Smaller Isoform Research Articles

Rap1A Modulates Store-Operated Calcium Entry in the Lung Endothelium: A Novel Mechanism Controlling NFAT-Mediated Vascular Inflammation and Permeability.

Store-operated calcium entry mediated by STIM (stromal interaction molecule)-1-Orai1 is essential in endothelial cell (EC) functions, affecting signaling, NFAT (nuclear factor for activated T cells)-induced transcription, and metabolic programs. While the small GTPase Rap1 isoforms, including the predominant Rap1B, are known for their role in cadherin-mediated adhesion, EC deletion of Rap1A after birth uniquely disrupts lung endothelial barrier function. Here, we elucidate the specific mechanisms by which Rap1A modulates lung vascular integrity and inflammation. The role of EC Rap1A in lung inflammation and permeability was examined using in vitro and in vivo approaches. We explored Ca2+ signaling in human ECs following siRNA-mediated knockdown of Rap1A or Rap1B. Rap1A knockdown, unlike Rap1B, significantly increased store-operated calcium entry in response to a GPCR (G-protein-coupled receptor) agonist, ATP (500 µmol/L), or thapsigargin (250 nmol/L). This enhancement was attenuated by Orai1 channel blockers 10 μmol/L BTP2, 10 μmol/L GSK-7975A, and 5 μmol/L Gd3+. Whole-cell patch clamp measurements revealed enhanced Ca2+ release-activated Ca2+ current density in siRap1A ECs. Rap1A depletion in ECs led to increased NFAT1 nuclear translocation and activity and elevated levels of proinflammatory cytokines (CXCL1, CXCL11, CCL5 [chemokine (C-C motif) ligand 5], and IL-6 [interleukin-6]). Notably, reducing Orai1 expression in siRap1A ECs normalized store-operated calcium entry, NFAT activity, and endothelial hyperpermeability in vitro. EC-specific Rap1A knockout (Rap1AiΔEC) mice displayed an inflammatory lung phenotype with increased lung permeability and inflammation markers, along with higher Orai1 expression. Delivery of siRNA against Orai1 to lung endothelium using lipid nanoparticles effectively normalized Orai1 levels in lung ECs, consequently reducing hyperpermeability and inflammation in Rap1AiΔEC mice. Our findings uncover a novel role of Rap1A in regulating Orai1-mediated Ca2+ entry and expression, crucial for NFAT-mediated transcription and endothelial inflammation. This study distinguishes the unique function of Rap1A from that of the predominant Rap1B isoform and highlights the importance of normalizing Orai1 expression in maintaining lung vascular integrity and modulating endothelial functions.

Paroxysmal dystonia results from the loss of RIM4 in Purkinje cells.

Full-length RIM1 and 2 are key components of the presynaptic active zone that ubiquitously control excitatory and inhibitory neurotransmitter release. Here, we report that the function of the small RIM isoform RIM4, consisting of a single C2 domain, is strikingly different from that of the long isoforms. RIM4 is dispensable for neurotransmitter release but plays a postsynaptic, cell type-specific role in cerebellar Purkinje cells that is essential for normal motor function. In the absence of RIM4, Purkinje cell intrinsic firing is reduced and caffeine-sensitive, and dendritic integration of climbing fibre input is disturbed. Mice lacking RIM4, but not mice lacking RIM1/2, selectively in Purkinje cells exhibit a severe, hours-long paroxysmal dystonia. These episodes can also be induced by caffeine, ethanol or stress and closely resemble the deficits seen with mutations of the PNKD (paroxysmal non-kinesigenic dystonia) gene. Our data reveal essential postsynaptic functions of RIM proteins and show non-overlapping specialized functions of a small isoform despite high homology to a single domain in the full-length proteins.

Novel Identification of Ankyrin-R in Cardiac Fibroblasts and a Potential Role in Heart Failure.

