Regulatory Proteins Research Articles

Changes of m6A Regulatory Proteins and Nrf2 Signaling Molecules in Liver Tissue of Type 2 Diabetes Mellitus Rats.

Both dysregulation of N6-methyladenosine (m6A) regulatory proteins and Nrf2 signaling molecules are involved in the process of injury to multiple tissues. However, changes of m6A regulatory proteins and Nrf2 signaling molecules in liver tissue of T2DM remain unclear. In present study, changes of m6A regulatory proteins (Mettl3, Mettl16, Fto, Alkbh5 and Ythdc2) and Nrf2 signaling molecules (Nrf2, Sod1, Ho-1, Gclc) were detected in the liver tissues of T2DM rats, which constructed by high fat-diet feeding and intraperitoneal injection of streptozotocin. Our results indicated that the morphology of liver tissues from T2DM rats showed obvious abnormalities, as well as levels of liver function indicators and expressions of Nrf2 signaling molecules Nrf2, Sod1, Ho-1 were significantly increased in T2DM rats when compared with those in corresponding control rats. More importantly, m6A regulatory proteins such as Mettl3, Mettl16, Fto, Alkbh5 and Ythdc2 were dramatically higher than those in control rat. In a word, m6A regulatory proteins and Nrf2 signaling molecules may significantly change in liver tissue of T2DM rats. And This provides clues and ideas for the study of liver injury in T2DM from the perspective of RNA epigenetics in the future.

High β-sitosterol-D-glucoside content in sweet potato varieties and its anti-breast cancer potential through multiple metastasis-associated signals

β-Sitosterol-D-glucoside (β-SDG) is a phytosterol compound whose antitumor activity has been confirmed by previous studies. However, its suppression on breast cancer remains unclear. To that purpose, we isolated β-SDG from sweet potato and investigated the breast-cancer-inhibiting mechanism using proteomic analysis. The sweet potato species S6 with high β-SDG content were chosen form 36 species and β-SDG was isolated by HPLC. Afterwards, an in situ animal model of breast cancer was established, and β-SDG significantly reduced the tumor volume of MCF-7 xenograft mice. Proteomic analysis of tumor tissues revealed that 127 of these proteins were upregulated and 80 were downregulated. Gene ontology and network analysis showed that regulatory proteins were mainly associated with epithelial-mesenchymal transition (EMT), myogenesis, cholesterol homeostasis, oxidative phosphorylation and reactive oxygen pathways, while Vimentin, NDUF, VDAC1, PPP2CA and SNX9 were the most significant 5 node degree genes. Meanwhile, in vitro and in vivo results showed that the protein expression of PPP2CA and Vimentin, which are markers of EMT, were involved in breast cancer cell metastasis and could be reversed by β-SDG. This work highlights β-SDG as a bioactive compound in sweet potato and the potential therapeutic effect of β-SDG for the treatment of breast cancer by inhibiting metastasis.

Two-pore channel 2 is required for soluble adenylyl cyclase-dependent regulation of melanosomal pH and melanin synthesis.

Melanosomal pH is important for the synthesis of melanin as the rate-limiting enzyme, tyrosinase, is very pH-sensitive. The soluble adenylyl cyclase (sAC) signaling pathway was recently identified as a regulator of melanosomal pH in melanocytes; however, the melanosomal proteins critical for sAC-dependent regulation of melanosomal pH were undefined. We now systematically examine four well-characterized melanosomal membrane proteins to determine whether any of them are required for sAC-dependent regulation of melanosomal pH. We find that OA1, OCA2, and SLC45A2 are dispensable for sAC-dependent regulation of melanosomal pH. In contrast, TPC2 activity is required for sAC-dependent regulation of melanosomal pH and melanin synthesis. In addition, activation of TPC2 by NAADP-AM rescues melanosomal pH alkalinization and reduces melanin synthesis following pharmacologic or genetic inhibition of sAC signaling. These studies establish TPC2 as a critical melanosomal protein for sAC-dependent regulation of melanosomal pH and pigmentation.

Melatonin regulates mitochondrial dynamics and mitophagy: Cardiovascular protection.

