Box-binding Protein Research Articles

Cell-based cccDNA reporter assay combined with functional genomics identifies YBX1 as HBV cccDNA host factor and antiviral candidate target

ObjectivesChronic hepatitis B virus (HBV) infection is a leading cause of liver disease and hepatocellular carcinoma. A key feature of HBV replication is the synthesis of the covalently close circular...

The IRE1α-XBP1 Signaling Axis Promotes Glycolytic Reprogramming in Response to Inflammatory Stimuli.

Immune cells must be able to adjust their metabolic programs to effectively carry out their effector functions. Here, we show that the endoplasmic reticulum (ER) stress sensor Inositol-requiring enzyme 1 alpha (IRE1α) and its downstream transcription factor X box binding protein 1 (XBP1) enhance the upregulation of glycolysis in classically activated macrophages (CAMs). The IRE1α-XBP1 signaling axis supports this glycolytic switch in macrophages when activated by lipopolysaccharide (LPS) stimulation or infection with the intracellular bacterial pathogen Brucella abortus. Importantly, these different inflammatory stimuli have distinct mechanisms of IRE1α activation; while Toll-like receptor 4 (TLR4) supports glycolysis under both conditions, TLR4 is required for activation of IRE1α in response to LPS treatment but not B. abortus infection. Though IRE1α and XBP1 are necessary for maximal induction of glycolysis in CAMs, activation of this pathway is not sufficient to increase the glycolytic rate of macrophages, indicating that the cellular context in which this pathway is activated ultimately dictates the cell's metabolic response and that IRE1α activation may be a way to fine-tune metabolic reprogramming. IMPORTANCE The immune system must be able to tailor its response to different types of pathogens in order to eliminate them and protect the host. When confronted with bacterial pathogens, macrophages, frontline defenders in the immune system, switch to a glycolysis-driven metabolism to carry out their antibacterial functions. Here, we show that IRE1α, a sensor of ER stress, and its downstream transcription factor XBP1 support glycolysis in macrophages during infection with Brucella abortus or challenge with Salmonella LPS. Interestingly, these stimuli activate IRE1α by independent mechanisms. While the IRE1α-XBP1 signaling axis promotes the glycolytic switch, activation of this pathway is not sufficient to increase glycolysis in macrophages. This study furthers our understanding of the pathways that drive macrophage immunometabolism and highlights a new role for IRE1α and XBP1 in innate immunity.

Structural basis of SNAPc-dependent snRNA transcription initiation by RNA polymerase II

RNA polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes, such as small nuclear RNA (snRNA) genes, is less well understood at the structural level. Here, we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol IIpre-initiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE), located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc, called wing-1 and wing-2, bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.

CD248 induces a maladaptive unfolded protein response in diabetic kidney disease

Dysfunction of mesangial cells plays a major role in the pathogenesis of diabetic kidney disease (DKD), the leading cause of kidney failure. However, the underlying molecular mechanisms are incompletely understood. By unbiased gene expression analysis of glucose-exposed mesangial cells, we identified the transmembrane receptor CD248 as the most upregulated gene, and the maladaptive unfolded protein response (UPR) as one of the most stimulated pathways. Upregulation of CD248 was further confirmed in glucose-stressed mesangial cells invitro, in kidney glomeruli isolated from diabetic mice (streptozotocin; STZ and db/db models, representing type 1 and type 2 diabetes mellitus, respectively) invivo, and in glomerular kidney sections from patients with DKD. Time course analysis revealed that glomerular CD248 induction precedes the onset of albuminuria, mesangial matrix expansion and maladaptive UPR activation (hallmarked by transcription factor C/EBP homologous protein (CHOP) induction) but is paralleled by loss of the adaptive UPR regulator spliced X box binding protein (XBP1). Mechanistically, CD248 promoted maladaptive UPR signaling via inhibition of the inositol requiring enzyme 1α (IRE1α)-mediated transcription factor XBP1 splicing invivo and invitro. CD248 induced a multiprotein complex comprising heat shock protein 90, BH3 interacting domain death agonist (BID) and IRE1α, in which BID impedes IRE1α-mediated XBP1 splicing and induced CHOP mediated maladaptive UPR signaling. While CD248 knockout ameliorated DKD-associated glomerular dysfunction and reverses maladaptive unfolded protein response signaling, concomitant XBP1 deficiency abolished the protective effect in diabetic CD248 knockout mice, supporting a functional interaction of CD248 and XBP1 invivo. Hence, CD248 is a novel mesangial cell receptor inducing maladaptive UPR signaling in DKD.

