Multifunctional Adaptor Research Articles

Decoding poly (RC)-binding protein 1 (PCBP1), the underrated guard at the foothill of ferroptosis.

PCBP1 is a multifunctional adaptor protein, whose function as an iron chaperone and epigenetic regulator of several chemical messengers involved in ferroptosis has garnered much attention. Herein, this review, several attempts have been made to simplify our understanding of the complex roles of PCBP1. The review begins by elucidating the relevance of PCBP1 in key events governing ferroptosis. We expeditiously shed light on some of the important mechanisms that have critical implications for the ferroptosis landscape. For instance, senescence, EMT, hypoxia, and regulation of the cell cycle and immune checkpoints, among others, have been demonstrated to influence ferroptosis sensitivity to varying degrees. Thus, this review entails a conscious attempt to carefully examine the relevance of PCBP1 in such potential mechanisms. Furthermore, we investigated the therapeutic relevance of PCBP1 in tumor biology and autoimmunity, while underscoring the contrasting perspective of ferroptosis targeting across the disease spectrum. Finally, we debate the different strategies that can be exploited to target PCBP1 in promoting or inhibiting ferroptosis.

SQSTM1/p62 predicts prognosis and upregulates the transcription of CCND1 to promote proliferation in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a rare, highly invasive non-Hodgkin's lymphoma. The main pathogenesis of MCL is associated with the formation of the IgH/CCND1 fusion gene and nuclear overexpression of cyclin D1, which accelerates the cell cycle, leading to tumorigenesis. The prognosis with current standard chemotherapy is still unsatisfactory. SQSTM1/p62 is a multifunctional adaptor that plays an important role in various tumors. Here, we found that the expression of p62 in MCL tissues was higher than that in hyperplastic lymphadenitis patients. Patients with low p62 expression in MCL cells had better overall survival and progression-free survival rates than those with high expression (p = 0.024 and p = 0.025, respectively). Multivariate Cox analysis indicated that the calculated death risk (hazard ratio [HR]) in patients with high expression levels of p62 increased to 2.742 (95% confidence interval (CI) of 1.268-5.852, p = 0.01), which was higher than those with low levels. Silencing p62 impaired Jeko-1 and Granta519 cell proliferation while downregulating CCND1 mRNA and protein expression, thereby inducing G0/G1 cell cycle arrest. However, silencing p62 does not affect the fusion of IgH and CCND1. Luciferase reporter gene analysis and chromatin immunoprecipitation analysis demonstrated that p62 may regulate CCND1 gene expression through Nrf2. These results provide evidence that p62 can predict poor prognosis in MCL. The precise targeting of p62 therapy reduces the promoting effect of Nrf2 on CCND1, thereby preventing cell cycle progression and effectively inhibiting tumor proliferation. Therefore, p62 may provide a potential target for MCL.

Molecular Basis of the Recognition of the Active Rab8a by Optineurin

Optineurin (OPTN), a multifunctional adaptor protein in mammals, plays critical roles in many cellular processes, such as vesicular trafficking and autophagy. Notably, mutations in optineurin are directly associated with many human diseases, such as amyotrophic lateral sclerosis (ALS). OPTN can specifically recognize Rab8a and the GTPase-activating protein TBC1D17, and facilitate the inactivation of Rab8a mediated by TBC1D17, but with poorly understood mechanism. Here, using biochemical and structural approaches, we systematically characterize the interaction between OPTN and Rab8a, revealing that OPTN selectively recognizes the GTP-bound active Rab8a through its leucine-zipper domain (LZD). The determined crystal structure of OPTN LZD in complex with the active Rab8a not only elucidates the detailed binding mechanism of OPTN with Rab8a but also uncovers a unique binding mode of Rab8a with its effectors. Furthermore, we demonstrate that the central coiled-coil domain of OPTN and the active Rab8a can simultaneously interact with the TBC domain of TBC1D17 to form a ternary complex. Finally, based on the OPTN LZD/Rab8a complex structure and relevant biochemical analyses, we also evaluate several known ALS-associated mutations found in the LZD of OPTN. Collectively, our findings provide mechanistic insights into the interaction of OPTN with Rab8a, expanding our understanding of the binding modes of Rab8a with its effectors and the potential etiology of diseases caused by OPTN mutations.