Despite extensive progress in the knowledge and understanding of cardiovascular diseases and significant advances in pharmacological treatments and procedural interventions, cardiovascular diseases (CVD) remain the leading cause of death globally. Mitochondrial dynamics refers to the repetitive cycle of fission and fusion of the mitochondrial network. Fission and fusion balance regulate mitochondrial shape and influence physiology, quality and homeostasis. Mitophagy is a process that eliminates aberrant mitochondria. Melatonin (Mel) is a pineal-synthesized hormone with a range of pharmacological properties. Numerous nonclinical trials have demonstrated that Mel provides cardioprotection against ischemia/reperfusion, cardiomyopathies, atherosclerosis and cardiotoxicity. Recently, interest has grown in how mitochondrial dynamics contribute to melatonin cardioprotective effects. This review assesses the literature on the protective effects of Mel against CVD via the regulation of mitochondrial dynamics and mitophagy in both in-vivo and in-vitro studies. The signalling pathways underlying its cardioprotective effects were reviewed. Mel modulated mitochondrial dynamics and mitophagy proteins by upregulation of mitofusin, inhibition of DRP1 and regulation of mitophagy-related proteins. The evidence supports a significant role of Mel in mitochondrial dynamics and mitophagy quality control in CVD.

Impact of Epithelial Claudin-4 and Leukotriene B4 Receptor 2 in Normoganglionic Hirschsprung Disease Colon on Post Pull-through Enterocolitis

PurposeTo investigate whether Leukotriene B4 receptor 2 (BLT-2), an upstream regulator of tight junction protein (TJP) Claudin-4, and TJPs could be etiologic factors in Hirschsprung-associated enterocolitis (HAEC) after pull-through (PT) for Hirschsprung disease (HD). MethodsNormoganglionic colon (HD-N) and aganglionic rectum (HD-A) specimens from rectal/rectosigmoid (R/RS) or descending/transverse (D/T) HD were assessed using quantitative polymerase chain reaction (qPCR) for Occludin, TJP-1, TJP-2, Junctional adhesion molecule (JAM)-1, JAM-2, Claudin-1, Claudin-3, Claudin-4, and BLT-2 and immunoblotting for Claudin-4 using fresh specimens obtained intraoperatively (2021–2024; n = 17; R/RS = 15 and D/T = 2). Claudin-4 immunohistochemistry was also evaluated quantitatively using preserved (n = 29; R/RS = 20 and D/T = 9; 2009–2021) and fresh HD specimens for comparison with anorectal malformation patients having colostomy closure as controls (n = 42) and between HD-A versus HD-N, R/RS versus D/T, and HAEC (+) versus HAEC (−). Technically inadequate or transitional zone PT were excluded. ResultsSubjects were 123 PT cases. Mean ages at PT/colostomy closure (years) were R/RS: 2.7 ± 2.9, D/T: 1.6 ± 2.2, and controls: 1.4 ± 0.7. Postoperative HAEC occurred 18 times in 14 PT cases (grade I = 5, grade II = 13). Post-PT HAEC was significantly more frequent in D/T (50.0% versus 6.4%; p < 0.001); Claudin-4 was significantly lower in HD-N from post-PT HAEC cases, especially D/T (p < 0.05) on immunohistochemistry. Claudin-4 was significantly lower in HD-N/HD-A compared with controls on immunoblotting (p < 0.05) and immunohistochemistry (p < 0.001). qPCR showed TJP-1, TJP-2, JAM-1, JAM-2, Claudin-4, and BLT-2 were significantly lower in HD-N/HD-A compared with controls. ConclusionsLower Claudin-4 and BLT2 in post-PT HAEC HD-N (especially D/T) suggests generalized epithelial barrier derangement with possible etiologic implications for HAEC. Level of EvidenceⅡ

Proteomic characterization of Tenacibaculum dicentrarchi under iron limitation reveals an upregulation of proteins related to iron oxidation and reduction metabolism, iron uptake systems and gliding motility.

A strategy for vaccine design involves identifying proteins that could be involved in pathogen-host interactions. The aim of this proteomic study was to determine how iron limitation affects the protein expression of Tenacibaculum dicentrarchi, with a primary focus on virulence factors and proteins associated with iron uptake. The proteomic analysis was carried out using two strains of T. dicentrarchi grown under normal (control) and iron-limited conditions, mimicking the host environment. Our findings revealed differences in the proteins expressed by the type strain CECT 7612T and the Chilean strain TdCh05 of T. dicentrarchi. Nonetheless, both share a common response to iron deprivation, with an increased expression of proteins associated with iron oxidation and reduction metabolism (e.g., SufA, YpmQ, SufD), siderophore transport (e.g., ExbD, TonB-dependent receptor, HbpA), heme compound biosynthesis, and iron transporters under iron limitation. Proteins involved in gliding motility, such as GldL and SprE, were also upregulated in both strains. A negative differential regulation of metabolic proteins, particularly those associated with amino acid biosynthesis, was observed under iron limitation, reflecting the impact of iron availability on bacterial metabolism. Additionally, the TdCh05 strain exhibited unique proteins associated with gliding motility machinery and phage infection control compared to the type strain. These groups of proteins have been identified as virulence factors within the Flavobacteriaceae family, including the genus Tenacibaculum. These results build upon our previous report on iron acquisition mechanisms and could lay the groundwork for future studies aimed at elucidating the role of some of the described proteins in the infectious process of tenacibaculosis, as well as in the development of potential vaccines.