Endoplasmic reticulum stress and unfolded protein accumulation correlate to seizure recurrence in focal cortical dysplasia patients.

Epileptic seizures occur due to an imbalance between excitatory and inhibitory neurosignals. The excitotoxic insults promote the accumulation of reactive oxygen species (ROS), unfolded proteins (UFP) aggregation, and sometimes even cell death. The epileptic brain samples in our study showed significant changes in the quantity of UFP accumulation. This part explored the efficiency of ER stress and autophagy responses at neutralizing the UFP using resected epileptic brain tissue samples. Meanwhile, we regularly observed these patients' post-surgical clinical data to find the recurrence of seizures. According to International League against Epilepsy (ILAE) suggestions, we classified the patients (n = 26) as class 1 (completely seizure-free), class 2 (less frequent seizures or auras), and class 3 (auras with < 3 seizures per year). The classification helped us understand the reason for variations in the UFP accumulation in patient samples. We have observed the protein levels of ER chaperone, glucose-regulated protein 78kDa (GRP78/BiP), inositol-requiring enzyme 1α (IRE1α), X box-binding protein 1s (XBP1s), eukaryotic translation initiation factor 2α (peIF2α), C/EBP homologous protein (CHOP), NADPH oxidase (NOX2), and autophagy proteins like BECLIN1, ATG 7, 12, 5, 16, p62, and LC3. Our results suggested that ER stress response limitation may contribute to seizure recurrence in epilepsy patients, particularly in classes 2 and 3. In addition, we have observed significant upregulation of ER stress-dependent apoptosis initiation factor CHOP in these patients. These results indicate that understanding the ER stress response pattern infers the possibility of post-surgical outcomes in focal cortical dysplasia (FCD) patients.

Deficiency of p53 Causes the Inadequate Expression of miR-1246 in B Cells of Systemic Lupus Erythematosus.

Underexpression of p53 is considered the leading cause of the decreased miR-1246 expression in B cells of systemic lupus erythematosus (SLE) patients, yet the exact mechanism of action still remains unclear. To further explore the molecular mechanism of p53 upregulating miR-1246 expression, we targeted the methylation and acetylation of histone H3 in the miR-1246 promoter region of SLE B cells. We found that increased histone H3 trimethylation at Lys27 (H3K27me3) and decreased histone H3 acetylation at Lys9 and Lys14 (H3K9/K14ac) in the miR-1246 promoter region are essential for the low expression of miR-1246 in SLE B cells. p53 can promote miR-1246 transcription by recruiting Jumonji domain-containing protein 3 (JMJD3), E1A-binding protein p300 (EP300), and CREB-binding protein (CBP) to bind to the miR-1246 promoter, downregulating H3K27me3 and upregulating H3K9/K14ac. Furthermore, early B cell factor 1 (EBF1), CD40, CD38, and X box binding protein-1 (XBP-1) expression levels in SLE B cells transfected with p53 expression plasmid were significantly decreased, whereas autoantibody IgG production in autologous CD4+ T cells cocultured with overexpressed p53 SLE B cells was reduced. Collectively, our data suggest that the reduction of p53 decreases miR-1246 expression via upregulation of H3K27me3 and downregulation of H3K9/14ac, which in turn results in SLE B cell hyperactivity.