Myeloid beta-arrestin 2 depletion attenuates metabolic dysfunction-associated steatohepatitis via the metabolic reprogramming of macrophages

Macrophage-mediated inflammation has been implicated in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH); however, the immunometabolic program underlying the regulation of macrophage activation remains unclear. Beta-arrestin 2, a multifunctional adaptor protein, is highly expressed in bone marrow tissues and macrophages and is involved in metabolism disorders. Here, we observed that β-arrestin 2 expression was significantly increased in the liver macrophages and circulating monocytes of patients with MASH compared with healthy controls and positively correlated with the severity of metabolic dysfunction-associated steatotic liver disease (MASLD). Global or myeloid Arrb2 deficiency prevented the development of MASH in mice. Further study showed that β-arrestin 2 acted as an adaptor protein and promoted ubiquitination of immune responsive gene 1 (IRG1) to prevent increased itaconate production in macrophages, which resulted in enhanced succinate dehydrogenase activity, thereby promoting the release of mitochondrial reactive oxygen species and M1 polarization. Myeloid β-arrestin 2 depletion may be a potential approach for MASH.

Mechanism of multifunctional adaptor protein SHARPIN regulating myocardial fibrosis and how SNP mutation affect the prognosis of myocardial infarction

Myocardial fibrosis (MF) is characterized by the excessive deposition of extracellular matrix within the heart, often following a cardiovascular insult. SHARPIN, a protein implicated in fibrosis, has emerged as a potential therapeutic target. This study aimed to elucidate the molecular mechanisms of SHARPIN in MF and to investigate the influence of its single nucleotide polymorphism (SNP), rs117299156, on myocardial infarction (MI) patients. A mouse model of Angiotensin II (AngII)-induced MF was established in SHARPIN heterozygous (SHARPIN+/−) and wild-type mice. Adult mouse cardiac fibroblasts (CFs) were isolated and subjected to adenovirus-encapsulated SHARPIN short hairpin RNA (shRNA) infection. Transcriptomic analysis was performed on CFs from SHARPIN+/− and wild-type (WT) mice, complemented by single-cell sequencing data from human cardiac tissues. Additionally, the association between the rs117299156 mutation and cardiovascular events in MI patients was assessed. Our findings indicate that SHARPIN is predominantly expressed in CFs and is upregulated in fibrotic myocardium. Partial knockdown of SHARPIN in murine hearts mitigated AngII-induced cardiac dysfunction and MF. Furthermore, reduced SHARPIN expression in CFs attenuated TGF-β1-induced collagen synthesis, cell proliferation, and myofibroblast transformation. Notably, MI patients carrying the rs117299156_C allele exhibited a reduced incidence of stroke events compared to those without the mutation.

Iridium(III) carbene complexes as potent girdin inhibitors against metastatic cancers

Many cancer-driving protein targets remain undruggable due to a lack of binding molecular scaffolds. In this regard, octahedral metal complexes with unique and versatile three-dimensional structures have rarely been explored as inhibitors of undruggable protein targets. Here, we describe antitumor iridium(III) pyridinium-N-heterocyclic carbene complex 1a, which profoundly reduces the viability of lung and breast cancer cells as well as cancer patient-derived organoids at low micromolar concentrations. Compound 1a effectively inhibits the growth of non-small-cell lung cancer and triple-negative breast cancer xenograft tumors, impedes the metastatic spread of breast cancer cells, and can be modified into an antibody-drug conjugate payload to achieve precise tumor delivery in mice. Identified by thermal proteome profiling, an important molecular target of 1a in cellulo is Girdin, a multifunctional adaptor protein that is overexpressed in cancer cells and unequivocally serves as a signaling hub for multiple pivotal oncogenic pathways. However, specific small-molecule inhibitors of Girdin have not yet been developed. Notably, 1a exhibits high binding affinity to Girdin with a Kd of 1.3 μM and targets the Girdin-linked EGFR/AKT/mTOR/STAT3 cancer-driving pathway, inhibiting cancer cell proliferation and metastatic activity. Our study reveals a potent Girdin-targeting anticancer compound and demonstrates that octahedral metal complexes constitute an untapped library of small-molecule inhibitors that can fit into the ligand-binding pockets of key oncoproteins.