Oxygen-dependent regulation of F-box proteins in Toxoplasma gondii is mediated by Skp1 glycosylation

A dynamic proteome is required for cellular adaption to changing environments including levels of O2, and the SKP1/CULLIN-1/F-box protein/RBX1 (SCF) family of E3 ubiquitin ligases contributes importantly to proteasome-mediated degradation. We examine, in the apicomplexan parasite Toxoplasma gondii, the influence on the interactome of SKP1 by its novel glycan attached to a hydroxyproline generated by PHYa, the likely ortholog of the HIFα PHD2 oxygen-sensor of human host cells. Strikingly, the representation of several putative F-box proteins (FBPs) is substantially reduced in PHYaΔ parasites grown in fibroblasts. One, FBXO13, is a predicted lysyl hydroxylase related to the human JmjD6 oncogene except for its F-box domain. The abundance of FBXO13, epitope-tagged at its genetic locus, was reduced in PHYaΔ parasites thus explaining its diminished presence in the SKP1 interactome. A similar effect was observed for FBXO14, a cytoplasmic protein of unknown function that may have co-evolved with PHYa in apicomplexans. Similar findings in glycosylation-mutant cells, rescue by proteasomal inhibitors, and unchanged transcript levels, suggested the involvement of the SCF in their degradation. The effect was selective, because FBXO1 was not affected by loss of PHYa. These findings are physiologically significant because the effects were phenocopied in parasites reared at 0.5% O2. Modest impact on steady-state SKP1 modification levels suggests that effects are mediated during a lag phase in hydroxylation of nascent SKP1. The dependence of FBP abundance on O2-dependent SKP1 modification likely contributes to the reduced virulence of PHYaΔ parasites owing to impaired ability to sense O2 as an environmental signal.

Protofilament-specific nanopatterns of tubulin post-translational modifications regulate the mechanics of ciliary beating

Controlling ciliary beating is essential for motility and signaling in eukaryotes. This process relies on the regulation of various axonemal proteins that assemble in stereotyped patterns onto individual microtubules of the ciliary structure. Additionally, each axonemal protein interacts exclusively with determined tubulin protofilaments of the neighboring microtubule to carry out its function. While it is known that tubulin post-translational modifications (PTMs) are important for proper ciliary motility, the mode and extent to which they contribute to these interactions remain poorly understood. Currently, the prevailing understanding is that PTMs can confer functional specialization at the level of individual microtubules. However, this paradigm falls short of explaining how the tubulin code can manage the complexity of the axonemal structure where functional interactions happen in defined patterns at the sub-microtubular scale. Here, we combine immuno-cryo-electron tomography (cryo-ET), expansion microscopy, and mutant analysis to show that, in motile cilia, tubulin glycylation and polyglutamylation form mutually exclusive protofilament-specific nanopatterns at a sub-microtubular scale. These nanopatterns are consistent with the distributions of axonemal dyneins and nexin-dynein regulatory complexes, respectively, and are indispensable for their regulation during ciliary beating. Our findings offer a new paradigm for understanding how different tubulin PTMs, such as glycylation, glutamylation, acetylation, tyrosination, and detyrosination, can coexist within the ciliary structure and specialize individual protofilaments for the regulation of diverse protein complexes. The identification of a ciliary tubulin nanocode by cryo-ET suggests the need for high-resolution studies to better understand the molecular role of PTMs in other cellular compartments beyond the cilium.

