Characterization of N-glycosylation and its functional role in SIDT1-Mediated RNA uptake.

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications.

Cell surface RNAs control neutrophil recruitment.

RNAs localizing to the outer cell surface have been recently identified in mammalian cells, including RNAs with glycan modifications known as glycoRNAs. However, the functional significance of cell surface RNAs and their production are poorly known. We report that cell surface RNAs are critical for neutrophil recruitment and that the mammalian homologs of the sid-1 RNA transporter are required for glycoRNA expression. Cell surface RNAs can be readily detected in murine neutrophils, the elimination of which substantially impairs neutrophil recruitment to inflammatory sites in vivo and reduces neutrophils' adhesion to and migration through endothelial cells. Neutrophil glycoRNAs are predominantly on cell surface, important for neutrophil-endothelial interactions, and can be recognized by P-selectin (Selp). Knockdown of the murine Sidt genes abolishes neutrophil glycoRNAs and functionally mimics the loss of cell surface RNAs. Our data demonstrate the biological importance of cell surface glycoRNAs and highlight a noncanonical dimension of RNA-mediated cellular functions.

Structural insight into the human SID1 transmembrane family member 2 reveals its lipid hydrolytic activity.

The systemic RNAi-defective (SID) transmembrane family member 2 (SIDT2) is a putative nucleic acid channel or transporter that plays essential roles in nucleic acid transport and lipid metabolism. Here, we report the cryo-electron microscopy (EM) structures of human SIDT2, which forms a tightly packed dimer with extensive interactions mediated by two previously uncharacterized extracellular/luminal ß-strand-rich domains and the unique transmembrane domain (TMD). The TMD of each SIDT2 protomer contains eleven transmembrane helices (TMs), and no discernible nucleic acid conduction pathway has been identified within the TMD, suggesting that it may act as a transporter. Intriguingly, TM3-6 and TM9-11 form a large cavity with a putative catalytic zinc atom coordinated by three conserved histidine residues and one aspartate residue lying approximately 6 Å from the extracellular/luminal surface of the membrane. Notably, SIDT2 can hydrolyze C18 ceramide into sphingosine and fatty acid with a slow rate. The information presented advances the understanding of the structure-function relationships in the SID1 family proteins.

SLC35E1 promotes keratinocyte proliferation in psoriasis by regulating zinc homeostasis.

Keratinocyte hyperproliferation is a key pathogenic factor in psoriasis. However, the mechanisms that regulate keratinocyte hyperproliferation in this condition remain unclear. Here, we found that SLC35E1 was highly expressed in keratinocytes of patients with psoriasis and that Slc35e1-/- mice displayed a less severe imiquimod (IMQ)-induced psoriasis-like phenotype than their wild-type siblings. In addition, SLC35E1 deficiency inhibited keratinocyte proliferation in both mice and cultured cells. On a molecular level, SLC35E1 was found to regulate zinc ion concentrations and subcellular localization, while zinc ion chelation reversed the IMQ-induced psoriatic phenotype in Slc35e1-/- mice. Meanwhile, epidermal zinc ion levels were decreased in patients with psoriasis and zinc ion supplementation alleviated the psoriatic phenotype in an IMQ-induced mouse model of psoriasis. Our results indicated that SLC35E1 can promote keratinocyte proliferation by regulating zinc ion homeostasis and zinc ion supplementation has potential as a therapy for psoriasis.

[Lysosomal membrane protein Sidt2 knockout induces apoptosis of human hepatocytes in vitro independent of the autophagy-lysosomal pathway].

OBJECTIVE: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout. METHODS: The Sidt2 knockout (Sidt2-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed. RESULTS: Sidt2-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 µmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2-/- HL7702 cells. CONCLUSION: Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.

Inherited Thrombocytopenia Caused by Variants in Crucial Genes for Glycosylation.

Protein glycosylation, including sialylation, involves complex and frequent post-translational modifications, which play a critical role in different biological processes. The conjugation of carbohydrate residues to specific molecules and receptors is critical for normal hematopoiesis, as it favors the proliferation and clearance of hematopoietic precursors. Through this mechanism, the circulating platelet count is controlled by the appropriate platelet production by megakaryocytes, and the kinetics of platelet clearance. Platelets have a half-life in blood ranging from 8 to 11 days, after which they lose the final sialic acid and are recognized by receptors in the liver and eliminated from the bloodstream. This favors the transduction of thrombopoietin, which induces megakaryopoiesis to produce new platelets. More than two hundred enzymes are responsible for proper glycosylation and sialylation. In recent years, novel disorders of glycosylation caused by molecular variants in multiple genes have been described. The phenotype of the patients with genetic alterations in GNE, SLC35A1, GALE and B4GALT is consistent with syndromic manifestations, severe inherited thrombocytopenia, and hemorrhagic complications.