Altered ankyrin-R (AnkR; encoded by ANK1) expression is associated with diastolic function, left ventricular remodeling, and heart failure with preserved ejection fraction (HFpEF). First identified in erythrocytes, the role of AnkR in other tissues, particularly the heart, is less studied. Here, we identified the expression of both canonical and small isoforms of AnkR in the mouse myocardium. We demonstrate that cardiac myocytes primarily express small AnkR (sAnkR), whereas cardiac fibroblasts predominantly express canonical AnkR. As canonical AnkR expression in cardiac fibroblasts is unstudied, we focused on expression and localization in these cells. AnkR is expressed in both the perinuclear and cytoplasmic regions of fibroblasts with considerable overlap with the trans-Golgi network protein 38, TGN38, suggesting a potential role in trafficking. To study the role of AnkR in fibroblasts, we generated mice lacking AnkR in activated fibroblasts (Ank1-ifKO mice). Notably, Ank1-ifKO mice fibroblasts displayed reduced collagen compaction, supportive of a novel role of AnkR in normal fibroblast function. At the whole animal level, in response to a heart failure model, Ank1-ifKO mice displayed an increase in fibrosis and T-wave inversion compared with littermate controls, while preserving cardiac ejection fraction. Collagen type I fibers were decreased in the Ank1-ifKO mice, suggesting a novel function of AnkR in the maturation of collagen fibers. In summary, our findings illustrate the novel expression of AnkR in cardiac fibroblasts and a potential role in cardiac function in response to stress.

Cancer-associated snaR-A noncoding RNA interacts with core splicing machinery and disrupts processing of mRNA subpopulations.

RNA polymerase III (Pol III) activity in cancer is linked to the production of small noncoding (nc)RNAs that are otherwise silent in most tissues. snaR-A (small NF90-associated RNA isoform A) - a hominid-specific ncRNA shown to enhance cell proliferation, migration, and invasion - is a cancer-emergent Pol III product that remains largely uncharacterized despite promoting growth phenotypes. Here, we applied a combination of genomic and biochemical approaches to study the biogenesis and subsequent protein interactions of snaR-A and to better understand its role as a putative driver of cancer progression. By profiling the chromatin landscapes across a multitude of primary tumor types, we show that predicted snaR-A upregulation is broadly linked with unfavorable outcomes among cancer patients. At the molecular level, we unexpectedly discover widespread interactions between snaR-A and mRNA splicing factors, including SF3B2 - a core component of the U2 small nuclear ribonucleoprotein (snRNP). We find that SF3B2 levels are sensitive to high snaR-A abundance and that depletion of snaR-A alone is sufficient to decrease intron retention levels across subpopulations of mRNA enriched for U2 snRNP occupancy. snaR-A sensitive genes are characterized by high GC content, close spatial proximity to nuclear bodies concentrated in pre-mRNA splicing factors, and functional enrichment for proteins involved in deacetylation and autophagy. We highlight examples of splicing misregulation and increased protein levels following snaR-A depletion for a wide-ranging set of factors, suggesting snaR-A-driven splicing defects may have far-reaching effects that re-shape the cellular proteome. These findings clarify the molecular activities and consequences of snaR-A in cancer, and altogether establish a novel mechanism through which Pol III overactivity may promote tumorigenesis.

1243 FOXM1*3 Overexpression Correlates to Staging of Astrocytic Glioma

INTRODUCTION: Diffuse gliomas are a group of primary brain tumors characterized by the presence of neoplastic glial-lineage cells. WHO 2021 classification of gliomas recognizes the mutational status of the isocitrate dehydrogenase-1(IDH-1,G395A) gene and the 1p/19q codeletion as identification of cell lineage and a prognosis marker. Transcription factors are binding proteins of genomic DNA and can activate or suppress gene expression. Forkhead box M1(FOXM1) is a transcription factor for Forkhead box(Fox) proteins, a superfamily which is defined by a conserved winged helix DNA binding domain. FOXM1 is a key regulator of both G1/S and G2/M phases of cell cycle and mitotic spindle integrity, it also plays an important role in angiogenesis, DNA damage repair and tissue regeneration. The human FOXM1 gene consists of 10 exons, two of them alternatively spliced. This splicing gives rise to three isoforms: FOXM1*1,*2 and *3. FOXM1*3 is the smallest isoform and is overexpressed in proliferative tissue such as malignant tumors. METHODS: A control group of 10 brain cortex, 22 A2, 13 O2, 11 A3, 3 O3, 6 A4 and 46 GBM with histological and molecular diagnosis of astrocytoma, oligodendroglioma and glioblastoma from 2011 to 2022 were included. The diagnosis correlated with patient outcome. mRNA FOXM1*3 and its housekeeping gen IPO8 was quantified by qPCR with Taqman probes. Data analysis was conducted by MannWhitney U test. RESULTS: There was a statistically significant difference between expression of mRNA FOXM1*3 in normal brain tissue compared to tumor tissue (p < 0.001). The transcript allowed differentiation between grade 2, 3 and 4 astrocytomas (p < 0.005), nevertheless there was no difference between astrocytic versus oligodendrocytic lineage, and grade 4 astrocytoma versus glioblastoma. CONCLUSIONS: The normalized expression of transcript FOXM1*3 allows differentiation of astrocytic gliomas grades.

