Full-length Form Research Articles

Development and Evaluation of Short-Form Measures of the HIV/AIDS Knowledge Assessment Tool Among Sexual and Gender Minorities in Brazil: Cross-sectional Study.

BackgroundIn theoretical models of health behavior, knowledge about disease transmission and self-protective behaviors are conceptualized as important drivers of behavior change. Several studies conducted in Brazil point to an unfortunate convergence of sexual and gender minority (SGM) populations with low levels of HIV knowledge and younger age, lower education, engagement in higher-risk sexual behavior, and never having tested for HIV. Measures to assess level of HIV knowledge have been previously published, including the 12-item HIV/AIDS Knowledge Assessment (HIV-KA) tool. However, measure length can be a barrier to assessment.ObjectiveWe started from the 12-item HIV-KA tool and developed candidate short forms using statistical procedures, evaluated their psychometric properties, and tested the equivalency of their associations with other measures of HIV knowledge compared to the 12-item version.MethodsA convenience sample of SGM was recruited during September 2020 to complete an online survey through advertisements on two social networking apps (Grindr and Hornet). The survey instrument included items on sociodemographic information, prior HIV testing and HIV test results, preexposure prophylaxis (PrEP) and antiretroviral treatment use, sexual behavior, and 3 HIV knowledge measures: the HIV-KA, World Health Organization Knowledge About HIV Transmission Prevention Indicator, and the Brief HIV Knowledge Questionnaire. We used exploratory factor analysis and confirmatory factor analysis (CFA) to assess the factor structure of the of the HIV-KA. We used optimal test assembly (OTA) methods to develop candidate short forms of the HIV-KA and evaluated them based on prespecified reliability, concurrent validity, and statistically equivalent convergent validity criteria.ResultsAmong 2552 SGM individuals from Brazil, mean age was 35.1 years, 98.2% (2507/2552) cisgender men and 1.8% (45/2552) transgender/nonbinary, 56.5% (1441/2552) White, and 31.0% (792/2552) self-reported HIV positive. CFA indicated a 1-factor structure for the 12-item HIV-KA. Concurrent validity correlations were high for all short forms with 6 items, but only versions with 9 items were as reliable as the full-length form and demonstrated equivalency for convergent validity correlations. Suggesting post hoc convergent validity, HIV knowledge scores using the 9- and 10-item short forms were higher for participants who perceived the Undetectable Equals Untransmittable (U=U) slogan as completely accurate versus not accurate. Suggesting post hoc concurrent validity, participants of younger age, of Black, Pardo or indigenous race, and reporting lower education and lower income scored lower on HIV knowledge. Participants who never tested for HIV scored lower than those who tested negative or positive, while those currently using PrEP scored higher than those reporting past or never use.ConclusionsOTA methods were used to shorten the 12-item HIV-KA to 9-item and 10-item versions while maintaining comparable reliability and validity among a large sample of Brazilian SGM. However, these short forms did not shorten sufficiently to justify deviation from the full measure.

Discovery and Characterization of an Aberrant Small Form of Glycoprotein I of Herpes Simplex Virus Type I in Cell Culture.

ABSTRACTThe 380-to-393-amino-acid glycoprotein I (gI) encoded by herpes simplex virus 1 (HSV-1) is a critical mediator for viral cell-to-cell spread and syncytium formation. Here we report a previously unrecognized aberrant form of gI in HSV-1-infected cells. Production of this molecule is independent of cell type and viral strains. It had an unexpected gel migration size of approximately 23 kDa, was packaged into viral particles, and could be coimmunoprecipitated by antibodies to both N and C termini of gI. Deep sequencing failed to detect alternative RNA splicing, and the in vitro transcribed full-length mRNA gave rise to the 23 kDa protein in transfected cells. Combined mass spectrometry and antibody probing analyses detected peptide information across different regions of gI, suggesting the possibility of a full-length gI but with abnormal migration behavior. In line with this notion, the HA insertion mutagenesis revealed a stable fold in the gI extracellular region aa.38-196 resistant to denaturing conditions, whereas small deletions within this region failed the antibodies to detect the fast, but not the slow-moving species of gI. It is also intriguing that the structure could be perturbed to some extent by a gBsyn mutation, leading to exposure or shielding of the gI epitopes. Thus, the HSV-1 gI apparently adopts a very stable fold in its natural form, rendering it an unusual biophysical property. Our findings provide novel insight into the biological properties of HSV gI and have important implications in understanding the viral spread and pathogenesis.IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but its behavior during infection has remained poorly defined. Along with the classic 66 kDa product, here we report a previously unrecognized, approximately 23 kDa form of gI. Biochemical and genetics analyses revealed that this molecule represents the full-length form of gI but adopts a stable fold in its extracellular domain that is resistant to denatured conditions, thus contributing to the aberrant migration rate. Our results revealed a novel property of HSV-1 gI and have important implications in understanding viral pathogenesis.