Abstract P197: Identification and Characterization of Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Proteins in Placenta

Preeclampsia is life threatening condition affecting about 10% of all pregnancies. A potential mechanism is via malfunctioning syncytiotrophoblasts in the placenta. RhoBTB1 is an adapter protein for the CUL3 E3 ubiquitin ligase which delivers protein substrates for proteasomal degradation. An analysis of gene expression databases revealed that the highest level of RhoBTB1 expression is in the placenta. RNAscope in situ hybridization data reveals that RhoBTB1 expression is specifically localized to syncytiotrophoblasts. In blood vessels, RhoBTB1 controls the state of nitric oxide mediated vasodilation because it acts as a substrate adaptor for Phosphodiesterase 5. Thus, we wanted to identify potential protein targets of RhoBTB1 in the placenta to assess if RhoBTB1 plays a role in placental function. To simplify the first identification of RhoBTB1 binding proteins, we employed HTR-8 /SVneo (HTR-8) cells as a model of placental trophoblast cells. We biotinylated the HTR-8 proteome utilizing ascorbate peroxidase (APEX2) proximity labelling system followed by mass spectrometry to identify RhoBTB1 targets. We employed a fusion protein consisting of the C-terminal half of RhoBTB1 (B1B2C), the region required for the binding of other protein substrates, and a control lacking the most C-terminal region (B1B2). We next chose proteins with binding to B1B2C &gt; B1B2 of 2-fold or greater that were expressed in the placenta based on the Human Protein Atlas. Rho GTPase Activating Protein 29 (ARHGAP29), which controls the regulation of small GTP binding proteins, and S-phase Kinase associated Protein 2 (SKP2), a regulator of cell cycle progression was studied further. SKP2 is interesting because it exhibits a tissue specific tropism for placental expression with its highest expression in extravillous trophoblasts, cytotrophoblasts, and syncytiotrophoblasts. Expression of ARHGAP29 and SKP2 in HTR-8 cells were markedly upregulated by MLN4924 suggesting they are regulated by the CUL pathway. They were also modestly upregulated after partial inhibition of RhoBTB1 by siRNA suggesting they may be RhoBTB1-CUL3 targets. Studies are ongoing to validate the physical interaction of ARHGAP29 and SKP2 with RhoBTB1 by co-immunoprecipitation. We are also using RNAscope to assess the co-expression of RhoBTB1, ARHGAP29 and SKP2 in the human placenta from normal and preeclamptic pregnancies.

Piperlongumine, a natural alkaloid from Piper longum L. ameliorates metabolic-associated fatty liver disease by antagonizing the thromboxane A2 receptor

Metabolic dysfunction-associated fatty liver disease (MAFLD) encompasses a broad spectrum of hepatic disorders, including hyperglycemia, hepatic steatosis, and insulin resistance. Piperlongumine (PL), a natural amide alkaloid extracted from the fruits of Piper longum L., exhibited hepatoprotective effects in zebrafish and liver injury mice. This study aimed to investigate the therapeutic potential of PL on MAFLD and its underlying mechanisms. The findings demonstrate that PL effectively combats MAFLD induced by a high-fat diet (HFD) and improves metabolic characteristics in mice. Additionally, our results suggest that the anti-MAFLD effect of PL is attributed to the suppression of excessive hepatic gluconeogenesis, inhibition of de novo lipogenesis, and alleviation of insulin resistance. Importantly, the results indicate that, on the one hand, the hypoglycemic effect of PL is closely associated with CREB-regulated transcriptional coactivators (CRTC2)-dependent cyclic AMP response element binding protein (CREB) phosphorylation; on the other hand, the lipid-lowering effect of PL is attributed to reducing the nuclear localization of sterol regulatory element-binding proteins 1c (Srebp-1c). Mechanistically, PL could alleviate insulin resistance induced by endoplasmic reticulum stress by antagonizing the thromboxane A2 receptor (TP)/Ca2+ signaling, and the TP receptor serves as the potential target for PL in the treatment of MAFLD. Therefore, our results suggested PL effectively improved the major hallmarks of MAFLD induced by HFD, highlighting a potential therapeutic strategy for MAFLD.

Fatty acid synthesis is indispensable for Kupffer cells to eliminate bacteria in ALD progression.