Nicotinamide deficiency promotes imidacloprid resistance via activation of ROS/CncC signaling pathway-mediated UGT detoxification in Nilaparvata lugens

Metabolic alternation is a typical characteristic of insecticide resistance in insects. However, mechanisms underlying metabolic alternation and how altered metabolism in turn affects insecticide resistance are largely unknown. Here, we report that nicotinamide levels are decreased in the imidacloprid-resistant strain of Nilaparvata lugens, may due to reduced abundance of the symbiotic bacteria Arsenophonus. Importantly, the low levels of nicotinamide promote imidacloprid resistance via metabolic detoxification alternation, including elevations in UDP-glycosyltransferase enzymatic activity and enhancements in UGT386B2-mediated metabolism capability. Mechanistically, nicotinamide suppresses transcriptional regulatory activities of cap ‘n’ collar isoform C (CncC) and its partner small muscle aponeurosis fibromatosis isoform K (MafK) by scavenging the reactive oxygen species (ROS) and blocking the DNA binding domain of MafK. In imidacloprid-resistant N. lugens, nicotinamide deficiency re-activates the ROS/CncC signaling pathway to provoke UGT386B2 overexpression, thereby promoting imidacloprid detoxification. Thus, nicotinamide metabolism represents a promising target to counteract imidacloprid resistance in N. lugens.

Abstract 5974: Targeting VEGFR on tumor cells and activated vasculature through delivery of granzyme B

Abstract Anti-VEGF therapy is a key therapeutic in several cancer treatment regimens. However, none of the current therapeutics are directly cytotoxic against tumor cells. VEGF121 is the smallest isoform in the VEGF-A family of cytokines that binds to VEGF receptors R-1 and R-2. Both receptors are over-expressed on several tumor cells and the endothelium of tumor vasculature but not normal vasculature. We hypothesized that delivery of a cytotoxic payload through VEGFR-2 into tumor cells and tumor neovasculature would significantly enhance cancer therapy. Granzyme B is a key effector in immune-mediated cell killing through both caspase-dependent and -independent mechanisms. We previously reported the development of a fusion protein composed of granzyme B (GrB) and VEGF121 (GrB/VEGF121). Here, we report on GrB-Fc-VEGF121, a fusion protein incorporating a human IgG heavy chain Fc fragment for dimerization to increase the molecular weight and improve in vivo circulation and targeting potential. We expressed and purified GrB-Fc-VEGF121 from HEK-293E cells under serum-free conditions with a final yield of approximately 40 mg/L. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. We characterized this construct against both human and mouse VEGFR+ cell lines as this ligand cross-reacts to both species. GrB-Fc-VEGF121 showed in vitro cytotoxicity in the nanomolar range against tumor and endothelial cell lines expressing high levels of VEGFR-1 or VEGFR-2, while receptor-negative cells demonstrated IC50 levels in the high micromolar range. GrB-Fc-VEGF121 internalized into VEGFR-2+ tumor and endothelial cells within 2 hours of treatment while untargeted GrB did not internalize, suggesting internalization was receptor-mediated. Treatment of VEGFR2+ cells at the respective IC50 dose resulted in >50% cell death via apoptosis and/or necrosis within 48 h. Ex vivo serum stability studies indicated a gradual protein loss of GrB-Fc-VEGF121 with an overall stability of about 50% over 96 hours. No toxicity was observed in mice treated with a total dose of 475 mg/kg (IP, QOD x 5), indicating that the maximum tolerated dose of GrB-Fc-VEGF121 exceeds this dose level. In vivo efficacy studies in an OVCAR8 tumor xenograft model (20 mg/kg GrB-Fc-VEGF121 x 5) resulted in significant growth inhibition of established tumors compared to vehicle controls. The number of CD31+ blood vessels and Ki-67+ proliferating tumor cells decreased significantly in treated tumors compared to controls. Pharmacokinetic studies are underway and will be reported. Thus, GrB-Fc-VEGF121 significantly reduces tumor growth in vivo with no observed toxicity against normal tissues. This tumor- and vascular-targeting agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Targeting VEGFR on tumor cells and activated vasculature through delivery of granzyme B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5974.

Calcium Induces the Cleavage of NopA and Regulates the Expression of Nodulation Genes and Secretion of T3SS Effectors in Sinorhizobium fredii NGR234.

The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR_b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC, genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia-legume interactions.

