although it traffics to the endosome. This lack of dimerization in the endosome correlates with strongly decreased receptor maturation, expression at the cell surface, and shedding of its luminal sSORLA domain.ConclusionSORLA dimer formation is required for its association with retromer, which is important for efficient receptor maturation and delivery to the cell surface. Decreased cell surface expression of mutated protein with impaired dimerization leads to less shedding, showing how maturation and sSORLA production can reliably inform on the pathogenicity of SORL1 genetic variants in Alzheimer’s disease.

Impaired Efficacy and Durability of Hematopoietic Stem Cell Engraftment Caused By Samd9l-Deficiency within Bone Marrow Niches

Germline mutations in the SAMD9 and SAMD9L (SAMD9/L) genes on chromosome 7q drive both childhood myelodysplastic syndrome (MDS)/secondary acute myeloid leukemia (sAML) associated with monosomy 7, and adult-onset MDS/sAML associated with distinct somatic genetic alterations. The ortholog for SAMD9/L in mice is Samd9l. Gain of Function (GoF) and Loss of Function (LoF) Samd9l alleles predispose to MDS at distinct ages. LoF models show that Samd9l-deficient mice, like humans, are more likely to develop MDS during adulthood, while GoF models result in early onset bone marrow (BM) dysfunction and MDS/sAML. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for bone marrow failure (BMF), MDS, and AML arising from germline genetic syndromes. Our previous research demonstrated that deficits in host BM microenvironment (ME) niches contribute to hematopoietic failure and poor HSC engraftment in murine models of many MDS predisposition syndromes associated with BMF. As we have observed clinical cases where poor engraftment impaired outcomes in patients with SAMD9/L mutations, we recently hypothesized, BM niche functions critical to facilitating HSC engraftment may be impaired due to niche-intrinsic deficits caused by SAMD9/L mutations. Herein, we present data testing this hypothesis, demonstrating Samd9l deficiency alters BM ME composition, extracellular matrix (ECM) regulation, and proinflammatory cytokine signaling resulting in impaired donor HSC engraftment after HSCT. We performed primary (1 ry) and secondary (2 ry) HSCT to model impacts of SAMD9/L deficiency on BM niche function during HSCT using WT ( Samd9l+/+), Samd9l+/-, and Samd9l-/- mice as HSCT recipients of healthy WT GFP + donor BM to measure the capacity of Samd9l LoF niches to engraft donor HSCs. To assess changes in niche composition and gene expression during HSCT, flow cytometry, multiplex ELISA, and RNASeq analysis were performed on WT, Samd9+/- and Samd9-/- mice. Compared to WT control niches, Samd9+/- and Samd9-/- niches exhibited impaired efficiency of donor HSC engraftment at 3 weeks post-1 ry HSCT, as measured by competitive 2 ry-HSCT performed with 1 ry recipient BM harvested at this timepoint. Beyond 30 weeks post-2 ry-HSCT in this model, GFP + reconstitution of GR1 + myeloid, RBC's, and platelets was reduced by 25%, 18%, and 35% in 2 ry recipients of Samd9+/-1 ry recipient BM and by 26%, 28%, and 32% in 2 ry recipients of Samd9-/- 1 ry recipient BM. Durability of HSC engraftment by 8 months was even more significantly impaired in Samd9+/- and Samd9-/- niches, with Samd9-/- mice exhibiting low survival by this timepoint. Competitive 2 ry HSCT performed 8 months post-1 ry HSCT showed reduction in trilineage GFP + blood cells out to 24 weeks post 2 ry-HSCT, including reductions in GFP + Gr1 + myeloid cells by 50% and 83% in 2 ry recipients of Samd9l+/-and Samd9l-/-1 ry recipient BM, respectively, compared to control 1 ry recipient BM. Similar reductions in BM long-term HSC and Lin -Sca-1 +cKit + HSC/progenitor cells in 2 ry recipients of Samd9l-/-compared to Samd9l+/+ 1 ryrecipient BM 8 months post 1 ry-HSCT. Following TBI, Samd9l+/-and Samd9l-/- BM niches contained similar percentages of osteoprogenitors, mesenchymal stem cells, and endothelial cells compared to WT controls. Multiplex ELISA on BM plasma collected 48hrs post-TBI revealed that Samd9l+/-and Samd9l-/-deletion in the BM ME led to markedly reduced expression of IFN-γ (43% and 55% reduction in Samd9l+/-and Samd9l-/-), Eotaxin (33%/36%), GM-CSF (47%/47%), and IL-1a (48%/65%). In contrast, expression of niche cell extracts showed markedly increased levels of IL-17 (4 fold) and CXCL11 (1.4 fold) in Samd9l+/-and of IL-17 (5.3 fold) IL-12p70 (1.3 fold), and CXCL11 (1.5 fold) in Samd9l-/-, suggesting that aberrantly active Th17 responses caused by niche Samd9l deficiency may be responsible for impairing HSC function and maintenance post-HSCT. RNASeq analysis showed downregulation of ECM genes in both Samd9l-/-and Samd9l+/-models. Our data demonstrates BM niche function is impaired by Samd9l deficiency in a gene dosage-dependent manner. These deficits are associated with increased expression of a specific inflammatory cytokine pathway, suggesting that aberrant growth factor receptor recycling due to Samd9l deficiency may drive stress hematopoiesis and alter HSC cell fate decisions leading to failure of durable donor HSC maintenance post-HSCT.

