Related Protein Complex Research Articles

AP3B1 facilitates PDIA3/ERP57 function to regulate rabies virus glycoprotein selective degradation and viral entry

ABSTRACT Rabies virus causes an estimated 59,000 annual fatalities worldwide and promising therapeutic treatments are necessary to develop. In this study, affinity tag-purification mass spectrometry was employed to delineate RABV glycoprotein and host protein interactions, and PDIA3/ERP57 was identified as a potential inhibitor of RABV infection. PDIA3 restricted RABV infection with follow mechanisms: PDIA3 mediated the degradation of RABV G protein by targeting lysine 332 via the selective macroautophagy/autophagy pathway; The PDIA3 interactor, AP3B1 (adaptor related protein complex 3 subunit beta 1) was indispensable in PDIA3-triggered selective degradation of the G protein; Furthermore, PDIA3 competitively bound with NCAM1/NCAM (neural cell adhesion molecule 1) to block RABV G, hindering viral entry into host cells. PDIA3 190-199 aa residues bound to the RABV G protein were necessary and sufficient to defend against RABV. These results demonstrated the therapeutic potential of biologics that target PDIA3 or utilize PDIA3 190-199 aa peptide to treat clinical rabies. Abbreviation: aa: amino acids; ANXA2: annexin A2; AP-MS: affinity tag purification-mass spectrometry; AP3B1: adaptor related protein complex 3 subunit beta 1; ATP6V1A: ATPase H+ transporting V1 subunit A; ATP6V1H: ATPase H+ transporting V1 subunit H; BafA1: bafilomycin A1; CHX: cycloheximide; co-IP: co-immunoprecipitation; DDX17: DEAD-box helicase 17; DmERp60: drosophila melanogaster endoplasmic reticulum p60; EBOV: Zaire ebolavirus virus; EV: empty vector; GANAB: glucosidase II alpha subunit; G protein: glycoprotein; GRM2/mGluR2: glutamate metabotropic receptor 2; HsPDIA3: homo sapiens protein disulfide isomerase family A member 3; IAV: influenza virus; ILF2: interleukin enhancer binding factor 2; KO: knockout; MAGT1: magnesium transporter 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MmPDIA3: mus musculus protein disulfide isomerase associated 3; NCAM1/NCAM: neural cell adhesion molecule 1; NGFR/p75NTR: nerve growth factor receptor; NGLY1: N-glycanase 1; OTUD4: OTU deubiquitinase 4; PDI: protein disulfide isomerase; PPIs: protein-protein interactions; RABV: rabies virus; RUVBL2: RuvB like AAA ATPase 2; SCAMP3: secretory carrier membrane protein 3; ScPdi1: Saccharomyces cerevisiae s288c protein disulfide isomerase 1; SLC25A6: solute carrier family 25 member 6; SQSTM1/p62: sequestosome 1; VSV: vesicular stomatitis virus.

A clathrin mediated endocytosis scaffolding protein, Intersectin 1, changes in an isoform, brain region, and sex specific manner in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by the accumulation of amyloid-beta (Aβ) plaques and neurofibrillary Tau tangles in the brain. We previously identified a set of candidate AD microRNAs (miRNAs) in human cerebrospinal fluid (CSF) and used a target prediction pipeline to identify mRNAs and pathways that could potentially be regulated by the miRNAs. Of these pathways, clathrin mediated endocytosis (CME) was selected for further investigation. CME is altered in multiple brain cell types in AD and is implicated in early cellular phenotypes such as enlarged early endosomes and pathogenic processing of Aβ. However, a comprehensive evaluation of major CME hub proteins in humans with AD across multiple brain regions is lacking. Thus, we used immunoblots to evaluate human post-mortem AD and control (CTL) frontal cortex (FC; AD n = 22, CTL n = 23) and hippocampus (HP; AD n = 34, CTL n = 22) for changes in Intersectin 1 (ITSN1), Phosphatidylinositol Binding Clathrin Assembly Protein gene (PICALM), Clathrin Light Chain (CLT), FCH and Mu Domain Containing Endocytic Adaptor 1 (FCHO1), Adaptor Related Protein Complex 2 (AP2) Subunit Alpha 1 (AP2A1), and Dynamin 2 (DNM2). Of these, we found that in AD, ITSN1-long (ITSN1-L) was decreased in the FC of males and HP of females, while ITSN1-short was increased in the HP of both males and females. We further evaluated ITSN1-L levels in cortex (CTX) and HP of the 5xFAD mouse model of Aβ pathology at different timepoints during aging and disease progression by immunoblot (n = 5-8 per group). At 3 months, female 5xFAD exhibited an increase of ITSN1-L in CTX but a decrease at 6 and 9 months. Additionally, immunofluorescent staining of 5xFAD primary HP neurons showed an increase of ITSN1-L in matured 5xFAD neurons at 21 and 28 days in vitro. Together, our studies show that in AD, isoforms of ITSN1 change in a brain region-and sex-dependent manner. Further, changes in ITSN1-L are transient with levels increasing during early Aβ accumulation and decreasing during later progression. These findings suggest that ITSN1 expression, and consequently CME activity, may change depending on the stage of disease progression.