Single Particle Analysis of H3N2 Influenza Entry Differentiates the Impact of the Sialic Acids (Neu5Ac and Neu5Gc) on Virus Binding and Membrane Fusion.

Entry of influenza A viruses (IAVs) into host cells is initiated by binding to sialic acids (Sias), their primary host cell receptor, followed by endocytosis and membrane fusion to release the viral genome into the cytoplasm of the host cell. Host tropism is affected by these entry processes, with a primary factor being receptor specificity. Sias exist in several different chemical forms, including the hydroxylated N-glycolylneuraminic acid (Neu5Gc), which is found in many hosts; however, it has not been clear how modified Sias affect viral binding and entry. Neu5Gc is commonly found in many natural influenza hosts, including pigs and horses, but not in humans or ferrets. Here, we engineered HEK293 cells to express the hydoxylase gene (CMAH) that converts Neu5Ac to Neu5Gc, or knocked out the Sia-CMP transport gene (SLC35A1), resulting in cells that express 95% Neu5Gc or minimal level of Sias, respectively. H3N2 (X-31) showed significantly reduced infectivity in Neu5Gc-rich cells compared to wild-type HEK293 (>95% Neu5Ac). To determine the effects on binding and fusion, we generated supported lipid bilayers (SLBs) derived from the plasma membranes of these cells and carried out single particle microscopy. H3N2 (X-31) exhibited decreased binding to Neu5Gc-containing SLBs, but no significant difference in H3N2 (X-31)'s fusion kinetics to either SLB type, suggesting that reduced receptor binding does not affect subsequent membrane fusion. This finding suggests that for this virus to adapt to host cells rich in Neu5Gc, only receptor affinity changes are required without further adaptation of virus fusion machinery. IMPORTANCE Influenza A virus (IAV) infections continue to threaten human health, causing over 300,000 deaths yearly. IAV infection is initiated by the binding of influenza glycoprotein hemagglutinin (HA) to host cell sialic acids (Sias) and the subsequent viral-host membrane fusion. Generally, human IAVs preferentially bind to the Sia N-acetylneuraminic acid (Neu5Ac). Yet, other mammalian hosts, including pigs, express diverse nonhuman Sias, including N-glycolylneuraminic acid (Neu5Gc). The role of Neu5Gc in human IAV infections in those hosts is not well-understood, and the variant form may play a role in incidents of cross-species transmission and emergence of new epidemic variants. Therefore, it is important to investigate how human IAVs interact with Neu5Ac and Neu5Gc. Here, we use membrane platforms that mimic the host cell surface to examine receptor binding and membrane fusion events of human IAV H3N2. Our findings improve the understanding of viral entry mechanisms that can affect host tropism and virus evolution.

Effects of SIDT2 on the miR-25/NOX4/HuR axis and SIRT3 mRNA stability lead to ROS-mediated TNF-α expression in hydroquinone-treated leukemia cells.

Our previous studies indicated that the benzene metabolite hydroquinone (HQ) evokes the ROS/p38 MAPK/protein phosphatase 2A/tristetraprolin axis, leading to increased TNF-α expression in human acute myeloid leukemia cell lines U937 and HL-60. In this study, we aimed to identify the upstream pathway involved in ROS-mediated TNF-α expression. HQ treatment increased SIDT2 expression, which subsequently decreased miR-25 and SIRT3 expression in U937 cells. Notably, miR-25 downregulation promoted SIDT2 expression in HQ-treated U937 cells. SIDT2 induced lysosomal degradation of SIRT3 mRNA, but inhibited miR-25 expression through a lysosome-independent pathway. MiR-25 inhibition reduced NOX4 mRNA turnover, resulting in increased NOX4 protein levels. NOX4 induces mitochondrial ROS production and HuR downregulation. Restoration of HuR expression increased SIRT3 expression, suggesting that NOX4-mediated HuR downregulation promotes SIDT2-mediated degradation of SIRT3 mRNA. Inhibition of NOX4 or SIRT3 overexpression abolished HQ-induced ROS production, thereby abolishing TNF-α upregulation. Overall, these results indicate that SIDT2 regulates the miR-25/NOX4/HuR axis and SIRT3 mRNA destabilization, leading to ROS-mediated TNF-α upregulation in HQ-treated U937 cells. HQ-induced increase in TNF-α expression in HL-60 cells was also mediated through a similar pathway.