The Human-Restricted Isoform of the α7 nAChR, CHRFAM7A: A Double-Edged Sword in Neurological and Inflammatory Disorders.

CHRFAM7A is a relatively recent and exclusively human gene arising from the partial duplication of exons 5 to 10 of the α7 neuronal nicotinic acetylcholine receptor subunit (α7 nAChR) encoding gene, CHRNA7. CHRNA7 is related to several disorders that involve cognitive deficits, including neuropsychiatric, neurodegenerative, and inflammatory disorders. In extra-neuronal tissues, α7nAChR plays an important role in proliferation, differentiation, migration, adhesion, cell contact, apoptosis, angiogenesis, and tumor progression, as well as in the modulation of the inflammatory response through the “cholinergic anti-inflammatory pathway”. CHRFAM7A translates the dupα7 protein in a multitude of cell lines and heterologous systems, while maintaining processing and trafficking that are very similar to the full-length form. It does not form functional ion channel receptors alone. In the presence of CHRNA7 gene products, dupα7 can assemble and form heteromeric receptors that, in order to be functional, should include at least two α7 subunits to form the agonist binding site. When incorporated into the receptor, in vitro and in vivo data showed that dupα7 negatively modulated α7 activity, probably due to a reduction in the number of ACh binding sites. Very recent data in the literature report that the presence of the duplicated gene may be responsible for the translational gap in several human diseases. Here, we will review the studies that have been conducted on CHRFAM7A in different pathologies, with the intent of providing evidence regarding when and how the expression of this duplicated gene may be beneficial or detrimental in the pathogenesis, and eventually in the therapeutic response, to CHRNA7-related neurological and non-neurological diseases.

ChREBPβ is dispensable for the control of glucose homeostasis and energy balance.

Impaired glucose metabolism is observed in obesity and type 2 diabetes. Glucose controls gene expression through the transcription factor ChREBP in liver and adipose tissues. Mlxipl encodes 2 isoforms: ChREBPα, the full-length form (translocation into the nucleus is under the control of glucose), and ChREBPβ, a constitutively nuclear shorter form. ChREBPβ gene expression in white adipose tissue is strongly associated with insulin sensitivity. Here, we investigated the consequences of ChREBPβ deficiency on insulin action and energy balance. ChREBPβ-deficient male and female C57BL6/J and FVB/N mice were produced using CRISPR/Cas9-mediated gene editing. Unlike global ChREBP deficiency, lack of ChREBPβ showed modest effects on gene expression in adipose tissues and the liver, with variations chiefly observed in brown adipose tissue. In mice fed chow and 2 types of high-fat diets, lack of ChREBPβ had moderate effects on body composition and insulin sensitivity. At thermoneutrality, ChREBPβ deficiency did not prevent the whitening of brown adipose tissue previously reported in total ChREBP-KO mice. These findings revealed that ChREBPβ is dispensable for metabolic adaptations to nutritional and thermic challenges.

Zic5 stabilizes Gli3 via a non-transcriptional mechanism during retinal development.

The Zic family of zinc finger transcription factors plays a critical role in multiple developmental processes. Using loss-of-function studies, we find that Zic5 is important for the differentiation of retinal pigmented epithelium (RPE) and the rod photoreceptor layer through suppressing Hedgehog (Hh) signaling. Further, Zic5 interacts with the critical Hh signaling molecule, Gli3, through the zinc finger domains of both proteins. This Zic5-Gli3 interaction disrupts Gli3/Gli3 homodimerization, resulting in Gli3 protein stabilization via a reduction in Gli3 ubiquitination. During embryonic Hh signaling, the activator form of Gli is normally converted to a repressor form through proteosome-mediated processing of Gli3, and the ratio of Gli3 repressor to full-length (activator) form of Gli3 determines the Gli3 repressor output required for normal eye development. Our results suggest Zic5 is a critical player in regulating Gli3 stability for the proper differentiation of RPE and rod photoreceptor layer during Xenopus eye development.