Neither alpha-synuclein fibril strain nor host murine genotype influences seeding efficacy

Parkinson’s disease (PD) is a neurodegenerative disease characterized by progressive motor symptoms and alpha-synuclein (αsyn) aggregation in the nervous system. For unclear reasons, PD patients with certain GBA1 mutations (GBA-PD) have a more aggressive clinical progression. Two testable hypotheses that can potentially account for this phenomenon are that GBA1 mutations promote αsyn spread or drive the generation of highly pathogenic αsyn polymorphs (i.e., strains). We tested these hypotheses by treating homozygous GBA1 D409V knockin (KI) mice with human α-syn-preformed fibrils (PFFs) and treating wild-type mice (WT) with several αsyn-PFF polymorphs amplified from brain autopsy samples collected from patients with idiopathic PD and GBA-PD patients with either homozygous or heterozygous GBA1 mutations. Robust phosphorylated-αsyn (PSER129) positive pathology was observed at the injection site (i.e., the olfactory bulb granule cell layer) and throughout the brain six months following PFF injection. The PFF seeding efficiency and degree of spread were similar regardless of the mouse genotype or PFF polymorphs. We found that PFFs amplified from the human brain, regardless of patient genotype, were generally more effective seeders than wholly synthetic PFFs (i.e., non-amplified); however, PFF concentration differed between these two studies, which might also account for the observed differences. To investigate whether the molecular composition of pathology differed between different seeding conditions, we performed Biotinylation by Antibody Recognition on PSER129 (BAR-PSER129). We found that for BAR-PSER129, the endogenous PSER129 pool dominated identified interactions, and thus, very few potential interactions were explicitly identified for seeded pathology. However, we found Dynactin Subunit 2 (Dctn2) interaction was shared across all PFF conditions, and NCK Associated Protein 1 (Nckap1) and Adaptor Related Protein Complex 3 Subunit Beta 2 (Ap3b2) were unique to PFFs amplified from GBA-PD brains of heterozygous mutation carriers. In conclusion, both the genotype and αsyn strain had little effect on overall seeding efficacy and global PSER129-interactions.

Host proteins interact with viral elements and affect the life cycle of highly pathogenic avian influenza A virus H7N9