NLRP3 Inflammasome Activation Through Heart-Brain Interaction Initiates Cardiac Inflammation and Hypertrophy During Pressure Overload.

BACKGROUND: Mechanical stress on the heart, such as high blood pressure, initiates inflammation and causes hypertrophic heart disease. However, the regulatory mechanism of inflammation and its role in the stressed heart remain unclear. IL-1ß (interleukin-1ß) is a proinflammatory cytokine that causes cardiac hypertrophy and heart failure. Here, we show that neural signals activate the NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3) inflammasome for IL-1ß production to induce adaptive hypertrophy in the stressed heart. METHODS: C57BL/6 mice, knockout mouse strains for NLRP3 and P2RX7 (P2X purinoceptor 7), and adrenergic neuron-specific knockout mice for SLC17A9, a secretory vesicle protein responsible for the storage and release of ATP, were used for analysis. Pressure overload was induced by transverse aortic constriction. Various animal models were used, including pharmacological treatment with apyrase, lipopolysaccharide, 2'(3')-O-(4-benzoylbenzoyl)-ATP, MCC950, anti-IL-1ß antibodies, clonidine, pseudoephedrine, isoproterenol, and bisoprolol, left stellate ganglionectomy, and ablation of cardiac afferent nerves with capsaicin. Cardiac function and morphology, gene expression, myocardial IL-1ß and caspase-1 activity, and extracellular ATP level were assessed. In vitro experiments were performed using primary cardiomyocytes and fibroblasts from rat neonates and human microvascular endothelial cell line. Cell surface area and proliferation were assessed. RESULTS: Genetic disruption of NLRP3 resulted in significant loss of IL-1ß production, cardiac hypertrophy, and contractile function during pressure overload. A bone marrow transplantation experiment revealed an essential role of NLRP3 in cardiac nonimmune cells in myocardial IL-1ß production and cardiac phenotype. Pharmacological depletion of extracellular ATP or genetic disruption of the P2X7 receptor suppressed myocardial NLRP3 inflammasome activity during pressure overload, indicating an important role of ATP/P2X7 axis in cardiac inflammation and hypertrophy. Extracellular ATP induced hypertrophic changes of cardiac cells in an NLRP3- and IL-1ß-dependent manner in vitro. Manipulation of the sympathetic nervous system suggested sympathetic efferent nerves as the main source of extracellular ATP. Depletion of ATP release from sympathetic efferent nerves, ablation of cardiac afferent nerves, or a lipophilic ß-blocker reduced cardiac extracellular ATP level, and inhibited NLRP3 inflammasome activation, IL-1ß production, and adaptive cardiac hypertrophy during pressure overload. CONCLUSIONS: Cardiac inflammation and hypertrophy are regulated by heart-brain interaction. Controlling neural signals might be important for the treatment of hypertensive heart disease.

SIDT2 Inhibits Phosphorothioate Antisense Oligonucleotide Activity by Regulating Cellular Localization of Lysosomes.

Phosphorothioate (PS)-modified antisense oligonucleotide (ASO) drugs enter cells through endocytic pathways where a majority are entrapped within membrane-bound endosomes and lysosomes, representing a limiting step for antisense activity. While late endosomes have been identified as a major site for productive PS-ASO release, how lysosomes regulate PS-ASO activity beyond macromolecule degradation remains not fully understood. In this study, we reported that SID1 transmembrane family, member 2 (SIDT2), a lysosome transmembrane protein, can robustly regulate PS-ASO activity. We showed that SIDT2 is required for the proper colocalization between PS-ASO and lysosomes, suggesting an important role of SIDT2 in the entrapment of PS-ASOs in lysosomes. Mechanistically, we revealed that SIDT2 regulates lysosome cellular location. Lysosome location is largely determined by its movement along microtubules. Interestingly, we also observed an enrichment of proteins involved in microtubule function among SIDT2-binding proteins, suggesting that SIDT2 regulates lysosome location via its interaction with microtubule-related proteins. Overall, our data suggest that lysosome protein SIDT2 inhibits PS-ASO activity potentially through its interaction with microtubule-related proteins to place lysosomes at perinuclear regions, thus, facilitating PS-ASO's localization to lysosomes for degradation.