Cis-Acting Splicing-Associated Variants Can Redefine the Molecular Signature of Genes Commonly Mutated in Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is a blood malignancy that occurs as a result of genomic alterations acquired in hematopoietic stem cells (HSCs). Several studies have recognized the importance of these alterations, including chromosomal rearrangements and single nucleotide variations (SNVs), for the classification and risk stratification of patients. However, none of these studies have thoroughly explored the functional impact of these lesions, potentially leading to the oversight or misinterpretation of certain driver mutations. In this study, we focused on the splicing process, as we aim to comprehensively characterize cis-acting splicing-associated variants (SAVs) in a large cohort of AML patients. Methods: We obtained recurrent driver gene variants (n= 3,847) from Table S5 reported by Papaemmanuil et al. and selected unique SNVs (n= 915) for our analysis. Among them, 628 (69%) were missense variants, 232 (25%) were nonsense variants, and 55 (6%) were located within splice sites. To assess their potential impact on splicing, we employed three splicing predictor tools (MaxEntScan, regSNP-splicing, and SpliceAI) and considered variants with two favorable predictions for further functional studies. Firstly, we attempted to locate the SNVs within the exomes of the TCGA-LAML (n= 149) and the BeatAML (n=342) datasets available in the Genomic Data Commons repository. If an SNV was identified, we obtained the RNA sequencing bam file from the corresponding patient's sample and utilized the rest of the cohort as a control for statistical analysis (p<0.05 for significance). In cases where the SNV was not found in these datasets, we employed the pSPL3-based splicing reporter minigene assay to study the variant. Results: The predictors unanimously identified all 55 splice-site affecting variants as true positives and reclassified 20 missense and nonsense variants as likely SAVs. Functional validation experiments confirmed that 11 of these variants (55%) indeed had an impact on splicing. Analysis of RNA sequencing data revealed interesting findings in commonly observed AML hotspots. For instance, in cases of U2AF1 c.470A>G and IDH1 c.394C>A, novel donor splice sites were created at distances of one (p=0.04522) and 34 nucleotides from the mutations (p=0.01878), respectively. Whereas, the TP53 c.395A>G change was found to activate a cryptic acceptor splice site located XX nucleotides away (p=0). Among private AML variants, minigene assays showed that eight variants resulted in aberrant splicing compared to the wild-type. Three variants led to exon skipping due to the loss of canonical donor splice sites ( EZH2 c.363G>A, FLT3 c.2523C>A, and TET2 c.3500G>A), four variants caused the activation of a novel donor splice site ( PTPN11 c.1508G>T, WT1 c.1157C>A, STAG2 c.545T>G, and KDM6A c.1516C>T), and one variant resulted in both exon skipping and the creation of a donor splice site ( RAD21 c.688G>A). Consequently, the predicted impact on the protein varied, with FLT3 and TET2 variants potentially leading to an in-frame deletion of the ATP-binding site and TET2-oxygenase domain, respectively, with the EZH2 variant likely resulting in an expression switch from the large to small isoform. The remaining SNVs were anticipated to trigger Nonsense-Mediated Decay or shorten proteins. Conclusions: This study revealed that approximately 7% of SNVs found in a large group of AML patients have the potential to impact splicing. Notably, 1% of these SNVs had been classified as missense or nonsense changes. These findings provide support for the significant role of mis-splicing in the development of leukemia and emphasize the importance of devising novel approaches to effectively characterize driver mutations.

Abstract 17153: Mitochondrial Cx43 and Gja1-20k Contribute to Sex-Specific Differences of Mitochondrial Response in Septic Cardiomyopathy