Host-virus interactions can significantly impact the viral life cycle and pathogenesis; however, our understanding of the specific host factors involved in highly pathogenic avian influenza A virus H7N9 (HPAI H7N9) infection is currently restricted. Herein, we designed and synthesized 65 small interfering RNAs targeting host genes potentially associated with various aspects of RNA virus life cycles. Afterward, HPAI H7N9 viruses were isolated and RNA interference was used to screen for host factors likely to be involved in the life cycle of HPAI H7N9. Moreover, the research entailed assessing the associations between host proteins and HPAI H7N9 proteins. Twelve key host proteins were identified: Annexin A (ANXA)2, ANXA5, adaptor related protein complex 2 subunit sigma 1 (AP2S1), adaptor related protein complex 3 subunit sigma 1 (AP3S1), ATP synthase F1 subunit alpha (ATP5A1), COPI coat complex subunit alpha (COP)A, COPG1, heat shock protein family A (Hsp70) member 1A (HSPA)1A, HSPA8, heat shock protein 90 alpha family class A member 1 (HSP90AA1), RAB11B, and RAB18. Co-immunoprecipitation revealed intricate interactions between viral proteins (hemagglutinin, matrix 1 protein, neuraminidase, nucleoprotein, polymerase basic 1, and polymerase basic 2) and these host proteins, presumably playing a crucial role in modulating the life cycle of HPAI H7N9. Notably, ANXA5, AP2S1, AP3S1, ATP5A1, HSP90A1, and RAB18, were identified as novel interactors with HPAI H7N9 proteins rather than other influenza A viruses (IAVs). These findings underscore the significance of host-viral protein interactions in shaping the dynamics of HPAI H7N9 infection, while highlighting subtle variations compared with other IAVs. Deeper understanding of these interactions holds promise to advance disease treatment and prevention strategies.

Prognostic and immunological role of adaptor related protein complex 3 subunit mu2 in colon cancer

The expression levels and prognostic role of AP3M2 in colorectal adenocarcinoma (CRAC) have yet to be fully unveiled. Our study comprehensively investigated the clinical significance of AP3M2 in colorectal cancer through an extensive bioinformatics data mining process (TCGA, GEO, GEPIA, Timer, Ualcan, ROCPLOT, and David), followed by experimental validation. We found AP3M2 is a cancer gene, which can be used to distinguish between colorectal cancer and colorectal adenomas, liver metastasis, lung metastasis, colorectal polyp. Higher AP3M2 expression levels were associated with longer overall survival in colon adenocarcinoma. AP3M2 might be the primary biomarker for oxaliplatin in colon cancer and an acquired resistance biomarker for oxaliplatin and 5-fu. AP3M2 was positively associated with CD274, CTLA4. AP3M2 might be associated with T-cell, NF-kappaB transcription factor activity, and response to hypoxia. AP3M2 could predict chemotherapy effectiveness and prognosis for colon cancer patients. AP3M2 might inhibit tumor growth via influencing tumor-infiltrating immune cells in the context of Tumor microenvironment. AP3M2 plays as an oncogene in CRAC and is suggested as a new potential biotarget for therapy.

Heterozygosity alters Msh5 binding to meiotic chromosomes in the baker's yeast.

Meiotic crossovers are initiated from programmed DNA double-strand breaks. The Msh4-Msh5 heterodimer is an evolutionarily conserved mismatch repair-related protein complex that promotes meiotic crossovers by stabilizing strand invasion intermediates and joint molecule structures such as Holliday junctions. In vivo studies using homozygous strains of the baker's yeast Saccharomyces cerevisiae (SK1) show that the Msh4-Msh5 complex associates with double-strand break hotspots, chromosome axes, and centromeres. Many organisms have heterozygous genomes that can affect the stability of strand invasion intermediates through heteroduplex rejection of mismatch-containing sequences. To examine Msh4-Msh5 function in a heterozygous context, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) analysis in a rapidly sporulating hybrid S. cerevisiae strain (S288c-sp/YJM789, containing sporulation-enhancing QTLs from SK1), using SNP information to distinguish reads from homologous chromosomes. Overall, Msh5 localization in this hybrid strain was similar to that determined in the homozygous strain (SK1). However, relative Msh5 levels were reduced in regions of high heterozygosity, suggesting that high mismatch densities reduce levels of recombination intermediates to which Msh4-Msh5 binds. Msh5 peaks were also wider in the hybrid background compared to the homozygous strain (SK1). We determined regions containing heteroduplex DNA by detecting chimeric sequence reads with SNPs from both parents. Msh5-bound double-strand break hotspots overlap with regions that have chimeric DNA, consistent with Msh5 binding to heteroduplex-containing recombination intermediates.