Mutation analysis of the TATA box-binding protein (TBP) gene in Russian patients with spinocerebellar ataxia and Huntington disease-like phenotype

ObjectiveWe aimed to analyze the occurrence and clinical and genetic characteristics of spinocerebellar ataxia type 17 (SCA17) among Russian patients with progressive cerebellar ataxia or Huntington disease-like phenotype. MethodsGenetic analysis of CAG/CAA repeats in TBP gene was carried out in 217 patients, including 153 patients with progressive unspecified ataxia and 64 patients with Huntington disease-like phenotype. SCA types 1, 2, 3, 6 and 8, Friedreich's ataxia, CANVAS and Huntington disease were preliminarily excluded. ResultsSix unrelated patients with SCA17 (2.8 %) were identified (43–57 CAG/CAA repeats in TBP gene). Two patients had a positive family history. Age at the disease onset ranged from 15 to 47 years. The core clinical syndrome included progressive cerebellar ataxia, dysarthria, movement disorders, cognitive impairment, and psychiatric symptoms. One patient had epilepsy with rare generalized tonic-clonic seizures. Another patient with diffuse muscle atrophy and small expansion size (43 CAG/CAA repeats) had myopathic changes in skeletal muscles on EMG study. We also described a patient with a large expansion size of 57 CAA/CAG repeats with early onset and rapid disease progression. ConclusionSCA17 is a relatively rare cause of progressive disorders with ataxia and chorea, but it should be considered in the spectrum of differential diagnosis in such patients. Most of our SCA17 cases were sporadic which should be kept in mind when planning genetic testing in patients with spinocerebellar ataxia and chorea.

TIF1γ inhibits lung adenocarcinoma EMT and metastasis by interacting with the TAF15/TBP complex.

The molecular underpinnings of lung adenocarcinoma (LUAD) metastasis remain poorly defined. Here, using human LUAD cell lines, we find that transcriptional intermediary factor 1 γ (TIF1γ) binds to TATA box binding protein (TBP) in competition with TBP-associated factor 15 (TAF15) and impedes TAF15/TBP-mediated interleukin 6 (IL-6) transactivation. TIF1γ modifies TAF15 through multi-mono-ubiquitylation and drives nuclear export of TAF15. Functionally, TAF15 accelerates epithelial-mesenchymal transition (EMT) and metastasis of LUAD cells, acting in just the opposite way as TIF1γ. Low TIF1γ or high TAF15 expression levels are shown in metastatic LUAD specimens and correlate with poor survival of individuals with LUAD. Our findings suggest that the TAF15/TBP complex is required for IL-6 activation-induced EMT and invasion, which are inhibited by TIF1γ. This study highlights the crucial interaction between TIF1γ and the TAF15/TBP complex for regulating EMT and metastasis in LUAD.

Reduction in Insulin Mediated ERK Phosphorylation by Palmitate in Liver Cells Is Independent of Fatty Acid Induced ER Stress.

Saturated free fatty acids (FFAs) such as palmitate in the circulation are known to cause endoplasmic reticulum (ER) stress and insulin resistance in peripheral tissues. In addition to protein kinase B (AKT) signaling, extracellular signal-regulated kinase (ERK) has been implicated in the development of insulin resistance. However, there are conflicting data regarding role of ERK signaling in ER stress-induced insulin resistance. In this study, we investigated the effects of ER stress on insulin resistance and ERK phosphorylation in Huh-7 cells and evaluated how oleate prevents palmitate-mediated ER stress. Treatment with insulin resulted in an increase of 38–45% in the uptake of glucose in control cells compared to non-insulin-treated control cells, along with an increase in the phosphorylation of AKT and ERK. We found that treatment with palmitate increased the expression of ER stress genes, including the splicing of X box binding protein 1 (XBP1) mRNA. At the same time, we observed a decrease in insulin-mediated uptake of glucose and ERK phosphorylation in Huh-7 cells, without any change in AKT phosphorylation. Supplementation of oleate along with palmitate mitigated the palmitate-induced ER stress but did not affect insulin-mediated glucose uptake or ERK phosphorylation. The findings of this study suggest that palmitate reduces insulin-mediated ERK phosphorylation in liver cells and this effect is independent of fatty-acid-induced ER stress.