Dysregulated fatty acid metabolism is closely linked to the development of alcohol-associated liver disease (ALD). KCs, which are resident macrophages in the liver, play a critical role in ALD pathogenesis. However, the effect of alcohol on fatty acid metabolism in KCs remains poorly understood. The current study aims to investigate fatty acid metabolism in KCs and its potential effect on ALD development. Wild-type C57BL/6 mice were fed a Lieber-DeCarli ethanol liquid diet for 3 days. Then, the liver injury and levels of intrahepatic bacteria were assessed. Next, we investigated the effects and underlying mechanisms of ethanol exposure on fatty acid metabolism and the phagocytosis of KCs, both in vivo and in vitro. Finally, we generated KCs-specific Fasn knockout and overexpression mice to evaluate the impact of FASN on the phagocytosis of KCs and ethanol-induced liver injury. Using Bodipy493/503 to stain intracellular neutral lipids, we found significantly reduced lipid levels in KCs from mice fed an alcohol-containing diet for 3 days and in RAW264.7 macrophages exposed to ethanol. Mechanistically, alcohol exposure suppressed sterol regulatory element-binding protein 1 transcriptional activity, thereby inhibiting fatty acid synthase (FASN)-mediated de novo lipogenesis in macrophages both in vitro and in vivo. We show that genetic ablation and pharmacologic inhibition of FASN significantly impaired KC's ability to take up and eliminate bacteria. Conversely, KCs-specific Fasn overexpression reverses the impairment of macrophage phagocytosis caused by alcohol exposure. We also revealed that KCs-specific Fasn knockout augmented KCs apoptosis and exacerbated liver injury in mice fed an alcohol-containing diet for 3 days. Our findings indicate the crucial role of de novo lipogenesis in maintaining effective KCs phagocytosis and suggest a therapeutic target for ALD based on fatty acid synthesis in KCs.

Ameliorative effects of Trichosanthes kirilowii Maxim. seed oil on hyperlipidemia rats associated with the regulation of gut microbiology and metabolomics

The mechanisms underlying the ameliorative effects of polyunsaturated fatty acids (PUFAs) on metabolic disorders induced by a high-fat diet (HFD) remain poorly unclear. In this study, we investigated the anti-hyperlipidemic effects of Trichosanthes kirilowii Maxim. (T. kirilowii) seed oil rich in conjugated linolenic acid in HFD-induced hyperlipidemic rats, by the gut microbiome, cecum bile acids (BAs), and serum metabolomics. The results showed that T. kirilowii seed oil improved dyslipidemia, hepatic steatosis, oxidative stress, and inflammatory responses in HFD-induced rats. Meanwhile, T. kirilowii seed oil inhibited sterol regulatory element-binding protein 1c (SREBP-1c) mediated fatty acid synthesis and upregulated cholesterol 7-alpha hydroxylase (CYP7A1) mediated hepatic cholesterol metabolism to exert hypolipidemic effects. The administration of high dose T. kirilowii seed oil (THD) improved gut microbiota dysbiosis, increased the relative abundance of beneficial bacteria Romboutsia and unidentified_Oscillospiraceae, and decreased the relative abundance of Christensenellaceae_R-7 group, Phascolarctobacterium, and Bacteroides in HFD-induced rats. T. kirilowii seed oil reduced the accumulation of cecum primary BAs in HFD-induced rats. In addition, THD reversed the HFD-induced changes in 24 serum metabolites including leucine, isoleucine, acetylcarnitine, and glucose. Metabolic pathway enrichment analysis of the differential metabolites revealed that valine, leucine and isoleucine metabolism, butanoate metabolism, citrate cycle, and glycolysis were potential metabolic pathways involved in the anti-hyperlipidemic effects of T. kirilowii seed oil. In conclusion, this study found that dietary T. kirilowii seed oil alleviated gut microbiota dysbiosis and improved metabolic disorders in hyperlipidemic rats. This provides new insights into the anti-hyperlipidemic mechanism by which other families of PUFAs are derived from different plants.

Ameliorative effects of Monascus-fermented hawthorn extract on a high-fat diet-induced rat model of non-alcoholic fatty liver disease

ObjectiveThe aim of this study was to elucidate the effects of fermented hawthorn extract on high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in rats, and explore the possible underlying mechanisms. MethodsA total of 42 male adult Sprague-Dawley rats were randomly divided into five groups: normal control group (given a normal feed diet and distilled water by gavage), NAFLD model (given HFD and distilled water by gavage), low-, medium-, and high-dose fermented hawthorn extract treatment groups (given HFD and different doses of fermented hawthorn extract by gavage). After 12 weeks of gavage administration, changes in body weight, liver/body weight ratio, serum liver enzymes, as well as triglyceride (TG) content and oxidative stress levels in rat liver tissueswere detected. Histological evaluation was performed to observe the degree of fat accumulation (steatosis). qRT-PCR and western blotting were performed to detect the mRNA and protein expression of cytochrome P4502E1 (CYP2E1, a key enzyme associated with lipid peroxidation), and lipogenic factors (sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS)) in rat liver tissues. ResultsFermented hawthorn extract significantly reduced the body weight, decreased the levels of liver enzymes, improved hepatic steatosis, and exhibited obvious antioxidant effects. Fermented hawthorn extract also significantly down-regulated the mRNA and protein expression levels of CYP2E1, SREBP-1c and FAS. ConclusionOur findings suggested that fermented hawthorn extract can markedly reduce body weight, ameliorate HFD-induced NAFLD in rats, and exhibits significant antioxidant effects. Its underlying mechanism may depend on the inhibition of CYP2E1, SREBP-1c, and FAS expression.