Introduction: Cardiac dysfunction contributes to morbidity and mortality of severe sepsis. Preserving mitochondrial (mito) activity is key to maintaining cardiac function. While epidemiological studies consistently report that male sex is an important risk factor for severe sepsis, it is unknown if male heart mito are susceptible to septic injury. We have shown that male cardiomyocyte mito were more vulnerable to TNFα- or H 2 O 2 -induced stress than females. We have also demonstrated that mito connexin 43 (Cx43 or Gja1) and its smaller isoform - Gja1-20k are implicated in sex-related heart response following myocardial ischemia. Hypothesis : In this study, we hypothesize that during sepsis, female (F) hearts exhibit preserved mito Cx43 and Gja1-20k to confer mitochondrial protection, thus improving cardiac function compared to male (M) myocardium. Methods: Age-matched adult M & F C57BL/6 mice received LPS (7.5 mg/kg BW, i.p.) or PBS (n > 6 mice/group). Cardiac function was detected 20h post LPS by 2D echocardiography. Cx43, Gja1-20k, OXPHOS, & PGC-1 were evaluated in M & F mouse heart mito 24h after LPS by Western Blot (n>3 hearts/group). Mito Cx43 was also detected by Immuno-electron microscopy (EM). Mito membrane potential (ΔΨm) was analyzed in cardiomyocytes (from adult M & F mice, n>3 hearts/group) +/- 1h LPS (1 μg/ml) using JC1 fluorescent probe. Mito respiration capacity was further assessed in H9c2 cells transfected with Gja1 siRNA upon LPS exposure by Seahorse XF Mito Stress test. Results: Decreased cardiac function was observed in M mice after LPS injection, but not in F. Higher mito levels of Cx43, Gja1-20k, OXPHOS, & PGC1 were noticed in F mouse hearts compared to M after LPS. Despite no difference in total Cx43 particles between M & F hearts by EM, more gold Cx43 particles were observed in F heart mito compared to M mito following LPS. LPS reduced ΔΨm in M cardiomyocytes, but not in F. Cx43 knockdown decreased mito maximal respiration capacity & ATP production in H9c2 cells with LPS stress. Conclusions: The mitochondria-derived protective mechanism is preserved in F mice, leading to better cardiac function than M mice following LPS-induced sepsis. F cardiac mito are more resistant to LPS challenge than M mito, partially due to preserved mito Cx43 and/or Gja1-20k.

Identification and quantification of small exon-containing isoforms in long-read RNA sequencing data.

Small exons are pervasive in transcriptomes across organisms, and their quantification in RNA isoforms is crucial for understanding gene functions. Although long-read RNA-seq based on Oxford Nanopore Technologies (ONT) offers the advantage of covering transcripts in full length, its lower base accuracy poses challenges for identifying individual exons, particularly microexons (≤30 nucleotides). Here, we systematically assess small exons quantification in synthetic and human ONT RNA-seq datasets. We demonstrate that reads containing small exons are often not properly aligned, affecting the quantification of relevant transcripts. Thus, we develop a local-realignment method for misaligned exons (MisER), which remaps reads with misaligned exons to the transcript references. Using synthetic and simulated datasets, we demonstrate the high sensitivity and specificity of MisER for the quantification of transcripts containing small exons. Moreover, MisER enabled us to identify small exons with a higher percent spliced-in index (PSI) in neural, particularly neural-regulated microexons, when comparing 14 neural to 16 non-neural tissues in humans. Our work introduces an improved quantification method for long-read RNA-seq and especially facilitates studies using ONT long-reads to elucidate the regulation of genes involving small exons.

Abstract P2022: Ankyrin-R Isoforms Are Differentially Expressed In Cardiac Tissues Under Stress

Heart disease remains the leading cause of death in the United States with heart failure specifically accounting for 13.4% of all heart disease related deaths. In 2019, Andersson et al., utilized a multi-omics approach to evaluate participant samples from the Framingham Heart Study and showed that ankyrin-R (AnkR; encoded by ANK1 ) is significantly associated with diastolic function, left ventricular remodeling and heart failure with preserved ejection fraction. Ankyrins are a family of proteins that link integral membrane proteins with the actin/β-spectrin cytoskeleton. Ankyrins-B/G have been extensively studied and identified within the heart and their dysfunction is associated with cardiac structural and electrical phenotypes. Ankyrin-R was first identified in red blood cells and has yet to be studied in the context of cardiac function and heart failure or arrhythmia disease. To study AnkR in the context of the heart we isolated perfused tissues from adult wild-type mice and performed immunoblot and qPCR analysis on ankyrin-R protein and Ank1 mRNA expression. The large AnkR isoform is expressed in the heart, along with the brain, intestine, and spleen. Interestingly, only the heart showed expression of a small AnkR isoform that has previously been shown to interact with the sarcoplasmic protein obscurin. Notably, isolated cardiomyocytes express the small AnkR isoform while cardiac fibroblasts express the canonical large AnkR isoform at both the protein and mRNA level. Canonical AnkR is diffusely expressed in the fibroblast membrane, cytoplasm, cytoskeleton, and soluble nuclear fractions. Interestingly, this expression pattern is switched under stressed conditions where cardiomyocytes increase and cardiac fibroblasts decrease their canonical AnkR expression, respectively. These preliminary results are the first to show canonical AnkR expression in the mouse myocardium specifically within the cardiac fibroblasts and allude that AnkR’s utilization during cardiac stress is cell-type dependent. Future studies will seek to define molecular, cell, and organ phenotypes related to AnkR in the heart.