1722P The subtype-specific cell fitness of head and neck cancer is regulated by the YBX1 integration of oncogenic PI3K/mTOR signalling

Molecular subtyping of heterogeneous cancers has gained substantial biological interest although with limited translation to the clinic. In heterogeneous head and neck cancer (HNC), subtype-specific treatment regimens are currently missing. A multi-omic approach integrating whole-genome and single-cell transcriptome sequencing with reverse-phase protein array was employed in patient primary and metastatic tumours and human cell lines to investigate the subtype-specific molecular mechanisms as the therapeutic vulnerabilities in HNC. An integrated analysis of HNC subtypes using single-cell sequencing and proteome profiles revealed a hallmark epithelial-mesenchymal transition (EMT) signature within the epithelial cancer-cell population. The EMT signature was evident in the invasive mesenchymal subtype and surprisingly coincided with PI3K/mTOR pathway inactivation. Conversely, the EMT signature was suppressed in epithelial cancer cells of the basal subtype which exhibited hyperactive PI3K/mTOR oncogenic signalling. We further identified that phosphorylation of Y Box binding protein 1 (YBX1), downstream of the PI3K/mTOR pathway, restrained the basal-like cancer cell proliferation. In contrast, YBX1 acted as a safeguard against the proliferation to invasion switch in mesenchymal-like epithelial cancer cells, and its loss accentuated partial EMT acquisition and in vivo invasion. Interestingly, the proteomic analysis of patient HNC identified phospho-YBX1 as a prognostic marker for overall patient outcomes, the loss of which correlated with HNC metastasis and poor prognosis. Our results indicate that HNC cells adopt a PI3K-phospho-YBX1-dependent proliferation program that is mutually exclusive to partial EMT involvement which relies on the lack of phosphorylation of YBX1 in PI3K-inactive cancer cells. These findings create a unique opportunity to sensitise mesenchymal cancer cells to PI3K/mTOR inhibitors by shifting them towards a basal-like subtype, constituting a promising therapeutic approach against HNC.

Abstract P3063: Downregulation Of The T Cell Unfolded Protein Response Precedes T Cell Cardiac Infiltration In Experimental Heart Failure With Preserved Ejection Fraction

Introduction: Diastolic dysfunction in Heart Failure with Preserved Ejection Fraction (HFpEF) is associated with T cell systemic inflammation and downregulation of myocardial unfolded protein response (UPR) genes. The T cell UPR can modulate T cell effector function and immune responses, yet this is largely unexplored in HFpEF. Hypothesis: We hypothesized T cells contribute to cardiometabolic HFpEF and that metabolic and nitrosative stress-induced alterations of the T cell UPR modulate T cell activation and diastolic function. Methods: Male C57/BL6 (wild-type, WT) or T cell receptor alpha-deficient ( Tcra-/-) were fed a high-fat diet (HFD) and L-NAME for 1, 3, and 5 weeks, or standard chow (STD). We assessed diastolic function by invasive hemodynamic analyses. Immune cells were characterized in the heart and spleen by flow cytometry, and cardiac pathology was assessed by histology. The UPR gene expression was evaluated in splenic CD4 + T cells over time by qPCR. Results: Unlike WT mice, Tcra-/- mice fed HFD/L-NAME for 5 weeks did not develop diastolic dysfunction or cardiomyocyte hypertrophy, demonstrating a critical role for T cells in experimental cardiometabolic HFpEF. Indeed, cardiac CD4 + T cells were increased in WT mice fed HFD/L-NAME for 5 weeks compared to STD, concordant with an expansion of splenic CD44 hi CD62l lo interferon-gamma (IFNγ) + effector cells. Strikingly, splenic T cells also experienced downregulation of the UPR proteins activating transcription factor 4 ( Atf4 ), Atf6 , and X box-binding protein 1 ( Xbp1s/u ). We found that the Atf4 arm of the T cell UPR is the earliest responder to HFD/L-NAME, with observed downregulation of Atf4 and its downstream effector C/EBP homologous protein ( Chop ) as early as 3 weeks, prior to onset of diastolic dysfunction, followed by the subsequent downregulation of Atf6 and Xbp1s/u . Conclusions: T cells contribute to diastolic dysfunction and cardiomyocyte hypertrophy in experimental cardiometabolic HFpEF. Early modulation of T cell Atf4 precedes diastolic dysfunction, enhanced IFNγ + effector T cell activation, and global T cell UPR downregulation. Ongoing studies are focused on understanding the functional consequences of impaired T cell UPR in cardiometabolic HFpEF.