Potential and Metabolic Impacts of Double Enrichments of Docosahexaenoic Acid and 25-Hydroxy Vitamin D3 in Tissues of Broiler Chickens

BackgroundChicken may be enriched with 25-hydroxy D3 [25(OH)D3] and docosahexaenoic acid (DHA) to enhance the dietary intake of the public. ObjectivesTwo experiments (Expt.) were conducted to determine the potential and metabolic impacts of enriching both DHA and 25(OH)D3 in the tissues of broiler chickens. MethodsIn Expt. 1, 144 chicks (6 cages/treatment and 6 birds/cage) were fed a corn–soybean meal basal diet (BD), BD + 10,000 IU 25(OH)D3/kg [BD + 25(OH)D3], BD + 1% DHA-rich Aurantiochytrium (1.2 g DHA/kg; BD + DHA), or BD + 25(OH)D3+DHA for 6 wk. In Expt. 2, 180 chicks were fed the BD, BD + DHA-rich microalgal oil (1.5–3.0 g DHA/kg, BD + DHA), BD + DHA + eicosapentaenoic acid (EPA)-rich microalgae (0.3–0.6 g EPA/kg, BD + DHA + EPA), BD + DHA + 25(OH)D3 [6000 to 12,000 IU/kg diet; BD + DHA + 25(OH)D3], and BD + DHA + EPA + 25(OH)D3 for 6 wk. ResultsSupranutrition of these 2 nutrients resulted in 57–62 mg DHA and 1.9–3.3 μg of 25(OH)D3/100 g of breast or thigh muscles. The DHA enrichment was independent of dietary EPA or 25(OH)D3, but that of 25(OH)D3 in the liver was decreased (68%, P < 0.05) by dietary DHA in Expt. 1. Compared with BD, BD + 25(OH)D3 enhanced (P < 0.05) gene expression related to D3 absorption (scavenger receptor class B type 1 and Niemann-pick c1 like 1) in the liver and D3 degradation (cytochrome P450 24A1) in the breast, and decreased mRNA or protein concentrations of vitamin D binding protein in the adipose tissue or thigh muscle. Supranutrition of DHA decreased mRNA concentrations of lipid metabolism-related genes (fatty acid desaturase 1,2, ELOVL fatty acid elongase 5, fatty acid desaturase 2, fatty acid synthase, and sterol regulatory element-binding protein 1). ConclusionsBoth DHA and 25(OH)D3 were enriched in the muscles up to meeting 50%–100% of the suggested intakes of these nutrients by consuming 2 servings of 100 g of fortified chicken. The enrichments altered gene expression related to lipid biosynthesis and vitamin D transport or storage.

Specific kinesin and dynein molecules participate in the unconventional protein secretion of transmembrane proteins.

Secretory proteins, including plasma membrane proteins, are generally known to be transported to the plasma membrane through the endoplasmic reticulum- to-Golgi pathway. However, recent studies have revealed that several plasma membrane proteins and cytosolic proteins lacking a signal peptide are released via an unconventional protein secretion (UcPS) route, bypassing the Golgi during their journey to the cell surface. For instance, transmembrane proteins such as the misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein and the Spike protein of coronaviruses have been observed to reach the cell surface through a UcPS pathway under cell stress conditions. Nevertheless, the precise mechanisms of the UcPS pathway, particularly the molecular machineries involving cytosolic motor proteins, remain largely unknown. In this study, we identified specific kinesins, namely KIF1A and KIF5A, along with cytoplasmic dynein, as critical players in the unconventional trafficking of CFTR and the SARS-CoV-2 Spike protein. Gene silencing results demonstrated that knockdown of KIF1A, KIF5A, and the KIF-associated adaptor protein SKIP, FYCO1 significantly reduced the UcPS of △F508-CFTR. Moreover, gene silencing of these motor proteins impeded the UcPS of the SARS-CoV-2 Spike protein. However, the same gene silencing did not affect the conventional Golgimediated cell surface trafficking of wild-type CFTR and Spike protein. These findings suggest that specific motor proteins, distinct from those involved in conventional trafficking, are implicated in the stress-induced UcPS of transmembrane proteins.