Abstract 3026: Genetic Deletion Of Skeletal Muscle Bag3 Exacerbates Ischemic Myopathy

Background: Bcl2-associated athanogene 3 (BAG3) is a multifunctional adaptor protein that regulates diverse cellular functions. Mutations in BAG3 are causally linked to myofibrillar myopathy and dilated cardiomyopathy in humans and now appear to be one of the most common to cause human disease. We previously identified a murine coding variant in Bag3 that contributes to hindlimb ischemia (HLI) pathology, however, no cell specific data on Bag3’s role in PAD pathology exists. Hypothesis: We hypothesized that muscle specific BAG3 is required for both the restoration of blood flow and muscle recovery following HLI. Methods: We generated a genetic model of lifelong muscle cell (mature fiber and progenitor) Bag3 deletion (Pax7-Cre; Bag3 -/- ) on a BL6 parental background. Male and Female mice (12-20 weeks; WT: n=45; KO: n=32) were evaluated at baseline and HLI d7 and d28 for blood flow via LDPI, isometric muscle force production, vascular density and muscle morphology. Bag3 deletion was validated by recombination, qRT-PCR and Western Blotting in whole tissue, isolated myofibers, and FACS muscle progenitor cells. Results: KO reduced muscle force production (n=18; 145.8mN Force ± 28.6 vs 56.9 ± 16.8 SEM; p=0.0208) and capillary perfusion (n=7; 0.17 CD31+/Lectin+ vessels ± 0.02 vs 0.09 ± 0.01 SEM p=0.0491) indicative of the multi-compartmental effect of BAG3 loss in skeletal muscle at HLI d7. Extended HLI (d28) revealed a KO phenotype characterized by reduced blood flow recovery (LDPI; n= 7; .68 ischemic/non limb ± 0.5 vs .38 ± 0.6 SEM p=0.0011) and sustained myopathy including reduced myofiber size ( n =7; 1585um 2 ± 161 vs 1093 ± 169 SEM; p =0.05) and increased tissue lesion size ( n =7; 2.5% muscle area ± 2.0 vs 48.3% ± 21.6; p =0.03). Conclusion: Loss of skeletal muscle Bag3 exacerbates myopathy and reduces capillary perfusion and blood flow recovery following HLI and demonstrate a requirement for BAG3 in recovery from limb ischemia.

Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice.

p62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC). This study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing. No protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.

Elevated aerobic glycolysis driven by p62-mTOR axis promotes arsenic-induced oncogenic phenotypes in human mammary epithelial cells.

Chronic arsenic exposure is considered to increase the risk of breast cancer. p62 is a multifunctional adaptor protein that controls myriad cellular processes and is overexpressed in breast cancer tissues. Although previous studies have indicated the involvement of p62 accumulation in arsenic tumorigenesis, the underlying mechanism remains obscure. Here, we found that 0.1 µM or 0.5 µM arsenite exposure for 24 weeks induced oncogenic phenotypes in human mammary epithelial cells. Elevated aerobic glycolysis, cell proliferation capacity, and activation of p62-mTOR pathway, as indicated by increased protein levels of p62, phosphorylated-mTOR (p-mTOR) and hypoxia-inducible factor 1α (HIF1α), were observed in chronically arsenite-exposed cells, and of note in advance of the onset of oncogenic phenotypes. Moreover, p62 silencing inhibited acquisition of oncogenic phenotypes in arsenite-exposed cells. The protein levels of p-mTOR and HIF1α, as well as aerobic glycolysis and cell proliferation, were suppressed by p62 knockdown. In addition, re-activation of p‑mTOR reversed the inhibitory effects of p62 knockdown. Collectively, our data suggest that p62 exerts an oncogenic role via mTORC1 activation and acts as a key player in glucose metabolism during arsenite-induced malignant transformation, which provides a new mechanistic clue for the arsenite carcinogenesis.