Nucleotide sugar transporter SLC35A2 is involved in promoting hepatocellular carcinoma metastasis by regulating cellular glycosylation.

PURPOSE: Recently, aberrant glycosylation has been recognized to be relate to malignant behaviors of cancer and outcomes of patients with various cancers. SLC35A2 plays an indispensable role on glycosylation as a nucleotide sugar transporter. However, effects of SLC35A2 on malignant behaviors of cancer cells and alteration of cancer cells surface glycosylation profiles are still not fully understood, particularly in hepatocellular carcinoma (HCC). Hence, from a glycosylation perspective, we investigated the effects of SLC35A2 on metastatic behaviors of HCC cells. METHODS: SLC35A2 expression in clinical samples and HCC cells was examined by immunohistochemical staining or Western blot/quantitative PCR and was regulated by RNA interference or vectors-mediated transfection. Effects of SLC35A2 expression alteration on metastatic behaviors and membrane glycan profile of HCC cells were observed by using respectively invasion, migration, cell adhesion assay, in vivo lung metastatic nude mouse model and lectins microarray. Co-location among proteins in HCC cells was observed by fluorescence microscope and detected by an in vitro co-immunoprecipitation assay. RESULTS: SLC35A2 was upregulated in HCC tissues, and is associated with poor prognosis of HCC patients. SLC35A2 expression alteration significantly affected the invasion, adhesion, metastasis and membrane glycan profile and led to the dysregulated expressions or glycosylation of cell adhesion-related molecules in HCC cells. Mechanistically, the maintenance of SLC35A2 activity is critical for the recruitment of the key galactosyltransferase B4GalT1, which is responsible for complex glycoconjugate and lactose biosynthesis, to Golgi apparatus in HCC cells. CONCLUSION: SLC35A2 plays important roles in promoting HCC metastasis by regulating cellular glycosylation modification and inducing the cell adhesive ability of HCC cells.

Lysosomal membrane protein Sidt2 knockout induces apoptosis of human hepatocytes in vitro independent of the autophagy-lysosomal pathway / 南方医科大学学报

OBJECTIVE@#To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout.@*METHODS@#The Sidt2 knockout (Sidt2-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed.@*RESULTS@#Sidt2-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2-/- HL7702 cells.@*CONCLUSION@#Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.

Genome-Wide CRISPR/Cas9 Screen Reveals a Role for SLC35A1 in the Adsorption of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, not only causes diarrhea in piglets but also possesses the potential to infect humans. To better understand host-virus genetic dependencies and find potential therapeutic targets for PDCoV, we used a porcine single-guide RNA (sgRNA) lentivirus library to screen host factors related to PDCoV infection in LLC-PK1 cells. The solute carrier family 35 member A1 (SLC35A1), a key molecule in the sialic acid (SA) synthesis pathway, was identified as a host factor required for PDCoV infection. A knockout of SLC35A1 caused decreases in the amounts of cell surface sialic acid (SA) and viral adsorption; meanwhile, trypsin promoted the use of SA in PDCoV infection. By constructing and assessing a series of recombinant PDCoV strains with the deletion or mutation of possible critical domain or amino acid residues for SA binding in the S1 N-terminal domain, we found that S T182 might be a PDCoV SA-binding site. However, the double knockout of SLC35A1 and amino peptidase N (APN) could not block PDCoV infection completely. Additionally, we found that different swine enteric coronaviruses, including transmissible gastroenteritis coronavirus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome coronavirus, are differentially dependent on SA. Overall, our study uncovered a collection of host factors that can be exploited as drug targets against PDCoV infection and deepened our understanding of the relationship between PDCoV and SA. IMPORTANCE Identifying the host factors required for replication will be helpful to uncover the pathogenesis mechanisms and develop antivirals against the emerging coronavirus porcine deltacoronavirus (PDCoV). Herein, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout screen, the results of which revealed that the solute carrier family 35 member A1 (SLC35A1) is a host factor required for PDCoV infection that acts by regulating cell surface sialic acid (SA). We also identified the T182 site in the N-terminal domain of PDCoV S1 subunit as being associated with the SA-binding site and found that trypsin promotes the use of cell surface SA by PDCoV. Furthermore, different swine enteric coronaviruses use SLC35A1 differently for infection. This is the first study to screen host factors required for PDCoV replication using a genome-wide CRISPR-Cas9 functional knockout, thereby providing clues for developing antiviral drugs against PDCoV infection.

Vesicular nucleotide transporter is a molecular target of eicosapentaenoic acid for neuropathic and inflammatory pain treatment.

Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC50) of 67 nM, acting as an allosteric modulator through competition with Cl-. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT-/- mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT-/- mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT-/- mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.

Single-nucleotide polymorphisms of the SLC17A9 and P2RY12 genes are significantly associated with phantom tooth pain.

Phantom tooth pain (PTP) is a rare and specific neuropathic pain that occurs after pulpectomy and tooth extraction, but its cause is not understood. We hypothesized that there is a genetic contribution to PTP. We focused on solute carrier family 17 member 9 (SLC17A9)/vesicular nucleotide transporter (VNUT) and purinergic receptor P2Y12 (P2RY12), both of which have been associated with neuropathic pain and pain transduction signaling in the trigeminal ganglion in rodents. We sought to corroborate these associations in humans. We investigated gene polymorphisms that contribute to PTP. We statistically examined the association between genetic polymorphisms and PTP vulnerability in 150 patients with orofacial pain, including PTP, and 500 healthy subjects. We found that the rs735055 polymorphism of the SLC17A9 gene and rs3732759 polymorphism of the P2RY12 gene were associated with the development of PTP. Carriers of the minor allele of rs735055 and individuals who were homozygous for the major allele of rs3732759 had a higher rate of PTP. Carriers of the minor allele of rs735055 reportedly had high SLC17A9 mRNA expression in the spinal cord, which may increase the storage and release of adenosine triphosphate. Individuals who were homozygous for the major allele of rs3732759 may have higher P2RY12 expression that is more active in microglia. Therefore, these carriers may be more susceptible to PTP. These results suggest that specific genetic polymorphisms of the SLC17A9 and P2RY12 genes are involved in PTP. This is the first report on genes that are associated with PTP in humans.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening.

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

Pathology-associated change in levels and localization of SIDT2 in postmortem brains of Parkinson's disease and dementia with Lewy bodies patients.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are major neurodegenerative disorders that share commonalities in their pathology involving the formation of Lewy bodies, the main component of which is α-synuclein protein. Aberrancy and dysfunction in lysosomes have been suggested to play critical roles in the pathogenesis of Lewy body diseases. We recently identified a novel lysosomal degradation pathway in which various macromolecules, including α-synuclein protein, are directly imported into lysosomes and degraded. In this study, we analyzed the levels and localization of the lysosomal membrane protein SIDT2, a key factor in this pathway, in the postmortem brains of patients with PD and DLB. The levels of SIDT2 protein were significantly higher in the anterior cingulate cortex (ACC) of both PD and DLB cases than in age-matched control subjects, but this difference was not observed in the inferior frontal gyrus. The levels of SIDT2 also showed a strong correlation with α-synuclein levels in the ACC of all subjects, including controls. SIDT2 was colocalized with aggregates positive for phosphorylated α-synuclein protein, which is a hallmark of Lewy bodies, in all examined cases of both PD and DLB. These observations suggest that changes in the levels and localization of SIDT2 occur at the lesion site of Lewy body diseases in accordance with the progression of Lewy body pathology. Our findings provide mechanistic insights into the pathogenesis of Lewy body diseases, as well as other neurodegenerative disorders, and may provide clues for improved diagnosis, prevention, and therapeutic intervention for such diseases.

Docetaxel-triggered SIDT2/NOX4/JNK/HuR signaling axis is associated with TNF-α-mediated apoptosis of cancer cells.

Previous studies have confirmed that docetaxel (DTX) treatment increases TNF-α production in cancer cells, but its mechanism of action remains unclear. Therefore, this study aimed to determine the signaling axis by which DTX induced the expression of TNF-α in U937 leukemia and MCF-7 breast carcinoma cells. DTX treatment promoted Ca2+-controlled autophagy and SIDT2 expression, resulting in lysosomal degradation of miR-25 in U937 cells. Downregulation of miR-25 increased NOX4 mRNA stability and protein expression. NOX4-stimulated ROS generation led to JNK-mediated phosphorylation of cytosolic HuR at Ser221, thereby increasing TNF-α protein expression by stabilizing TNF-α mRNA. Consequently, DTX induced TNF-α-dependent death in U937 cells. Depletion of HuR using siRNA or abolishment of JNK activation reduced TNF-α expression and eliminated DTX-mediated cytotoxicity. Knockdown of SIDT2 or pretreatment with chloroquine (a lysosome inhibitor) reduced DTX-induced NOX4 and TNF-α expression and mitigated JNK-mediated HuR phosphorylation. Altogether, our data indicate that DTX triggers HuR-mediated TNF-α mRNA stabilization through the Ca2+/SIDT2/NOX4/ROS/JNK axis, thereby inducing TNF-α-dependent apoptosis in U937 cells. In addition, DTX induces apoptosis in MCF-7 cells through SIDT2/NOX4/JNK/HuR axis-mediated TNF-α expression.