Nuclear Small Dystrophin Isoforms during Muscle Differentiation.

Mutations in the DMD gene can cause Duchenne or Becker muscular dystrophy (DMD/BMD) by affecting the giant isoform of dystrophin, a protein encoded by the DMD gene. The role of small dystrophin isoforms is not well investigated yet, and they may play a role in muscle development and molecular pathology. Here, we investigated the nuclear localization of short carboxy-terminal dystrophin isoforms during the in vitro differentiation of human, porcine, and murine myoblast cultures. We could not only confirm the presence of Dp71 in the nucleoplasm and at the nuclear envelope, but we could also identify the Dp40 isoform in muscle nuclei. The localization of both isoforms over the first six days of differentiation was similar between human and porcine myoblasts, but murine myoblasts behaved differently. This highlights the importance of the porcine model in investigating DMD. We could also detect a wave-like pattern of nuclear presence of both Dp71 and Dp40, indicating a direct or indirect involvement in gene expression control during muscle differentiation.

Hepcidin analysis in pneumonia: Comparison of immunoassay and LC-MS/MS.

The iron-regulatory hormone hepcidin is a promising biomarker to differentiate anaemia of inflammation from iron deficiency. Plasma hepcidin concentrations increase substantially during inflammation, and the amount of smaller, non-biologically active isoforms of hepcidin increase in inflammatory conditions. These smaller isoforms are measured in some, but not all analytical methods. Thus, we evaluated the comparability of two analytical methods with different isoform selectivity during and after acute-phase pneumonia as a highly inflammatory model disease. Blood samples from a cohort of 267 hospitalized community-acquired pneumonia patients collected at admission and a 6-week follow-up were analysed. Hepcidin was measured in plasma by an immunoassay, which recognizes all hepcidin isoforms, and a liquid chromatography tandem mass spectrometry (LC-MS/MS), which selectively measures the bioactive hepcidin-25. Additionally, a subset of serum samples was analysed by LC-MS/MS. Hepcidin measurements by immunoassay were higher compared with LC-MS/MS. The relative mean difference of hepcidin plasma concentrations between the two analytical methods was larger in admission samples than in follow-up samples (admission samples <200ng/mL: 37%, admission samples >200ng/mL: 78%, follow-up samples >10ng/mL: 22%). During acute-phase pneumonia, serum concentrations were on average 22% lower than plasma concentrations when measured by LC-MS/MS. Immunoassay measured higher hepcidin concentrations compared with LC-MS/MS, with more pronounced differences in high-concentration samples during acute-phase pneumonia. These findings should be considered in local method validations and in future harmonization and standardization optimization of hepcidin measurements.

Emerging Role of SMILE in Liver Metabolism.

Small heterodimer partner-interacting leucine zipper (SMILE) is a member of the CREB/ATF family of basic leucine zipper (bZIP) transcription factors. SMILE has two isoforms, a small and long isoform, resulting from alternative usage of the initiation codon. Interestingly, although SMILE can homodimerize similar to other bZIP proteins, it cannot bind to DNA. As a result, SMILE acts as a co-repressor in nuclear receptor signaling and other transcription factors through its DNA binding inhibition, coactivator competition, and direct repression, thereby regulating the expression of target genes. Therefore, the knockdown of SMILE increases the transactivation of transcription factors. Recent findings suggest that SMILE is an important regulator of metabolic signals and pathways by causing changes in glucose, lipid, and iron metabolism in the liver. The regulation of SMILE plays an important role in pathological conditions such as hepatitis, diabetes, fatty liver disease, and controlling the energy metabolism in the liver. This review focuses on the role of SMILE and its repressive actions on the transcriptional activity of nuclear receptors and bZIP transcription factors and its effects on liver metabolism. Understanding the importance of SMILE in liver metabolism and signaling pathways paves the way to utilize SMILE as a target in treating liver diseases.