Mechanistic insights into the interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins

TAX1BP1, a multifunctional autophagy adaptor, plays critical roles in different autophagy processes. As an autophagy receptor, TAX1BP1 can interact with RB1CC1, NAP1, and mammalian ATG8 family proteins to drive selective autophagy for relevant substrates. However, the mechanistic bases underpinning the specific interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins remain elusive. Here, we find that there are two distinct binding sites between TAX1BP1 and RB1CC1. In addition to the previously reported TAX1BP1 SKICH (skeletal muscle and kidney enriched inositol phosphatase (SKIP) carboxyl homology)/RB1CC1 coiled-coil interaction, the first coiled-coil domain of TAX1BP1 can directly bind to the extreme C-terminal coiled-coil and Claw region of RB1CC1. We determine the crystal structure of the TAX1BP1 SKICH/RB1CC1 coiled-coil complex and unravel the detailed binding mechanism of TAX1BP1 SKICH with RB1CC1. Moreover, we demonstrate that RB1CC1 and NAP1 are competitive in binding to the TAX1BP1 SKICH domain, but the presence of NAP1's FIP200-interacting region (FIR) motif can stabilize the ternary TAX1BP1/NAP1/RB1CC1 complex formation. Finally, we elucidate the molecular mechanism governing the selective interactions of TAX1BP1 with ATG8 family members by solving the structure of GABARAP in complex with the non-canonical LIR (LC3-interacting region) motif of TAX1BP1, which unveils a unique binding mode between LIR and ATG8 family protein. Collectively, our findings provide mechanistic insights into the interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins and are valuable for further understanding the working mode and function of TAX1BP1 in autophagy.

Sequestosome 1 (p62) mitigates hypoxia-induced cardiac dysfunction by stabilizing hypoxia-inducible factor 1α and nuclear factor erythroid 2-related factor 2.

Heart failure due to ischaemic heart disease (IHD) is a leading cause of mortality worldwide. A major contributing factor to IHD-induced cardiac damage is hypoxia. Sequestosome 1 (p62) is a multi-functional adaptor protein with pleiotropic roles in autophagy, proteostasis, inflammation, and cancer. Despite abundant expression in cardiomyocytes, the role of p62 in cardiac physiology is not well understood. We hypothesized that cardiomyocyte-specific p62 deletion evokes hypoxia-induced cardiac pathology by impairing hypoxia-inducible factor 1α (Hif-1α) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling. Adult mice with germline deletion of cardiomyocyte p62 exhibited mild cardiac dysfunction under normoxic conditions. Transcriptomic analyses revealed a selective impairment in Nrf2 target genes in the hearts from these mice. Demonstrating the functional importance of this adaptor protein, adult mice with inducible depletion of cardiomyocyte p62 displayed hypoxia-induced contractile dysfunction, oxidative stress, and cell death. Mechanistically, p62-depleted hearts exhibit impaired Hif-1α and Nrf2 transcriptional activity. Because findings from these two murine models suggested a cardioprotective role for p62, mechanisms were evaluated using H9c2 cardiomyoblasts. Loss of p62 in H9c2 cells exposed to hypoxia reduced Hif-1α and Nrf2 protein levels. Further, the lack of p62 decreased Nrf2 protein expression, nuclear translocation, and transcriptional activity. Repressed Nrf2 activity associated with heightened Nrf2-Keap1 co-localization in p62-deficient cells, which was concurrent with increased Nrf2 ubiquitination facilitated by the E3 ligase Cullin 3, followed by proteasomal-mediated degradation. Substantiating our results, a gain of p62 in H9c2 cells stabilized Nrf2 and increased the transcriptional activity of Nrf2 downstream targets. Cardiac p62 mitigates hypoxia-induced cardiac dysfunction by stabilizing Hif-1α and Nrf2.