Comparative analysis of kinetic realizations of insulin signaling

Several studies have developed dynamical models to understand the underlying mechanisms of insulin signaling, a signaling cascade that leads to the translocation of glucose, the human body’s main source of energy. Fortunately, reaction network analysis allows us to extract properties of dynamical systems without depending on their model parameter values. This study focuses on the comparison of insulin signaling in healthy state (INSMS or INSulin Metabolic Signaling) and in type 2 diabetes (INRES or INsulin RESistance) using reaction network analysis. The analysis uses network decomposition to identify the different subsystems involved in insulin signaling (e.g., insulin receptor binding and recycling, GLUT4 translocation, and ERK signaling pathway, among others). Furthermore, results show that INSMS and INRES are similar with respect to some network, structo-kinetic, and kinetic properties. Their differences, however, provide insights into what happens when insulin resistance occurs. First, the variation in the number of species involved in INSMS and INRES suggests that when irregularities occur in the insulin signaling pathway, other complexes (and, hence, other processes) get involved, characterizing insulin resistance. Second, the loss of concordance exhibited by INRES suggests less restrictive interplay between the species involved in insulin signaling, leading to unusual activities in the signaling cascade. Lastly, GLUT4 losing its absolute concentration robustness in INRES may signify that the transporter has lost its reliability in shuttling glucose to the cell, inhibiting efficient cellular energy production. This study also suggests possible applications of the equilibria parametrization and network decomposition, resulting from the analysis, to potentially establish absolute concentration robustness in a species.

Abstract 11755: Efferocytic Regulator Rab11FIP1 Alleviates Inflammation and Cardiac Remodeling After Myocardial Infarction

Introduction: Defective efferocytosis drives cardiac dysfunction after myocardial infarction (MI); however, the underlying molecular mechanisms and regulators of macrophage efferocytosis remain unknown. Hypothesis: This study aimed to explore the modulator and its mechanisms of the continued clearance and degradation of phagolysosomal cargo during MI. Methods: Leveraging macrophage RAB11FIP1 knockout model, flow cytometry and PET-CT technologies, we examined the functional significance of RAB11FIP1 after MI. Moreover, a nanoliposome-linked ssDNA aptamer contained modified RAB11FIP1 mRNA was designed. Results: By comparing the transcriptomic signatures of our scRNA-seq data, we identified RAB11FIP1 was the most sensitively efferocytic gene among all phagosome-related signatures. RAB11FIP1 deficiency resulted in a considerable exacerbation in cardiac function, the accumulation of apoptotic cardiomyocytes and a reduced efferocytosis. RAB11FIP1-loss also led to increased infiltration of CCR2+ macrophages and neutrophils with upregulation of proinflammatory mediators, IL-1β, TNF-α, Ccl2 after MI. In mechanistic studies, RAB11FIP1 defect disabled the recycling of efferocytic receptor, Mertk, further impaired continued macrophages efferocytosis. Moreover, the scRNA-seq data revealed that the regulon activity of XBP1, which acted as the key ER-stress executor, was significantly activated in RAB11FIP1-null hearts, then, the activated XBP1 governed the transcriptional expression of pro-inflammatory mediators after MI. Finally, we constructed a nanoliposome-linked ssDNA aptamer to specifically target macrophages. RAB11FIP1-expressing phagocytes display a striking increase in efferocytosis and decline in inflammation, cardiac remodeling after MI. Conclusions: Collectively, these findings advance the relationship of RAB11FIP1 between macrophage efferocytosis, ER-stress response and the cardiac inflammation.

The Genetic Background of Abnormalities in Metabolic Pathways of Phosphoinositides and Their Linkage with the Myotubular Myopathies, Neurodegenerative Disorders, and Carcinogenesis.

Myo-inositol belongs to one of the sugar alcohol groups known as cyclitols. Phosphatidylinositols are one of the derivatives of Myo-inositol, and constitute important mediators in many intracellular processes such as cell growth, cell differentiation, receptor recycling, cytoskeletal organization, and membrane fusion. They also have even more functions that are essential for cell survival. Mutations in genes encoding phosphatidylinositols and their derivatives can lead to many disorders. This review aims to perform an in-depth analysis of these connections. Many authors emphasize the significant influence of phosphatidylinositols and phosphatidylinositols' phosphates in the pathogenesis of myotubular myopathies, neurodegenerative disorders, carcinogenesis, and other less frequently observed diseases. In our review, we have focused on three of the most often mentioned groups of disorders. Inositols are the topic of many studies, and yet, there are no clear results of successful clinical trials. Analysis of the available literature gives promising results and shows that further research is still needed.

Lipotoxicity, ER Stress, and Cardiovascular Disease: Current Understanding and Future Directions.