ALDH1A3 Segregated Expression and Nucleus-Associated Proteasomal Degradation Are Common Traits of Glioblastoma Stem Cells.

Aldehyde dehydrogenase 1 isoforms A1 and A3 have been implicated as functional biomarkers associated with distinct molecular subtypes of glioblastoma and glioblastoma stem cells. However, the exact roles of these isoforms in different types of glioma cells remain unclear. The purpose of this study was to dissect the association of A1 or A3 isoforms with stem and non-stem glioblastoma cells. This study has undertaken a systematic characterization of A1 and A3 proteins in glioblastoma tissues and a panel of glioblastoma stem cells using immunocytochemical and immunofluorescence staining, Western blot and the subcellular fractionation methodology. Our main findings are (i) human GSCs express uniformly ALDH1A3 but not the ALDH1A1 isoform whereas non-stem glioma cells comparably express both isoforms; (ii) there is an abundance of ALDH1A3 peptides that prevail over the full-length form in glioblastoma stem cells but not in non-stem glioma cells; (iii) full-length ALDH1A3 and ALDH1A3 peptides are spatially segregated within the cell; and (vi) the abundance of full-length ALDH1A3 and ALDH1A3 peptides is sensitive to MG132-mediated proteasomal inhibition. Our study further supports the association of ALDH1A3 with glioblastoma stem cells and provide evidence for the regulation of ALDH1A3 activities at the level of protein turnover.

A leader peptide of the extracellular cyanobacterial carbonic anhydrase ensures the efficient secretion of recombinant proteins in Escherichia coli

Several forms of EcaA protein, correspondent to the extracellular α-class carbonic anhydrase (CA) of cyanobacterium Crocosphaera subtropica ATCC 51142 were expressed in Escherichia coli. The recombinant proteins with no leader peptide (EcaA and its fusion with thioredoxin or glutathione S-transferase) were allocated inside cells in a full-length form; these cells did not display any extracellular CA activity. Soluble proteins (including that of periplasmic space) of E. coli cells that expressed both ЕсаА equipped with its native leader peptide (L-EcaA) as well as L-EcaA fused with thioredoxin or glutathione S-transferase at N-terminus, mainly contained the processed EcaA. The appearance of mature ЕсаА in outer layers of E. coli cells expressed leader peptide-containing forms of recombinant proteins, has been directly confirmed by immunofluorescent microscopy. Those cells also displayed high extracellular CA activity. In addition, the mature EcaA protein was detected in the culture medium. This suggests that cyanobacterial signal peptide is recognized by the secretory machinery and by the leader peptidase of E. coli even as a part of a fusion protein. The efficiency of EcaA leader peptide was comparable to that of PelB and TorA signal peptides, commonly used for biotechnological production of extracellular recombinant proteins in E. coli.

Adaptation and Cryptic Pseudogenization in Penguin Toll-Like Receptors.

Penguins (Sphenisciformes) are an iconic order of flightless, diving seabirds distributed across a large latitudinal range in the Southern Hemisphere. The extensive area over which penguins are endemic is likely to have fostered variation in pathogen pressure, which in turn will have imposed differential selective pressures on the penguin immune system. At the front line of pathogen detection and response, the Toll-like receptors (TLRs) provide insight into host evolution in the face of microbial challenge. TLRs respond to conserved pathogen-associated molecular patterns and are frequently found to be under positive selection, despite retaining specificity for defined agonist classes. We undertook a comparative immunogenetics analysis of TLRs for all penguin species and found evidence of adaptive evolution that was largely restricted to the cell surface-expressed TLRs, with evidence of positive selection at, or near, key agonist-binding sites in TLR1B, TLR4, and TLR5. Intriguingly, TLR15, which is activated by fungal products, appeared to have been pseudogenized multiple times in the Eudyptes spp., but a full-length form was present as a rare haplotype at the population level. However, in vitro analysis revealed that even the full-length form of Eudyptes TLR15 was nonfunctional, indicating an ancestral cryptic pseudogenization prior to its eventual disruption multiple times in the Eudyptes lineage. This unusual pseudogenization event could provide an insight into immune adaptation to fungal pathogens such as Aspergillus, which is responsible for significant mortality in wild and captive bird populations.