Transcriptomic Analysis Reveals That Granulocyte Colony-Stimulating Factor Trigger a Novel Signaling Pathway (TAF9-P53-TRIAP1-CASP3) to Protect Retinal Ganglion Cells after Ischemic Optic Neuropathy.

Optic nerve head (ONH) infarct can result in progressive retinal ganglion cell (RGC) death. The granulocyte colony-stimulating factor (GCSF) protects the RGC after ON infarct. However, protective mechanisms of the GCSF after ONH infarct are complex and remain unclear. To investigate the complex mechanisms involved, the transcriptome profiles of the GCSF-treated retinas were examined using microarray technology. The retinal mRNA samples on days 3 and 7 post rat anterior ischemic optic neuropathy (rAION) were analyzed by microarray and bioinformatics analyses. GCSF treatment influenced 3101 genes and 3332 genes on days 3 and 7 post rAION, respectively. ONH infarct led to changes in 702 and 179 genes on days 3 and 7 post rAION, respectively. After cluster analysis, the levels of TATA box-binding protein (TBP)-associated factor were significantly reduced after ONH infarct, but these significantly increased after GCSF treatment. The network analysis revealed that TBP associated factor 9 (TAF9) can bind to P53 to induce TP53-regulated inhibitor of apoptosis 1 (TRIAP1) expression. To evaluate the function of TAF9 in RGC apoptosis, GCSF plus TAF9 siRNA-treated rats were evaluated using retrograde labeling with FluoroGold assay, TUNEL assay, and Western blotting in an rAION model. The RGC densities in the GCSF plus TAF9 siRNA-treated rAION group were 1.95-fold (central retina) and 1.75-fold (midperipheral retina) lower than that in the GCSF-treated rAION group (p < 0.05). The number of apoptotic RGC in the GCSF plus TAF9 siRNA-treated group was threefold higher than that in the GCSF-treated group (p < 0.05). Treatment with TAF9 siRNA significantly reduced GCSF-induced TP53 and TRIAP1 expression by 2.4-fold and 4.7-fold, respectively, in the rAION model. Overexpression of TAF9 significantly reduced apoptotic RGC and CASP3 levels, and induced TP53 and TRIAP1 expression in the rAION model. Therefore, we have demonstrated that GCSF modulated a new pathway, TAF9-P53-TRIAP1-CASP3, to control RGC death and survival after ON infarct.

Conformational landscape of the yeast SAGA complex as revealed by cryo-EM

Spt-Ada-Gcn5-Acetyltransferase (SAGA) is a conserved multi-subunit complex that activates RNA polymerase II-mediated transcription by acetylating and deubiquitinating nucleosomal histones and by recruiting TATA box binding protein (TBP) to DNA. The prototypical yeast Saccharomyces cerevisiae SAGA contains 19 subunits that are organized into Tra1, core, histone acetyltransferase, and deubiquitination modules. Recent cryo-electron microscopy studies have generated high-resolution structural information on the Tra1 and core modules of yeast SAGA. However, the two catalytical modules were poorly resolved due to conformational flexibility of the full assembly. Furthermore, the high sample requirement created a formidable barrier to further structural investigations of SAGA. Here, we report a workflow for isolating/stabilizing yeast SAGA and preparing cryo-EM specimens at low protein concentration using a graphene oxide support layer. With this procedure, we were able to determine a cryo-EM reconstruction of yeast SAGA at 3.1 Å resolution and examine its conformational landscape with the neural network-based algorithm cryoDRGN. Our analysis revealed that SAGA adopts a range of conformations with its HAT module and central core in different orientations relative to Tra1.