P112 SUPPRESSION OF PROTEIN KINASE RNA-LIKE ENDOPLASMIC RETICULUM KINASE BY PROBIOTICS CIRCUMVENTS CARDIOVASCULAR RISK PROFILE IN EXPERIMENTALLY-INDUCED PCOS MODEL

Background and Objective: The global cause of death attributable to cardiovascular diseases (CVD). including in women of reproductive age is alarming. Individuals with PCOS usually present CVD as a long-term life-threatening effect of PCOS. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) has been implicated in cardiomyopathy. However, its role in PCOS-induced CVD risk has not been established. Studies have shown that probiotics improves metabolic-related disorders. The present study was designed to investigate the role of PERK in CVD risk associated with PCOS in experimental rat model, and the therapeutic benefits of probiotics. Methods: Eight-week-old female Wistar rats were assigned into four groups (n=6). Polycystic ovarian syndrome was induced by uninterrupted administration of letrozole (1 mg/kg) for 21 days, thereafter, probiotics (2 x 107 CFU) was administered for 6 weeks. Gene expression, biochemical parameters and histological evaluations were performed with Western blot, ELISA, and H&amp;E staining techniques Results: The present findings revealed that animals with PCOS were characterized with phenotypic features such as hyperandrogenemia and multiple cysts in the ovaries. In addition, PCOS rats manifested insulin resistance and increase in glucose regulatory protein (GRP78), together with increased levels of circulating corticosterone, cardiac triglyceride, inflammatory mediators (NF- kappaB/TNF-alpha), TGF-beta1, Caspase-6, and HDAC2, while a decrease in HIF-1alpha and NrF2 was observed when compared with control animals. These were accompanied by elevated expression of PERK. However, treatment with probiotics reversed these systemic, endocrine, metabolic and cardiac anomalies. Conclusion: The present study suggests that probiotics attenuates CVD risk profile in experimental PCOS rat model by suppression of PERK/HDAC2-dependent pathway.

Modification of Regulatory Tyrosine Residues Biases Human Hsp90α in its Interactions with Cochaperones and Clients

The highly conserved Hsp90 chaperones control stability and activity of many essential signaling and regulatory proteins including many protein kinases, E3 ligases and transcription factors. Thereby, Hsp90s couple cellular homeostasis of the proteome to cell fate decisions. High-throughput mass spectrometry revealed 178 and 169 posttranslational modifications (PTMs) for human cytosolic Hsp90α and Hsp90β, but for only a few of the modifications the physiological consequences are investigated in some detail. In this study, we explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90α. Replacement of three tyrosine residues known to be phosphorylated by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced yeast growth and the maturation of 7 different Hsp90 clients in distinct ways. Furthermore, wild-type and mutant Hsp90 differed in their ability to stabilize known clients when expressed in HepG2 HSP90AA1−/− cells. The purified mutant proteins differed in their interaction with the cochaperones Aha1, Cdc37, Hop and p23 and in their support of the maturation of glucocorticoid receptor ligand binding domain in vitro. In vivo and in vitro data correspond well to each other confirming that the yeast system is suitable for the identification of key regulatory sites in human Hsp90s. Our findings indicate that even closely related clients are affected differently by the amino acid replacements in the investigated positions, suggesting that PTMs could bias Hsp90s client specificity.

GLYAT suppresses liver cancer and clear cell renal cell carcinoma progression by downregulating ROCK1 expression.