Caspase activation and inflammation observed in the olfactory bulb of APOE4 transgenic mice

AbstractBackgroundAlzheimer disease (AD) is a chronic neurodegenerative disorder that affects millions of individuals worldwide. Olfactory dysfunction is a common symptom of several neurological disorders including AD. Studying the mechanisms underlying the olfactory dysfunction may lead to the discovery of biomarkers and/or treatments for neurodegenerative diseases. Objectives: To characterize the olfactory system in a murine model expressing a genetic risk factor for AD. We investigated olfactory brain region atrophy as well as caspase expression and tissue inflammation in the APOE4 mouse model of AD.MethodWe assessed the olfactory system in 12 month old wild type (WT) and APOE4 olfactory bulb (OB) for atrophy, caspase activation, markers of neurogenesis and inflammation. We used Western blotting, RT‐PCR and immunohistochemistry.ResultThere was a significant decrease in OB bulb weight in APOE4 mice compared to WT (p<0.01). There was a significant increase in the full length (p<0.05) form of caspase‐8 and in the p30 fragment (p<0.05) in the APOE4 OB. Moreover, a trend increase in caspase‐8 mRNA (p=0.08) and significant increase in caspase‐9 mRNA in the APOE4 OB vs. WT. Furthermore, there was a decrease in the expression of the mature neuronal marker NeuN in the OB of the APOE4 mice compared to WT. This was with both the 46kDa (p<0.05) and 48kDa (p<0.05) isoforms of NeuN. As atrophy was observed in the APOE4 OB we then assessed for inflammation. Using the IBA‐1 marker for microglia demonstrated that there was a significant increase in IBA‐1 expression (p<0.01) and a trend increased in IBA‐1 positive cells (p=0.054) in the APOE4 OB compared to WTConclusionThese data suggest that there are alterations in neurogenesis in the OB of human APOE4 transgenic mice in addition to inflammation. This corresponds well with what has been observed in human AD brain tissue. Caspase activities detected may play a role in the inflammation observed. Studying the olfactory system in the aging population may help to discover biomarkers in the early stages of AD

Development of a microplate duplex immunoassay to simplify detection of growth hormone doping: Proof of concept.

The World Anti-Doping Agency (WADA) prohibits athletes from using recombinant growth hormone (GH). The validated method used in antidoping laboratories for the direct detection of exogenous GH in serum requires two immunoluminometric assays (ILMAs): The first mainly measures the concentration of the full-length (22 kDa) form of GH (recGH), and the second measures concentrations of multiple GH fragments produced by the pituitary gland (22 kDa, 20 kDa and other forms) (pitGH). The tube-by-tube analysis is laborious. A recent development opened new possibilities to simplify the detection of recGH in serum: multiplexed immunoassays that detect multiple targets in a single well of a 96-well plate using an ELISA-like procedure with high sensitivity. Our aim was to evaluate this technology by developing a customized assay for GH detection. One pair of antibodies with specificities similar to those of the recGH assay and one pair of antibodies compatible with pitGH detection were selected for a single duplex assay. Forty-eight serum samples (negative athlete samples and positive samples following GH administration) were analyzed using the two methods. The microplate duplex assay discriminated between the negative athlete samples and the positive controls, although the rec/pit ratios from the duplex assay were lower than those obtained with the ILMAs. This new assay would offer a modern alternative to ILMAs, with fewer analytical steps and a smaller sample volume. However, an adaptation of the decision limits seems mandatory.

Loss of full-length DNA replication regulator Rif1 in two-cell embryos is associated with zygotic transcriptional activation

Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of two-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing, whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2C embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction and are likely nonfunctional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides may be involved in 2C-specific transcription program.

Sedentary Conditions Promote Subregionally Specific Changes in Brain-Derived Neurotrophic Factor in the Rostral Ventrolateral Medulla.