The endoplasmic reticulum (ER) is a sub-cellular organelle that is responsible for the correct folding of proteins, lipid biosynthesis, calcium storage, and various post-translational modifications. In the disturbance of ER functioning, unfolded or misfolded proteins accumulate inside the ER lumen and initiate downstream signaling called unfolded protein response (UPR). The UPR signaling pathway is involved in lipolysis, triacylglycerol synthesis, lipogenesis, the mevalonate pathway, and low-density lipoprotein receptor recycling. ER stress also affects lipid metabolism by changing the levels of enzymes that are involved in the synthesis or modifications of lipids and causing lipotoxicity. Lipid metabolism and cardiac diseases are in close association as the deregulation of lipid metabolism leads to the development of various cardiovascular diseases (CVDs). Several studies have suggested that lipotoxicity is one of the important factors for cardiovascular disorders. In this review, we will discuss how ER stress affects lipid metabolism and their interplay in the development of cardiovascular disorders. Further, the current therapeutics available to target ER stress and lipid metabolism in various CVDs will be summarized.

FRI091 Treatment Of Secondary Hyperlipidemia Due To Nephrotic Syndrome

Abstract Disclosure: J. Girard: None. H. Quadri: None. B. Jiang: None. B.K. Force: None. L. Abushamat: None. R.S. Aguirre: None. Introduction: Secondary Hyperlipidemia associated with nephrotic syndrome contributes to significant cardiovascular morbidity. The elevated atherogenic lipoprotein levels are caused by upregulation of HMG-CoA reductase and downregulation of lipoprotein lipase. More recently, studies in mice have linked PCSK9 and Inducible Degrader of the LDL Receptor (IDOL) as primary contributors to hypercholesterolemia in nephrotic syndrome due to the decrease in LDL receptor recycling (1). Treating nephrotic syndrome is the first-line treatment for the associated secondary hyperlipidemia. Since exposure time to atherogenic lipoproteins matters in the development of cardiovascular disease, statins, with or without ezetimibe, are utilized. PCSK9 inhibitors are initiated if LDL remains elevated despite these measures. This case report highlights a severe case of hypercholesterolemia associated with nephrotic syndrome and treatment response. Clinical Case: A 55-year-old male with no significant past medical history presented to the emergency department with shortness of breath and lower extremity edema. He denied a family and personal history of premature cardiovascular disease. Initial evaluation revealed a serum total cholesterol on 10x dilution of 833.9 (</=200mg/dL), calculated LDL 718mg/dL (<100mg/dL), triglycerides 381mg/dL (<150mg/dL), HDL 39.3mg/dL (40-60mg/dL), Apo B >400mg/dL (<90mg/dL), and Lp(a) 17.5mg/dL (<75mg/dL). His serum albumin was <1.5g/dL (4.2-4.5g/dL), creatinine 2mg/dL (0.7-1.3mg/dL), and 24-hour calculated urinary protein level 10,294mg/24-hour (30-150mg/24-hour). A kidney biopsy revealed a mixed Type III and Type V Lupus Nephritis. The patient started lupus treatment along with atorvastatin 80mg qHS, fenofibrate 45mg daily, and ezetimibe 10mg daily. His LDL decreased to 418mg/dL (<100mg/dL) after 15 days. The patient continued to have undetectable albumin although kidney function improved after 2 months. We planned to initiate PCSK9 inhibitor treatment for further LDL reduction pending treatment of lupus, but patient died from septic shock prior to initiation. Conclusion: This case illustrates that extremely elevated LDL levels from nephrotic syndrome resulted in a nearly 60% reduction of LDL with statin and ezetimibe treatment. Statins, ezetimibe and PCSK9 inhibitor therapy should be considered stepwise when treating secondary hyperlipidemia from nephrotic syndrome. 1. LIU S, VAZIRI ND. ROLE OF PCSK9 AND IDOL IN THE PATHOGENESIS OF ACQUIRED LDL RECEPTOR DEFICIENCY AND HYPERCHOLESTEROLEMIA IN NEPHROTIC SYNDROME. NEPHROL DIAL TRANSPLANT. 2014 MAR;29(3):538-43. DOI: 10.1093/NDT/GFT439. EPUB 2013 OCT 28. PMID: 24166456 Presentation: Friday, June 16, 2023

Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate

The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe peroxisome associated developmental disorders. Despite its pathophysiological importance, mechanistic details of the heterohexamer are not yet available. Here, we report cryoEM structures of Pex1/Pex6 from Saccharomyces cerevisiae, with an endogenous protein substrate trapped in the central pore of the catalytically active second ring (D2). Pairs of Pex1/Pex6(D2) subdomains engage the substrate via a staircase of pore-1 loops with distinct properties. The first ring (D1) is catalytically inactive but undergoes significant conformational changes resulting in alternate widening and narrowing of its pore. These events are fueled by ATP hydrolysis in the D2 ring and disengagement of a “twin-seam” Pex1/Pex6(D2) heterodimer from the staircase. Mechanical forces are propagated in a unique manner along Pex1/Pex6 interfaces that are not available in homo-oligomeric AAA-ATPases. Our structural analysis reveals the mechanisms of how Pex1 and Pex6 coordinate to achieve substrate translocation.