Therapeutic targets for endometriosis: Genome-wide Mendelian randomization and colocalization analyses

BackgroundEndometriosis (EM) greatly affects women's reproductive health, identifying new drug targets for EM is urgently needed. This study utilizes comprehensive genome-wide Mendelian randomization (MR) and colocalization analyses, using genomic data, to identify potential therapeutic approaches for EM. MethodsGenome-wide cis-expression quantitative trait loci (cis-eQTL) data were obtained from GTEx V8, which included 838 participants across 49 tissues or cells, and the eQTLGen consortium, which included 31,684 participants. Genome-wide association analysis (GWAS) data for EM were sourced from the FinnGen study, which consisted of 8,288 cases and 68,969 controls, as well as the UK Biobank study, which included 1,496 cases and 359,698 controls. This study utilized MR analysis to assess the correlation between genes and the risk of EM. Subsequently, colocalization analysis was conducted to investigate potential shared causal variants between the identified genes and EM. ResultsAfter conducting MR and colocalization analyses, we identified a total of 13 genes that showed significant evidence of colocalization. These genes are considered promising therapeutic candidates for treating EM. Among them, inner membrane mitochondrial protein (IMMT), src kinase associated phosphoprotein 1 (SKAP1), lysine methyltransferase 5A (KMT5A), KLF transcription factor 12 (KLF12), GRB10 interacting GYF protein 1 (GIGYF1), Wnt family member 7A (WNT7A), Sad1 and UNC84 domain containing 1 (SUN1), and poly (ADP-ribose) polymerase family member 3 (PARP3) were found to have positive associations with the risk of EM. On the other hand, progestin and adipoQ receptor family member 8 (PAQR8), adaptor related protein complex 3 subunit mu 1 (AP3M1), surfeit 6 (SURF6), TUB bipartite transcription factor (TUB), and DNA polymerase delta interacting protein 2 (POLDIP2) were found to have inverse relationships with the risk of EM. ConclusionsThrough genome-wide MR studies, a comprehensive set of genes associated with EM has been identified. Among them, IMMT, PAQR8, SKAP1, KMT5A, AP3M1, SURF6, KLF12, GIGYF1, TUB, WNT7A, SUN1, POLDIP2, and PARP3 show potential as therapeutic targets for EM treatment. Nonetheless, it is crucial to conduct further rigorous investigations to validate these prospects.

Current management of generalized pustular psoriasis.

Generalized pustular psoriasis (GPP) is a rare subset of psoriasis involving episodes of sterile pustules accompanied by inflammation and, often, systemic involvement. The inflammatory nature of GPP has potential for severe multisystem complications including high-output cardiac failure, infections, digestive system issues, and disfiguring or lethal acute flare episodes. The disease tends to have higher prevalence in females and Asians. The IL-1/IL-36 inflammatory pathway is a critical facet of GPP's pathology. Genetic mutations that are associated with GPP include modifications of Interleukin 36 Receptor Antagonist (IL36RN), Caspase Recruitment Domain Family Member 14 (CARD14), Adaptor Related Protein Complex 1 Subunit Sigma 3 (AP1S3), Myeloperoxidase (MPO) and Serpin Peptidase Inhibitor Clade A Member 3 (SERPINA3) genes. Treatment guidelines for GPP are not well-entrenched. Currently, only one GPP-specific treatment, the interleukin-36 receptor antagonist (IL-36Ra) spesolimab, has been approved for use in the United States. Additional anti-IL-36 pathway therapies are currently being developed. Other treatment options include other biologic therapies such as IL-17 inhibitors, IL-23 inhibitors and TNFα inhibitors. Non-biologic therapeutic options include retinoids, cyclosporine and methotrexate. Treatment options differ throughout the world; most countries utilize retinoids, cyclosporine and methotrexate as first-line non-biologic options. China and United Kingdom have no GPP-specific biologic therapies approved for use, while several biologic therapies are approved for use in Japan. This review aims to serve as an update on the current global management of GPP while also including relevant aspects of disease pathogenesis, diagnosis, clinical presentation, histopathology, aetiology and epidemiology.