A sedentary lifestyle is the top preventable cause of death and accounts for substantial socioeconomic costs to society. The rostral ventrolateral medulla regulates blood pressure under normal and pathophysiological states, and demonstrates inactivity-related structural and functional neuroplasticity, which is subregionally specific. The purpose of this study was to examine pro- and mature forms of brain-derived neurotrophic factor (BDNF) and their respective receptors in the male rat rostral ventrolateral medulla (RVLM) and its rostral extension following sedentary vs. active (running wheels) conditions (10–12weeks). We used subregionally specific Western blotting to determine that the mature form of BDNF and its ratio to its pro-form were lower in more caudal subregions of the rostral ventrolateral medulla of sedentary rats but higher in the rostral extension when both were compared to active rats. The full-length form of the tropomyosin receptor kinase B receptor and the non-glycosylated form of the 75 kilodalton neurotrophin receptor were lower in sedentary compared to active rats. The rostrocaudal patterns of expression of the mature form of BDNF and the full-length form of the tropomyosin receptor kinase B receptor were remarkably similar to the subregionally specific patterns of enhanced dendritic branching, neuronal activity, and glutamate-mediated increases in sympathetic nerve activity observed in previous studies performed in sedentary rats. Our studies suggest signaling pathways related to BDNF within subregions of both the rostral ventrolateral medulla and its rostral extension contribute to cardiovascular disease and premature death related to a sedentary lifestyle.

Experimental nephrotic syndrome leads to proteolytic activation of the epithelial Na+ channel in the mouse kidney.

Proteolytic activation of the renal epithelial Na+ channel (ENaC) involves cleavage events in its α- and γ-subunits and is thought to mediate Na+ retention in nephrotic syndrome (NS). However, the detection of proteolytically processed ENaC in kidney tissue from nephrotic mice has been elusive so far. We used a refined Western blot technique to reliably discriminate full-length α-ENaC and γ-ENaC and their cleavage products after proteolysis at their proximal and distal cleavage sites (designated from the NH2-terminus), respectively. Proteolytic ENaC activation was investigated in kidneys from mice with experimental NS induced by doxorubicin or inducible podocin deficiency with or without treatment with the serine protease inhibitor aprotinin. Nephrotic mice developed Na+ retention and increased expression of fragments of α-ENaC and γ-ENaC cleaved at both the proximal cleavage site and, more prominently, the distal cleavage site, respectively. Treatment with aprotinin but not with the mineralocorticoid receptor antagonist canrenoate prevented Na+ retention and upregulation of the cleavage products in nephrotic mice. Increased expression of cleavage products of α-ENaC and γ-ENaC was similarly found in healthy mice treated with a low-salt diet, sensitive to mineralocorticoid receptor blockade. In human nephrectomy specimens, γ-ENaC was found in the full-length form and predominantly cleaved at its distal cleavage site. In conclusion, murine experimental NS leads to aprotinin-sensitive proteolytic activation of ENaC at both proximal and, more prominently, distal cleavage sites of its α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.NEW & NOTEWORTHY This study demonstrates that murine experimental nephrotic syndrome leads to aprotinin-sensitive proteolytic activation of the epithelial Na+ channel at both the α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.

Evidence that class I glutamine amidotransferase, GAT1_2.1, acts as a glutaminase in roots of Arabidopsis thaliana

The glutamine amidotransferase gene GAT1_2.1 is a marker of N status in Arabidopsis root, linked to a shoot branching phenotype. The protein has an N-terminal glutamine amidotransferase domain and a C-terminal extension with no recognizable protein domain. A purified, recombinant version of the glutamine amidotransferase domain was catalytically active as a glutaminase, with apparent Km value of 0.66 mM and Vmax value of 2.6 μkatal per mg. This form complemented an E. coli glutaminase mutant, ΔYneH. Spiking of root metabolite extracts with either the N-terminal or full length form purified from transformed tobacco leaves led to reciprocal changes in glutamine and ammonia concentration. No product derived from amido-15N-labeled glutamine was identified. Visualization of GAT1_2.1-YPF transiently expressed in tobacco leaves confirmed its mitochondrial localization. gat1_2.1 exhibited reduced growth as compared with wild-type seedlings on media with glutamine as sole nitrogen source. Results of targeted metabolite profiling pointed to a possible activation of the GABA shunt in the mutant following glutamine treatments, with reduced levels of glutamic acid, 2-oxoglutarate and γ-aminobutyric acid and increased levels of succinic acid. GAT1_2.1 may act as a glutaminase, in concert with Glutamate Dehydrogenase 2, to hydrolyze glutamine and channel 2-oxoglutarate to the TCA cycle under high nitrogen conditions.

Germline ERBB3 mutation in familial non-small-cell lung carcinoma: expanding ErbB's role in oncogenesis.