Four-and-a-Half LIM-Domain Protein 2 (FHL2) Induces Neuropeptide Y (NPY) in Macrophages in Visceral Adipose Tissue and Promotes Diet-Induced Obesity.

Obesity is characterized by the expansion of the adipose tissue, usually accompanied by inflammation, with a prominent role of macrophages infiltrating the visceral adipose tissue (VAT). This chronic inflammation is a major driver of obesity-associated comorbidities. Four-and-a-half LIM-domain protein 2 (FHL2) is a multifunctional adaptor protein that is involved in the regulation of various biological functions and the maintenance of the homeostasis of different tissues. In this study, we aimed to gain new insights into the expression and functional role of FHL2 in VAT in diet-induced obesity. We found enhanced FHL2 expression in the VAT of mice with Western-type diet (WTD)-induced obesity and obese humans and identified macrophages as the cellular source of enhanced FHL2 expression in VAT. In mice with FHL2 deficiency (FHL2KO), WTD feeding resulted in reduced body weight gain paralleled by enhanced energy expenditure and uncoupling protein 1 (UCP1) expression, indicative of activated thermogenesis. In human VAT, FHL2 was inversely correlated with UCP1 expression. Furthermore, macrophage infiltration and the expression of the chemokine MCP-1, a known promotor of macrophage accumulation, was significantly reduced in WTD-fed FHL2KO mice compared with wild-type (wt) littermates. While FHL2 depletion did not affect the differentiation or lipid metabolism of adipocytes in vitro, FHL2 depletion in macrophages resulted in reduced expressions of MCP-1 and the neuropeptide Y (NPY). Furthermore, WTD-fed FHL2KO mice showed reduced NPY expression in VAT compared with wt littermates, and NPY expression was enhanced in VAT resident macrophages of obese individuals. Stimulation with recombinant NPY induced not only UCP1 expression and lipid accumulation but also MCP-1 expression in adipocytes. Collectively, these findings indicate that FHL2 is a positive regulator of NPY and MCP-1 expression in macrophages and herewith closely linked to the mechanism of obesity-associated lipid accumulation and inflammation in VAT. Thus, FHL2 appears as a potential novel target to interfere with the macrophage-adipocyte crosstalk in VAT for treating obesity and related metabolic disorders.

Bacterial Outer Membrane‐Based Biomimetic Immune Adaptors: Mild Immunomodulatory and Bacterial Targeted Delivery Strategy Against Implant‐Related Infections

AbstractImplant‐related infections (IRIs) are difficult to manage and are a major cause of surgical failure. The formation of biofilms with a solid barrier on the surface of implants and concomitant suppression of the local immune response constitute major challenges for treating IRIs. This study develops a multifunctional biomimetic immune adaptor (Hybrid Membrane@Levo@SiO2; HMLS) generated by loading the antibiotic levofloxacin (Levo) into silica nanoparticles and coating the particles with a hybrid membrane formed from outer membrane vesicles (OMVs) of Escherichia coli and red blood cell membranes (RBCMs). The HMLS nanoplatform is designed to address the challenges associated with IRIs through a dual‐approach strategy. First, the unique immunogenicity of OMVs regulates the antibacterial immune responses of innate immune cells and reverses the immunosuppressive microenvironment around IRIs. Second, the homotypic targeting effect of OMVs in the hybrid membrane provides HMLS with bacterial targeting functions and efficient bactericidal effects. Additionally, the RBCMs endow the hybrid membrane with mild immunogenicity, which prevents excessive inflammatory stimulation. The synergistic strategy of HMLS exhibits superior antibiofilm effects both in vivo and in vitro, providing a new option for the clinical treatment of refractory IRIs.