Human glycoprotein-2 expressed in Brunner glands – A putative autoimmune target and link between Crohn's and coeliac disease

Glycoprotein 2 (GP2) is an autoantigen in Crohn's (CD) and coeliac disease (CeD). We assessed GP2-isoform (GP21–4)-expression in intestinal biopsies of paediatric patients with CD, CeD, ulcerative colitis (UC), and healthy children (HC). Transcription of GP21–4 was elevated in proximal small intestine in CeD and CD patients (only GP22/4) compared to jejunum (CeD/CD) and large bowel (CD). CeD patients demonstrated higher duodenal GP22/4-mRNA levels compared to HC/UC patients whereas CD patients showed higher GP24-mRNA levels compared to UC patients. Duodenal synthesis of only small GP2 isoforms (GP23/4) was demonstrated in epithelial cells in patients/HC and in Brunner glands (also large isoforms) with a more frequent apical location in CD/CeD patients. All four GP2 isoforms interacted with gliadin and phosphopeptidomannan. Gliadin digestion improved binding to GP2 isoforms. GP21–4 binding to CeD/CD-related antigens, elevated duodenal GP21–4-mRNA transcription, and GP2-protein secretion in Brunner glands of CeD/CD patients suggest an autoimmune CeD/CD link.

Nicotiana benthamiana Kunitz peptidase inhibitor-like protein involved in chloroplast-to-nucleus regulatory pathway in plant-virus interaction.

Plant viruses use a variety of strategies to infect their host. During infection, viruses cause symptoms of varying severity, which are often associated with altered leaf pigmentation due to structural and functional damage to chloroplasts that are affected by viral proteins. Here we demonstrate that Nicotiana benthamiana Kunitz peptidase inhibitor-like protein (KPILP) gene is induced in response to potato virus X (PVX) infection. Using reverse genetic approach, we have demonstrated that KPILP downregulates expression of LHCB1 and LHCB2 genes of antenna light-harvesting complex proteins, HEMA1 gene encoding glutamyl-tRNA reductase, which participates in tetrapyrrole biosynthesis, and RBCS1A gene encoding RuBisCO small subunit isoform involved in the antiviral immune response. Thus, KPILP is a regulator of chloroplast retrograde signaling system during developing PVX infection. Moreover, KPILP was demonstrated to affect carbon partitioning: reduced glucose levels during PVX infection were associated with KPILP upregulation. Another KPILP function is associated with plasmodesmata permeability control. Its ability to stimulate intercellular transport of reporter 2xGFP molecules indicates that KPILP is a positive plasmodesmata regulator. Moreover, natural KPILP glycosylation is indispensable for manifestation of this function. During PVX infection KPILP increased expression leads to the reduction of plasmodesmata callose deposition. These results could indicate that KPILP affects plasmodesmata permeability via callose-dependent mechanism. Thus, virus entering a cell and starting reproduction triggers KPILP expression, which leads to downregulation of nuclear-encoded chloroplast genes associated with retrograde signaling, reduction in photoassimilates accumulation and increase in intercellular transport, creating favorable conditions for reproduction and spread of viral infection.

Abstract 12528: Variants in Solute Cation Transporters 3 Associate with High Lp(a) Levels Independent of Apo(a) Isoform Size

Introduction: Lipoprotein (a) [Lp(a)] consists of a low-density lipoprotein (LDL) molecule attached to apolipoprotein(a) [apo(a)]. Apo(a) comprises two kringle domains (KIV and KV) derived from the plasminogen gene and an inactive protease domain. The KIV domain is subdivided into 10 subtypes (KIV1-10), with KIV2 occurring as a copy number variation (CNV) that varies among individuals. Elevated Lp(a) levels are a major risk factor for CVDs and are inversely correlated to the KIV2 CNV determining apo(a) isoform size. Objectives: This study aimed to identify variants associated with a high Lp(a) phenotype that were not linked to apo(a) isoform size. Methods: Illumina sequencing of 13 candidate genes was performed on a discovery set comprising 54 high Lp(a) European samples (&gt;50mg/dL). The sequenced genes included LPA, adjacent genes, and those coding for receptors involved in Lp(a) uptake. Variants commonly found in high samples were genotyped in the Otago LPA European cohort (n=589) and their association with Lp(a) levels was checked via Kruskal-Wallis and linear regression statistical models. The associations were then replicated in the much bigger German Chronic Kidney Disease (GCKD) cohort (n=4930). Results: Nine variants were significantly associated with high Lp(a) levels. Two variants were in the LPA gene (rs3124784 and rs3798220) and seven were in neighbouring genes (rs2282143 in SLC22A1, rs3127592, rs3127593, rs3103353 and rs3127594 in SLC22A2 and rs1810126 and rs3088442 in SLC22A3). Interestingly, the minor allele of all nine variants was significantly associated with increased LPA expression in liver tissues in the GTEx database. Seven of the variants were significantly associated with small apo(a) isoform size (P=1x10-4 to 2.9x10-18), while rs1810126 and rs3088442 in SLC22A3 were not associated with the small isoform size (P=0.43 and 0.42). Conclusion: Nine variants in the LPA and nearby genes were significantly associated with elevated Lp(a) levels and increased LPA expression, with the majority being linked to a short apo(a) isoform. The association of two SLC22A3 gene variants with Lp(a) levels independent of apo(a) isoform size warrants further investigation to establish any functional connection between LPA and this gene.