Lung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable, with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2. Germline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and subcellular localization were assessed, and cellular proliferation, pAkt/Akt and pERK levels determined. A novel germline variant in ERBB3 (c.1946 T > G: p.Iso649Arg), coding for receptor tyrosine-protein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80 kDa form (which enhances proliferation) compared with the full-length (180 kDa) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localization pre- and post-epidermal growth factor stimulation; however, epidermal growth factor receptor (EGFR) levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared with wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 min post-stimulation compared with wtErbB3 transfection, demonstrating altered signalling pathway activation. Cumulatively, these results support this mutation as tumorogenic. This is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

Biophysical characterization of the interaction between the full-length XIAP and Smac/DIABLO

XIAP is multi-functional protein which regulates apoptosis acting as a direct caspase inhibitor. It is overexpressed in cancer cells, where it antagonizes the pro-apoptotic action of chemotherapeutics, and therefore it has become an important target for the treatment of cancer. In cells undergoing programmed cell death, the pro-apoptotic protein Smac is released by the mitochondria and binds to XIAP, thereby blocking caspase inhibition. Thus, Smac is considered a master regulator of apoptosis in mammals. In this regard, several Smac mimetic compounds have been developed to inhibit XIAP activity in cancer tissues. These compounds have shown low efficacy, partly due to the lack of structural knowledge of the XIAP-Smac interaction. In this work, through SEC-MALS and circular dichroism, we provide the first biophysical characterization of the interaction between the full-length form of XIAP and Smac, determining the stoichiometry of the complex and providing important information to develop more effective XIAP inhibitors.

Abstract 2856: GLI3-mediated HH regulates prostate cancer progression

Abstract Although prostate cancer patients initially respond to androgen deprivation therapy, they eventually progress to lethal castration resistant prostate cancer (CRPC), characterized by restoration of the androgen receptor (AR) signaling despite castrate levels of circulating androgens. Mechanisms proposed to underlie CRPC include AR crosstalk with alternative signaling pathways. Among these, the Sonic hedgehog (SHH)Hedgehog (HH) pathway, an essential signaling axis in prostate development and homeostasis, has been implicated in prostate cancerCRPC progression. SHH pathway is regulated by transcription factors and effectors Glioma-associated oncogene family zinc finger (GLI) proteins 1, 2, and 3. Previous studies reported that GLI1 and GLI2 drive androgen-independent growth of prostate cancer by upregulating androgen-stimulated genes. However, whether and how GLI3 plays a role in prostate cancer progression remains unclear. In the present study, we investigated the role of GLI3 in the progression of prostate cancer cells. Like GLI2, GLI3 is the a downstream transcriptional factor effector of the SHH signaling pathway that can exist as a full-length activator form or a cleaved repressor form. Our data showed an increase in GLI3 expression in prostate cancer cells growing under androgen deprived conditions. Thuserefore, we hypothesize that androgen deprivation leads to activation of the SHH signaling pathway which consequently activates GLI3 to promote androgen-independent growth of prostate cancer cells. We showed that knockdown of GLI3 repressed while overexpression of GLI3 promoted androgen-independent growth of prostate cancer cells. GLI3 knockdown also resulted in regression of castration-resistant outgrowth of xenograft tumors in mice. Additionally, transcriptomic analysis of GLI3 knockdown cells revealed that the SHH signaling pathway and epithelial mesenchymal transition (EMT) genes are altered suggesting that GLI3 regulates androgen-independent growth of prostate cancer cells through canonical SHH signaling pathway and likely contributes to prostate cancer metastasis. Lastly, our data demonstrated a physical and functional relationship between GLI3 and AR suggesting a functional crosstalk between SHH signaling and AR signaling pathways in advanced prostate cancer. Citation Format: Thu Minh Duong, Marieke Oldenbroek Burleson, J J. Deng, T Qin, Das D, L-z Sun, T G. Boyer. GLI3-mediated HH regulates prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2856.

A Common Polymorphism in the FADS1 Locus Links miR1908 to Low-Density Lipoprotein Cholesterol Through BMP1.