Astragalus polysaccharide alleviates transport stress-induced heart injury in newly hatched chicks via ERS-UPR-autophagy dependent pathway

Transport stress (TS) not only affects animal welfare but also eventually leads to higher morbidity and mortality. Moreover, TS could induce heart injury in animals, but the possible mechanism has yet to be fully explored. Astragalus polysaccharide (APS) is a main active component of Radix Astragali, which has an extensive anti-stress effect. However, the effect of APS on TS-induced heart injury has not yet been elucidated. In this study, a chick model of simulated TS was used. 240 newly hatched chicks were arranged into 4 groups: Control (Con), Transport group (T), Transport + water group (TW), and Transport + APS group (TA). Before transport, the chicks of the TW and TA groups were treated with deionized water and APS (0.25 mg/mL, 100 µL) by oral drops respectively. The histopathological analysis of myocardial tissue was assessed by hematoxylin and eosin staining. qRT-PCR and Western Blotting assays were employed to measure the expression of genes and proteins. Semiquantitative PCR was performed for the X box-binding protein-1 (XBP-1) mRNA splicing assay. The results indicated that APS significantly reduced TS-induced myocardial histopathological changes. Meanwhile, TS induced endoplasmic reticulum stress (ERS), evidenced by an activation of the unfolded protein response (UPR) signaling pathway and up-regulation of ERS-markers (P < 0.05). Moreover, TS markedly triggered autophagy induction by activating AMP-activated protein kinase (AMPK), reflected by augmented LC3-II/LC3-I, AMPK phosphorylation and autophagy-related genes (ATGs) expression (P < 0.05). Importantly, our study manifested that treatment of APS could reduce TS-induced ERS and AMPK-activated autophagy, accordingly alleviating heart injury of transported chicks. In summary, these findings indicate that TS induces heart injury in chicks via an ERS-UPR-autophagy-dependent pathway, and APS as an effective therapeutic method to alleviate it.

Induction of endoplasmic reticulum stress markers in an acromegaly model.

Acromegaly is a growth hormone (GH) excess pathological condition in humans. Acromegaly is associated with somatic disfigurement and a wide range of systemic manifestations such as arthritis, neuropathy, carpal tunnel syndrome, reproductive disorders, metabolic disorders, and gastrointestinal complications. The influence of excess GHon the cellular level could aid in understanding the root causes of acromegaly-related health complications. Previously, we found that GH excess induces DNA damage to somatic cells and reduces the stem cells number and causes premature aging. In this study, an in-depth analysis of the acromegaly RNAseq data revealed the disruption of important biological cellular processes. Gene set enrichment analysis, heatmap, and enrichment analysis of acromegaly RNAseq data revealed induction of endoplasmic reticulum (ER) stress markers in various organs. Interestingly, the induction of ER stress was even more apparent than in aged zebrafish. Splicing of box-binding protein-1 (XBP1) mRNA is a hallmark of ER stress. Therefore, we quantified spliced XBP1 mRNA in different organs of our acromegaly model. Thus, our study emphasizes the importance of ER stress in GH oversecretion, which is important for understanding the health complications of acromegaly.

Single trait versus principal component based association analysis for flowering related traits in pigeonpea