The liver and kidney are important metabolic organs in the body and common sites of tumor occurrence. Glycine-N-acyltransferase (GLYAT) is primarily expressed in the liver and kidney and downregulated in several tumors. But its specific functions and molecular mechanisms in liver cancer and clear cell renal cell carcinoma (ccRCC) have not yet been fully elucidated. The aim of this study was to explore the role and clinical significance of GLYAT in liver cancer and ccRCC. This study used proteomics technology to identify differentially expressed proteins in liver cancer. Western blot and immunohistochemistry (IHC) were used to analyze the protein expression pattern of GLYAT. assays were performed in liver cancer and ccRCC cells. Xenograft models in nude mice were used to confirm the roles of GLYAT in liver cancer. Moreover, the downstream regulatory proteins of GLYAT were identified by proteomics. GLYAT was lowly expressed in liver cancer and ccRCC. Immunofluorescence staining indicated that GLYAT was mainly expressed in the cytoplasm, particularly the mitochondria. Kaplan-Meier curves showed that the low protein expression of GLYAT was correlated with a poor prognosis in liver cancer and ccRCC patients. Moreover, GLYAT expression was associated with several clinical parameters in liver cancer. Cell experiments showed that the overexpression of GLYAT inhibited cell proliferation and migration abilities; however, interfering GLYAT protein expression rescued these abilities in GLYAT overexpression (GLYAT-OE) cells. In vivo assays confirmed the tumor-suppressor function of GLYAT in liver cancer. Moreover, our research showed that GLYAT downregulated Rho-associated coiled-coil-containing protein kinase 1 (ROCK1). Our study showed that GLYAT is lowly expressed in liver cancer and ccRCC, emphasizing its prognostic significance. It also showed that GLYAT inhibits the progression of liver cancer and ccRCC by downregulating ROCK1.

Circulating mRNA Expression of VEGF, PTEN, and SOCS1 as Potential Prognostic Predictor for Nasopharyngeal Carcinoma Progression.

The molecular mechanisms underlying nasopharyngeal carcinoma (NPC) progression remain poorly understood. In particular, the roles of circulating mRNAs encoding key regulatory proteins have yet to be explored. This study aimed to identify NPC-associated expression signatures of circulating VEGF, PTEN, and SOCS1 mRNAs and their potential as biomarkers. A case-control study was conducted comprising 160 nasopharyngeal carcinoma (NPC) patients and 80 controls, from whom peripheral blood samples. Total RNA was extracted and the levels of VEGF, PTEN, and SOCS1 mRNAs were quantified using reverse transcription quantitative PCR (RT-qPCR). Relative expression was calculated using the 2-ΔΔCt method. Bioinformatic analyses, including GeneMANIA, Gene Ontology (GO), and KEGG pathway analysis, were performed to predict the functional roles and interactions of these mRNAs. We identified significantly increased circulating VEGF mRNA in lymph node metastases (1.66-fold, p<0.05) and elevated SOCS1 mRNA in late-stage NPC (20-fold, p<0.05). PTEN mRNA was reduced 4.26-fold in NPC patients. These data suggest that circulating VEGF, PTEN, and SOCS1 mRNAs represent signatures of NPC progression and can potentially be biomarkers. Network analyses implicate these mRNAs in mechanisms enabling NPC pathogenesis. Our study reveals NPC-associated expression changes of circulating VEGF, PTEN, and SOCS1 mRNAs. These molecular signatures may serve as biomarkers during NPC progression and provide insights into underlying mechanisms. Further validation of their utility as prognostic indicators of NPC is warranted.

Establishment of Star-edited Y1 cells as a novel in vitro functional assay for STAR.

Genetic variants involving steroidogenic acute regulatory protein cause lipoid congenital adrenal hyperplasia, which is characterized by impaired steroidogenesis in the adrenal glands and gonads. Functional assessment of variant STAR proteins is necessary for accurate genetic diagnosis. Ideally, steroidogenic cells should be used to assess the functionality of STAR proteins, but the presence of endogenous STARs in steroidogenic cells precludes such a method. Here, we generated Star-edited cells from steroidogenic Y1 mouse adrenocortical tumor cells by genome editing. Star-edited Y1 cells exhibited very low but measurable cAMP-dependent pregnenolone production. Furthermore, stimulation of the cAMP pathway for two weeks resulted in the formation of lipid droplets in the cytoplasm of Star-edited Y1 cells, which resembled the histology of the adrenal glands of patients with lipoid congenital adrenal hyperplasia. The steroidogenic defect of Star-edited Y1 cells can be restored by transient over-expression of mouse Star. We found that human STAR can also restore defective steroidogenesis of Star-edited Y1 cells, and were able to construct a novel in vitro system to evaluate human STAR variants. Collectively, we established Star-edited Y1 cells that retain the steroidogenic pathway downstream the Star protein. Star-edited Y1 cells recapitulate the functional and morphological changes of lipoid congenital adrenal hyperplasia, and can be used to evaluate the functionality of human STAR variants.