Multi-functional adaptor SKAP1: regulator of integrin activation, the stop-signal, and the proliferation of T cells.

T-cell activation is a complex process involving a network of kinases and downstream molecular scaffolds or adaptors that integrate surface signals with effector functions. One key immune-specific adaptor is Src kinase-associated phosphoprotein 1 (SKAP1), which is also known as src kinase-associated protein of 55 kDa (SKAP55). This mini-review explains how SKAP1 plays multiple roles in regulating integrin activation, the "stop-signal", and the optimization of the cell cycling of proliferating T cells through interactions with various mediators, including the Polo-like kinase 1 (PLK1). Ongoing research on SKAP1 and its binding partners will likely provide important insights into the regulation of immune function and have implications for the development of new treatments for disease states such as cancer and autoimmunity.

Impact of stress on cardiac phenotypes in mice harboring an ankyrin-B disease variant

Encoded by ANK2, ankyrin-B (AnkB) is a multifunctional adapter protein critical for the expression and targeting of key cardiac ion channels, transporters, cytoskeletal-associated proteins, and signaling molecules. Mice deficient for AnkB expression are neonatal lethal, and mice heterozygous for AnkB expression display cardiac structural and electrical phenotypes. Human ANK2 loss-of-function variants are associated with diverse cardiac manifestations; however, human clinical 'AnkB syndrome' displays incomplete penetrance. To date, animal models for human arrhythmias have generally been knock-out or transgenic overexpression models and thus the direct impact of ANK2 variants on cardiac structure and function invivo is not clearly defined. Here, we directly tested the relationship of a single human ANK2 disease-associated variant with cardiac phenotypes utilizing a novel invivo animal model. At baseline, young AnkBp.E1458G+/+ mice lacked significant structural or electrical abnormalities. However, aged AnkBp.E1458G+/+ mice displayed both electrical and structural phenotypes at baseline including bradycardia and aberrant heart rate variability, structural remodeling, and fibrosis. Young and old AnkBp.E1458G+/+ mice displayed ventricular arrhythmias following acute (adrenergic) stress. In addition, young AnkBp.E1458G+/+ mice displayed structural remodeling following chronic (transverse aortic constriction) stress. Finally, AnkBp.E1458G+/+ myocytes harbored alterations in expression and/or localization of key AnkB-associated partners, consistent with the underlying disease mechanism. In summary, our findings illustrate the critical role of AnkB in invivo cardiac function as well as the impact of single AnkB loss-of-function variants invivo. However, our findings illustrate the contribution and in fact necessity of secondary factors (aging, adrenergic challenge, pressure-overload) to phenotype penetrance and severity.