PMON52 Use of PDE5 Inhibitors as Potential Treatment for Isolated Growth Hormone Deficiency Caused by Alternate Splicing of GH1 Gene

Abstract Mutations in the GH1 gene cause isolated growth hormone deficiency (IGHD) by affecting production, secretion, and stability of growth hormone as well as its binding to GHR. Genetic mutations resulting from splicing errors are known to increase the production of a small 17.5 kD isoform of human growth hormone. This smaller isoform of GH1 is inactive and is linked to IGHD. While studying the impact of small GH isoform on GH production and secretion, we noticed that cells transfect with sequences carrying the mutated GH gene that resulted in splicing defects produce the short 17.5 kD version of GH, looked very different from the control cells with WT GH gene. We found that production of short GH isoform distorts the cell morphology, contributing to detrimental effects seen in the patients with mutations in GH1 gene causing alternate splicing. We hypothesized that cellular distortion caused by small GH isoform and overall GH secretion can be a target and a tool to experimental approaches for reversing the damaging effects of small GH protein, thereby providing potential treatments for IGHD caused by splicing errors. It is known for some time that increasing cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, and recently increase in cGMP levels has also been shown to regulate protein degradation. Inhibitors of phosphodiesterase 5 (PDE5; e.g., Viagra (sildenafil)), which raise cGMP by inhibiting its hydrolysis to GMP, are widely used for the treatment of erectile dysfunction and pulmonary hypertension, and stimulators of soluble guanylyl cyclases (riociguat), which stimulate cGMP synthesis, are used as treatments for pulmonary hypertension and heart failure. Using our model of cellular morphology changes, have investigated the impact of chemicals that enhance cGMP levels by inhibiting PDE5 or activating guanyl cyclases. The cells expressing small isoform of GH consistently show distorted rounded morphology. Treatment with BAY 41-2272, a cGMP inducer slightly improved the cell morphology in small isoform GH expressing cells. BAY 41-2272 did not show any adverse effects of normal and WT GH expressing cells. Treatment of sildenafil to cells expressing 17.5 kD isoform of GH markedly improved the cell morphology compared to untreated cells, especially after 48h when a larger percentage of cells that were distorted (rounder shapes) regained the shape of normal cells expressing WT GH and no damage to cells expressing WT-GH was observed. Another PDE5 inhibitor tadalafil also caused a marked improvement in cell morphology caused by expression of small GH isoform. Overall, it seems the strategy to regulate the cGMP levels in cells transfected in small GH isoform worked and needs further investigation. A detailed analysis of the impact of PDE5 inhibitors on GH expression and secretion will be presented. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Involvement of a BH3-only apoptosis sensitizer gene Blm-s in hippocampus-mediated mood control.

Mood disorders are an important public health issue and recent advances in genomic studies have indicated that molecules involved in neurodevelopment are causally related to mood disorders. BLM-s (BCL-2-like molecule, small transcript isoform), a BH3-only proapoptotic BCL-2 family member, mediates apoptosis of postmitotic immature neurons during embryonic cortical development, but its role in the adult brain is unknown. To better understand the physiological role of Blm-s gene in vivo, we generated a Blm-s-knockout (Blm-s−/−) mouse. The Blm-s−/− mice breed normally and exhibit grossly normal development. However, global depletion of Blm-s is highly associated with depression- and anxiety-related behaviors in adult mutant mice with intact learning and memory capacity. Functional magnetic resonance imaging of adult Blm-s−/− mice reveals reduced connectivity mainly in the ventral dentate gyrus (vDG) of the hippocampus with no alteration in the dorsal DG connectivity and in total hippocampal volume. At the cellular level, BLM-s is expressed in DG granule cells (GCs), and Blm-s−/− mice show reduced dendritic complexity and decreased spine density in mature GCs. Electrophysiology study uncovers that mature vGCs in adult Blm-s−/− DG are intrinsically more excitable. Interestingly, certain genetic variants of the human Blm homologue gene (VPS50) are significantly associated with depression traits from publicly resourced UK Biobank data. Taken together, BLM-s is required for the hippocampal mood control function. Loss of BLM-s causes abnormality in the electrophysiology and morphology of GCs and a disrupted vDG neural network, which could underlie Blm-s-null-associated anxiety and depression.