Sidt2 is a key protein in the autophagy-lysosomal degradation pathway and is essential for the maintenance of kidney structure and filtration function.

The regulation and homeostasis of autophagy are essential for maintaining organ morphology and function. As a lysosomal membrane protein, the effect of Sidt2 on kidney structure and renal autophagy is still unknown. In this study, we found that the kidneys of Sidt2-/- mice showed changes in basement membrane thickening, foot process fusion, and mitochondrial swelling, suggesting that the structure of the kidney was damaged. Increased urine protein at 24 h indicated that the kidney function was also damaged. At the same time, the absence of Sidt2 caused a decrease in the number of acidic lysosomes, a decrease in acid hydrolase activity and expression in the lysosome, and an increase of pH in the lysosome, suggesting that lysosomal function was impaired after Sidt2 deletion. The accumulation of autophagolysosomes, increased LC3-II and P62 protein levels, and decreased P62 mRNA levels indicated that the absence of the Sidt2 gene caused abnormal autophagy pathway flow. Chloroquine experiment, immunofluorescence autophagosome, and lysosome fusion assay, and Ad-mcherry-GFP-LC3B further indicated that, after Sidt2 deletion, the production of autophagosomes did not increase, but the fusion of autophagosomes and lysosomes and the degradation of autophagolysosomes were impaired. When incubating Sidt2-/- cells with the autophagy activator rapamycin, we found that it could activate autophagy, which manifested as an increase in autophagosomes, but it could not improve autophagolysosome degradation. Meanwhile, it further illustrated that the Sidt2 gene plays an important role in the smooth progress of autophagolysosome processes. In summary, the absence of the Sidt2 gene caused impaired lysosome function and a decreased number of acidic lysosomes, leading to formation and degradation disorders of the autophagolysosomes, which eventually manifested as abnormal kidney structure and function. Sidt2 is essential in maintaining the normal function of the lysosomes and the physiological stability of the kidneys.

Long non-coding RNA LIFR-AS1 suppressed the proliferation, angiogenesis, migration and invasion of papillary thyroid cancer cells via the miR-31-5p/SIDT2 axis.

Long non-coding RNA LIFR-AS1 is low-expressed in many cancers, but its functions in papillary thyroid carcinoma (PTC) were not defined and require further study. The relationship between LIFR-AS1 expression and clinicopathological characteristics of patients with PTC was statistically analyzed. The downregulation of LIFR-AS1 in PTC tissues and cell lines was predicted by bioinformatics analysis and verified by qRT-PCR. After overexpressing or silencing LIFR-AS1, the regulatory role of LIFR-AS1 in PTC was examined by performing MTT, colony formation, wound healing, Transwell, ELISA, tube formation and xenograft tumor experiment. MiR-31-5p and SID1 transmembrane family member 2 (SIDT2) expressions in PTC tissues or cell lines were detected by qRT-PCR, Western blot, or in situ hybridization. The relationship between miR-31-5p and LIFR-AS1/SIDT2 was predicted by LncBase, TargetScan or Pearson correlation test and then verified by Dual-Luciferase Reporter assay, RNA pull-down assay and qRT-PCR. The regulatory effect of LIFR-AS1/miR-31-5p/SIDT2 axis on the biological behaviors of PTC cells was confirmed by functional experiments and rescue experiments mentioned above. The tumor size and lymphatic metastasis were correlated with LIFR-AS1 overexpression. Overexpressed LIFR-AS1 suppressed tumorigenesis in vivo. LIFR-AS1 and SIDT2 expressions were suppressed in PTC tissues, while that of miR-31-5p was elevated in PTC tissues. LIFR-AS1 was negatively correlated with miR-31-5p. LIFR-AS1 sponged miR-31-5p to upregulate SIDT2, thereby inhibiting the viability, proliferation, migration, invasion, and the secretion of vascular endothelial growth factor (VEGF) of PTC cells and angiogenesis of human umbilical vein endothelial cells (HUVECs). This paper demonstrates that LIFR-AS1/miR-31-5p/SIDT2 axis modulated the development of PTC.