Abstract 553: Loss Of Endothelial Bag3 Does Not Worsen Ischemia Induced Myopathy

Chronic limb threatening ischemia (CLTI), the most severe manifestation of peripheral artery disease, isaccompanied by high rates of limb amputation and death. There is a pressing need for novel therapydevelopment in these patients, which will require a thorough understanding of the individual cellularcontributions to ischemic limb pathology. BAG3 is a multifunctional adaptor protein that regulatesdiverse cellular functions. Mutations in BAG3 now appear to be one of the most common to causedisease in the clinical setting, including predisposing Black patients to cardiovascular disease. Wepreviously identified a coding variant in Bag3 that contributes to ischemic muscle necrosis after murinehindlimb ischemia (HLI) that was rescued by gene therapy, although we do not know which cell typesrequire Bag3 individually to mount efficient responses to ischemia. We hypothesized that vascularendothelial cell Bag3 would be required for both the restoration of blood flow and muscle function afterHLI. To test this hypothesis, we generated an inducible genetic model of endothelial cell Bag3 deletion(Cdh5(PAC)-CreERT2;Bag3 -/- ). At 20 weeks of age, mice were administered either tamoxifen (KO; n =20) orvehicle (CON, n =28) and evaluated at baseline or subjected to HLI for 7-days. Endothelial cell specific Bag3 deletion was validated first by genotyping and then by qRT-PCR in flow sorted cells. KO did notalter animal body weights, heart or skeletal muscle wet weights, physiologic muscle function (extensordigitorum longus absolute or specific force) or muscle histology (no regression of limb muscle tissuevascular cells) at baseline when compared with CON. After HLI, KO did not cause limb tissue necrosis,reduce limb blood flow recovery (laser doppler perfusion imaging) or physiologic muscle function, anddid not worsen histological damage to the limb muscle when compared with CON. Our study providesevidence for the first time that loss of endothelial cell Bag3 does not exacerbate myopathy and delaylimb blood flow recovery after hindlimb ischemia and suggests that the benefits of Bag3 gene therapymay be driven by the muscle cell compartment of the ischemic limb.

Abstract P2-25-03: β-arrestin1, a potential tumour suppressor in breast cancer is downregulated in PERK/ATF4-dependent manner

Abstract Introduction: β-Arrestin1 (ARRB1) belongs to the arrestin family originally identified as a multifunctional adaptor protein that negatively regulates the desensitization and internalization of G-protein-coupled receptors. Recent studies have indicated that ARRB1 can form multiprotein complexes with transcription regulators, thereby indirectly inhibiting the function of some transcription factors, e.g., NF-kB, and promoting the activity of others, e.g., p53. Further, arrestins function as multifunctional adaptors in many signalling pathways, such as the Hedgehog (Hh), Wingless, Notch, and transforming growth factor-β (TGF-β) pathways. Expression of ARRB1 is downregulated in TNBC patients and ARRB1 expression level is inversely correlated with the histological grade of the breast cancer and positively associated with TNBC patient survival, suggestive of a tumour-suppressive function of ARRB1 in breast cancer. Loss of ARRB1 expression is associated with a poor prognosis for non-small cell lung cancer patients. However, the mechanisms regulating the expression of ARRB1 in human cancers remain unclear. We hypothesized that metabolic endoplasmic reticulum (EnR) stressors including low glucose and hypoxia that activate an unfolded protein response (UPR) in tumour cells might regulate the expression of ARRB1 in human cancers. Methods: Expression of ARRB1 was determined by a combination of real-time RT-PCR, western blotting along with promoter reporter assays. Computational target prediction analysis was done to identify miRNAs targeting ARRB1 as well as transcription factors regulating ARRB1 expression. ARRB1overexpressing breast cancer clones were generated by lentiviral transduction to investigate its functional role in tumour progression and drug sensitivity. Results: We observed that ARRB1 expression was significantly reduced in several cancers including breast tumours as compared to normal tissue and decreased expression of ARRB1 was associated with poor prognosis in Luminal A subtype. We found significant downregulation of ARRB1 mRNA and protein during conditions of EnR stress in multiple ER-positive breast cancer cell lines. Using PERK, ATF6 and XBP1 knockdown sub-clones of MCF7 we observed that PERK signalling was required for the downregulation of ARRB1. ATF4 and miR-204/211 downstream of PERK were identified as key regulators of ARRB1 expression. Conclusion: Our results show that ATF4 and miR-204/211 downstream of the PERK signalling pathway repress ARRB1 expression in breast cancer cells. Our results suggest that the downregulation of ARRB1 by EnR stressors in the tumour microenvironment may contribute to breast cancer progression. Citation Format: Afrin Sultana, Ananya Gupta, Sanjeev Gupta. β-arrestin1, a potential tumour suppressor in breast cancer is downregulated in PERK/ATF4-dependent manner [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-25-03.