Pigeonpea, a tropical photosensitive crop, harbors significant diversity for days to flowering, but little is known about the genes that govern these differences. Our goal in the current study was to use genome wide association strategy to discover the loci that regulate days to flowering in pigeonpea. A single trait as well as a principal component based association study was conducted on a diverse collection of 142 pigeonpea lines for days to first and fifty percent of flowering over 3 years, besides plant height and number of seeds per pod. The analysis used seven association mapping models (GLM, MLM, MLMM, CMLM, EMLM, FarmCPU and SUPER) and further comparison revealed that FarmCPU is more robust in controlling both false positives and negatives as it incorporates multiple markers as covariates to eliminate confounding between testing marker and kinship. Cumulatively, a set of 22 SNPs were found to be associated with either days to first flowering (DOF), days to fifty percent flowering (DFF) or both, of which 15 were unique to trait based, 4 to PC based GWAS while 3 were shared by both. Because PC1 represents DOF, DFF and plant height (PH), four SNPs found associated to PC1 can be inferred as pleiotropic. A window of ± 2 kb of associated SNPs was aligned with available transcriptome data generated for transition from vegetative to reproductive phase in pigeonpea. Annotation analysis of these regions revealed presence of genes which might be involved in floral induction like Cytochrome p450 like Tata box binding protein, Auxin response factors, Pin like genes, F box protein, U box domain protein, chromatin remodelling complex protein, RNA methyltransferase. In summary, it appears that auxin responsive genes could be involved in regulating DOF and DFF as majority of the associated loci contained genes which are component of auxin signaling pathways in their vicinity. Overall, our findings indicates that the use of principal component analysis in GWAS is statistically more robust in terms of identifying genes and FarmCPU is a better choice compared to the other aforementioned models in dealing with both false positive and negative associations and thus can be used for traits with complex inheritance.

Calpains as novel players in the molecular pathogenesis of spinocerebellar ataxia type 17

Spinocerebellar ataxia type 17 (SCA17) is a neurodegenerative disease caused by a polyglutamine-encoding trinucleotide repeat expansion in the gene of transcription factor TATA box-binding protein (TBP). While its underlying pathomechanism is elusive, polyglutamine-expanded TBP fragments of unknown origin mediate the mutant protein’s toxicity. Calcium-dependent calpain proteases are protagonists in neurodegenerative disorders. Here, we demonstrate that calpains cleave TBP, and emerging C-terminal fragments mislocalize to the cytoplasm. SCA17 cell and rat models exhibited calpain overactivation, leading to excessive fragmentation and depletion of neuronal proteins in vivo. Transcriptome analysis of SCA17 cells revealed synaptogenesis and calcium signaling perturbations, indicating the potential cause of elevated calpain activity. Pharmacological or genetic calpain inhibition reduced TBP cleavage and aggregation, consequently improving cell viability. Our work underlines the general significance of calpains and their activating pathways in neurodegenerative disorders and presents these proteases as novel players in the molecular pathogenesis of SCA17.

A Severe Dementia Syndrome Caused by Intron Retention and Cryptic Splice Site Activation in STUB1 and Exacerbated by TBP Repeat Expansions.

Heterozygous pathogenic variants in the STIP1 homologous and U-box containing protein 1 (STUB1) gene have been identified as causes of autosomal dominant inherited spinocerebellar ataxia type 48 (SCA48). SCA48 is characterized by an ataxic movement disorder that is often, but not always, accompanied by a cognitive affective syndrome. We report a severe early onset dementia syndrome that mimics frontotemporal dementia and is caused by the intronic splice donor variant c.524+1G>A in STUB1. Impaired splicing was demonstrated by RNA analysis and in minigene assays of mutated and wild-type constructs of STUB1. The most striking consequence of this splicing impairment was retention of intron 3 in STUB1, which led to an in-frame insertion of 63 amino acids (aa) (p.Arg175_Glu176ins63) into the highly conserved coiled-coil domain of its encoded protein, C-terminus of HSP70-interacting protein (CHIP). To a lesser extent, activation of two cryptic splice sites in intron 3 was observed. The almost exclusively used one, c.524+86, was not predicted by in silico programs. Variant c.524+86 caused a frameshift (p.Arg175fs*93) that resulted in a truncated protein and presumably impairs the C-terminal U-box of CHIP, which normally functions as an E3 ubiquitin ligase. The cryptic splice site c.524+99 was rarely used and led to an in-frame insertion of 33 aa (p.Arg175_Glu176ins33) that resulted in disruption of the coiled-coil domain, as has been previously postulated for complete intron 3 retention. We additionally detected repeat expansions in the range of reduced penetrance in the TATA box-binding protein (TBP) gene by excluding other genes associated with dementia syndromes. The repeat expansion was heterozygous in one patient but compound heterozygous in the more severely affected patient. Therefore, we concluded that the observed severe dementia syndrome has a digenic background, making STUB1 and TBP important candidate genes responsible for early onset dementia syndromes.