OBJECTIVE: Leveraging microRNA-Seq data and the 1000 Genomes imputed genotypes, we identified rs174561 as a strong microRNA quantitative trait loci for circulating microRNA-1908-5p with higher miR-1908-5p and reduced LDL (lowdensity lipoprotein)-cholesterol, fasting glucose and A1c concentrations in carriers of the rs-174561-C allele. Here, we have investigated the molecular mechanism(s) linking miR-1908-5p to LDL-C concentrations. APPROACH AND RESULTS: Transfection experiments demonstrate that the presence of the C allele significantly increases miR- 1908-5p abundance relative to the T allele. LDLR mRNA and low-density lipoprotein receptor (LDLR) total protein were unchanged in response to differential miR-1908-5p expression. However, the ratio of the cleaved to full-length form of LDLR decreased with miR-1908-5p mimic and increased with miR-1908-5p inhibitor treatment. BMP1 (bone morphogenetic protein 1) is a protease responsible for LDLR cleavage, and we show that miR-1908-5p mimic reduces BMP1 mRNA. Using a reporter array, we identified the TGF-β (transforming growth factor-beta) signaling pathway activity to be reduced by miR- 1908-5p mimic treatment, and this was associated with reduced TGFB1 expression. TGF-β signaling increases BMP1, and we further demonstrate that the effect of miR-1908-5p on LDLR cleavage is abolished by exogenous TGF-β treatment. CONCLUSIONS: These findings uncover a mechanism whereby miR-1908-5p reduces TGFB1 abundance resulting in lower expression of BMP1, ultimately leading to reduced LDLR cleavage. Cleavage of the mature LDLR is known to reduce cell surface affinity for LDL, thereby linking miR-1908-5p to lower circulating LDL-cholesterol levels.

Alternative Splicing of the Basic Helix-Loop-Helix Transcription Factor Gene CmbHLH2 Affects Anthocyanin Biosynthesis in Ray Florets of Chrysanthemum (Chrysanthemum morifolium).

Chrysanthemum is an important ornamental crop worldwide. Some white-flowered chrysanthemum cultivars produce red ray florets under natural cultivation conditions, but little is known about how this occurs. We compared the expression of anthocyanin biosynthetic and transcription factor genes between white ray florets and those that turned red based on cultivation conditions to comprehend the underlying mechanism. Significant differences in the expression of CmbHLH2 were detected between the florets of different colors. CmbHLH2 generated two alternatively spliced transcripts, designated CmbHLH2Full and CmbHLH2Short. Compared with CmbHLH2Full, CmbHLH2Short encoded a truncated protein with only a partial MYB-interaction region and no other domains normally present in the full-length protein. Unlike the full-length form, the splicing variant protein CmbHLH2Short localized to the cytoplasm and the nucleus and could not interact with CmMYB6. Additionally, CmbHLH2Short failed to activate anthocyanin biosynthetic genes and induce pigment accumulation in transiently transfected tobacco leaves, whereas CmbHLH2Full promoted both processes when simultaneously expressed with CmMYB6. Co-expressing CmbHLH2Full and CmMYB6 also enhanced the promoter activities of CmCHS and CmDFR. Notably, the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, could be complemented by the heterologous expression of CmbHLH2Full, which restored red pigmentation and resulted in red pigmentation in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively, whereas expression of CmbHLH2Short did not. Together, these results indicate that CmbHLH2 and CmMYB6 interaction plays a key role in the anthocyanin pigmentation changes of ray florets in chrysanthemum. Our findings highlight alternative splicing as a potential approach to modulate anthocyanin biosynthesis in specific tissues.

OASIS/CREB3L1 is a factor that responds to nuclear envelope stress

The nuclear envelope (NE) safeguards the genome and is pivotal for regulating genome activity as the structural scaffold of higher-order chromatin organization. NE had been thought as the stable during the interphase of cell cycle. However, recent studies have revealed that the NE can be damaged by various stresses such as mechanical stress and cellular senescence. These types of stresses are called NE stress. It has been proposed that NE stress is closely related to cellular dysfunctions such as genome instability and cell death. Here, we found that an endoplasmic reticulum (ER)-resident transmembrane transcription factor, OASIS, accumulates at damaged NE. Notably, the major components of nuclear lamina, Lamin proteins were depleted at the NE where OASIS accumulates. We previously demonstrated that OASIS is cleaved at the membrane domain in response to ER stress. In contrast, OASIS accumulates as the full-length form to damaged NE in response to NE stress. The accumulation to damaged NE is specific for OASIS among OASIS family members. Intriguingly, OASIS colocalizes with the components of linker of nucleoskeleton and cytoskeleton complexes, SUN2 and Nesprin-2 at the damaged NE. OASIS partially colocalizes with BAF, LEM domain proteins, and a component of ESCRT III, which are involved in the repair of ruptured NE. Furthermore, OASIS suppresses DNA damage induced by NE stress and restores nuclear deformation under NE stress conditions. Our findings reveal a novel NE stress response pathway mediated by OASIS.