Deletion of Arrb2 Down-regulates Autophagy in the Mouse Hippocampus via Akt-mTOR Pathway Activation

The cytoplasmic multifunctional adaptor protein β-arrestin 2 (Arrb2) is involved in the occurrence of various nervous system diseases, such as Alzheimer's disease and Parkinson's disease. Previous laboratory studies have shown that the expression and function of the Arrb2 gene was increased in valproic acid-induced autistic mice models. However, few reports have examined the possible role of Arrb2 in the pathogenesis of autism spectrum disorder. Therefore, Arrb2-deficient (Arrb2−/−) mice were further studied to uncover the physiological function of Arrb2 in the nervous system. In this study, we found that Arrb2−/− mice had normal behavioral characteristics compared with wild-type mice. The autophagy marker protein LC3B was decreased in the hippocampus of Arrb2−/− mice compared to wild-type mice. Western blot analysis revealed that deletion of Arrb2 caused hyperactivation of Akt-mTOR signaling in the hippocampus. In addition, abnormal mitochondrial dysfunction was observed in Arrb2−/− hippocampal neurons, which was characterized by a reduction in mitochondrial membrane potential and adenosine triphosphate production and an increase in reactive oxygen species levels. Therefore, this study elucidates the interaction between Arrb2 and the Akt-mTOR signaling pathway and provides insights into the role of Arrb2 in hippocampal neuron autophagy.

Mechanisms of action of cytoplasmic microRNAs. Part 6. MicroRNA-mediated translation activation

In the scientific review, the mechanisms of action of cytoplasmic miRNAs, namely miRNA-mediated activation of translation, are given. To write the article, information was searched using Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library databases. Examples of direct activation of mRNA translation by miRNA are presented. One of them is miRNA-mediated activation of translation, which is associated with the peculiarities of the state of the cell (resting cell effect). It has been shown that protein 1 of the fragile X mental retardation (FMR1) syndrome, depending on the stage of the cell cycle, can participate in both inhibition and enhancement of translation. It is known that microRNAs can influence the activity of RNP by binding to the RNA-binding sites of specific mRNAs or directly to RBP molecules, directly inhibiting their activity. Poly (rC) binding protein 2 (PCBP2) is a multifunctional adapter molecule that binds to RNA and DNA, competing with other RNA-binding factors. The PCBP2 protein limits translation initiation by preventing ribosome recruitment. The authors provided information on miR-346-mediated activation of the translation of receptor-interacting protein 140. It is emphasized that some miRNAs, preventing the degradation of the mRNA molecule, increasе the level of its stability, which is accompanied by an enhancement in their translation. MicroRNAs stabilize specific mRNA targets, preventing the association of the ARE element degradation factor, tristetraprolin, with mRNA. Data are presented on the activation of mRNA target translation by factors that sequester miRNAs or compete with miRNAs. Various intracellular factors and proteins can enter into a competitive relationship with miRNA and interfere with or remove it from the target mRNA. It is known that activation of translation can occur due to microRNA inhibition of repressor proteins. The authors indicate that increased miR-145 expression is accompanied by activation of myocardin translation, which induces the proliferation and migration of smooth muscle cells.

Generation of FOUR iPSC lines (CRICKi004-A; CRICKi005-A; CRICKi006-A, CRICKi007-A) from Spinal muscle atrophy patients with lower extremity dominant (SMALED) phenotype

Spinal muscular atrophy with lower extremity dominant (SMALED) is a hereditary neuromuscular disorder characterized by degeneration of spinal cord motor neurons resulting in lower limbs muscle weakness and paralysis. Mutations in DYNC1H1, which encodes BICD2, a multifunctional adaptor for microtubule motor proteins, cause the disorder. Here, we generated four induced pluripotent stem cell (iPSC) lines from patients with SMALED. Dermal fibroblasts were obtained from the MRC neuromuscular disease biobank and reprogrammed using non-integrating mRNA-based protocol. Characterization of the four iPSC lines included karyotyping and Sanger sequencing, while the expression of associated markers confirmed pluripotency and differentiation potential.