Selective sorting and secretion of hY4 RNA fragments into extracellular vesicles mediated by methylated YBX1 to promote lung cancer progression

BackgroundExtracellular vesicles (EVs) are emerging mediators of intercellular communication that have been shown to play important roles in tumor progression. YRNA fragments, a type of small non-coding RNA, are dysregulated in non-small cell lung cancer (NSCLC) cell-derived EVs, suggesting that they may be an effective biomarker for cancer diagnosis and treatment strategies.MethodsDifferentially expressed YRNA hY4 fragments (hY4F) in EVs from NSCLC cells and normal lung fibroblasts were isolated by differential ultra-centrifugation. RNA-binding proteins that interacted with hY4F were identified by screening with an RNA pulldown assay and mass spectrometry. The molecular mechanism of hY4F and the RNA-binding protein Y box binding protein 1 (YBX1) was demonstrated by qRT-PCR, western blot, RNA pulldown, and rescue experiments. Transcriptome sequencing, qRT-PCR validation, bioinformatics analysis and NF-κB pathway inhibitor assays elucidate the mechanism of YBX1 and hY4F inhibiting lung cancer. A peptide pulldown assay was performed to screen and identify a potential methyltransferase for YBX1. The roles of hY4F, YBX1, and SET domain containing 3 in biological functions, such as proliferation, migration, invasion, and apoptosis, in lung cancer cells were also examined by EdU incorporation assay, Transwell assay, flow cytometry, and other methods. Lastly, a mouse xenograft assay was used to assess the clinical relevance of YBX1 and hY4F in vivo.ResultsOur data demonstrate that hY4 RNA fragments were upregulated in lung cancer- derived EVs, hY4F inhibits tumor progression through downregulating MAPK/NF-κB signaling, and then the selective sorting and secretion of hY4F into lung cancer EVs is regulated by the RNA-binding protein YBX1. Furthermore, we identified lysine K264 within the YBX1 C-terminal domain as the necessary site for its interaction with hY4Fs. K264 is modified by methylation, which affects its binding to hY4F and subsequent selective sorting into EVs in lung cancer cells.ConclusionOur findings demonstrate that hY4F acts as a tumor suppressor and is selectively sorted into lung cancer cell-derived EVs by interacting with methylated YBX1, which in turn promotes lung cancer progression. hY4F is a promising circulating biomarker for non-small cell lung cancer diagnosis and prognosis and an exceptional candidate for further therapeutic exploration.

The Mechanism of Lipopolysaccharide Escaping the Intestinal Barrier in Megalobrama amblycephala Fed a High-Fat Diet.

With the popularity of western food characterized by excessive fat and sugars, obesity has currently been a public health issue. Low-grade chronic inflammation accompanied by obesity increases the risk of multiple epidemics such as diabetes, cancer and cardiovascular diseases. Here, we show that feeding Megalobrama amblycephala with a high-fat diet (HFD) drives obesity-related chronic inflammation and the penetration of lipopolysaccharide (LPS). Interference with antibiotics inhibits the produce of LPS and this alleviates the sustained release of pro-inflammatory factors induced by HFD. LPS penetration is attributed to weakened intestinal mucus barrier after high-fat exposure. Mechanically, the consumption of HFD inhibits the secretion of mucin 2 (MUC2) due to the induction of endoplasmic reticulum stress mediated by the inositol-requiring enzyme 1 (IRE1) /X box-binding protein 1 (XBP1) pathway in goblet cells. Furthermore, excessive lipid exacerbates the leakage of LPS across the intestinal epithelial cell barrier via the transcellular pathway. Mechanically, lipid increases the internalization of LPS in intestinal epithelial cells depending on the activation of fatty acid translocase (FAT/CD36). These results demonstrate that HFD causes the penetration of LPS due to the weakened intestinal mucosal barrier and the assistance of CD36.