High expression of AP2M1 correlates with worse prognosis by regulating immune microenvironment and drug resistance to R-CHOP in diffuse large B cell lymphoma.

First-line treatment with R-CHOP has cured 50%-60% patients of diffuse large B cell lymphoma (DLBCL), and more than one-third patients will eventually progressed to relapsed/refractory disease with dismal outcomes. Adaptor Related Protein Complex 2 Subunit Mu 1 (AP2M1) is required for the activity of a vacuolar ATPase and may also play an important role in regulating the intracellular trafficking and function of CTLA-4 protein. Herein, using both public databases and our own tumor samples, we aimed to demonstrate the prognostic role of AP2M1 and the potential tumor-promoting mechanisms in DLBCL. Using public datasets of DLBCL from both GEO and TCGA databases, we analyzed the role of AP2M1 in mediating chemoresistance to R-CHOP and its correlation with various clinical parameters and prognosis. By using various R packages, we evaluated the role of AP2M1 on regulating tumor immune microenvironment. Moreover, tumor samples of DLBCL from Beijing TongRen Hospital were used to validate our findings by immunohistochemistry staining. Expression of AP2M1 was significantly increased in DLBCL, which was correlated with poor prognosis and a variety of clinical indicators. On the basis of enrichment analysis, it was found that AP2M1 may be related to intracellular receptor signaling pathway. Through immune analysis and drug prediction, we found that the expression of AP2M1 affected the immune environment and drug response of DLBCL, which further revealed the important role of AP2M1 in DLBCL. By analyzing 61 patients treated uniformly with R-CHOP regimen in our center, we validated the above findings that high expression of AP2M1 correlated with inferior survival outcomes and affected sensitivity to R-CHOP treatment. Expression of AP2M1 may affect the prognosis of DLBCL patients probably by affecting the immune environment and the responses to many drugs in treating DLBCL, indicating AP2M1 as a potential therapy target in DLBCL.

PB2170: HIGH EXPRESSION OF AP2M1 CORRELATES WITH WORSE PROGNOSIS BY REGULATING IMMUNE MICROENVIRONMENT AND DRUG RESPONSE TO R-CHOP IN DLBCL

Background: DLBCL (diffuse large B cell lymphoma) is the most common subtype of non-Hodgkin lymphoma (NHL). After anthracycline-based chemotherapy, more than 70% of DLBCL patients can get complete response or partial response, but only 50% - 60% of patients can survive for a long time. Adaptor Related Protein Complex 2 Subunit Mu 1 (AP2M1) encodes a subunit of the hetero tetrameric coat assembly protein complex 2 (AP2), which required for the activity of a vacuolar ATPase and may also play an important role in regulating the intracellular trafficking and function of CTLA-4 protein. There are few reports on the relationship between DLBCL and AP2M1. Meanwhile, it is unclear whether it is related to the pathogenesis of DLBCL. Aims: Here, in this study, the relationship between DLBCL and AP2M1 and its effect in DLBCL chemotherapy were studied. Methods: Using public datasets of DLBCL from both GEO and TCGA databases, we analyzed the role of AP2M1 in mediating chemoresistance to R-CHOP and its correlation with various clinical parameters and prognosis. By using various R packages, we evaluated the role of AP2M1 on regulating tumor immune microenvironment. Moreover, FFPE samples of DLBCL from Beijing Tongren Hospital were used to valiate our findings. Results: We found that the expression of AP2M1 was significantly increased in DLBCL, which was correlated with poor prognosis and a variety of clinical indicators. On the basis of enrichment analysis, it was found that AP2M1 may be related to intracellular receptor signaling pathway. Through immune analysis and drug prediction, we found that the expression of AP2M1 affected the immune environment and drug response of DLBCL, which further revealed the important role of AP2M1 in DLBCL. Using FFPE DLBCL samples from patients treated in our center, we validated the above findings that high expression of AP2M1 correlated with inferior survival outcomes and affected sensitivity to R-CHOP treatment. Image:Summary/Conclusion: In this study, we found that the expression of AP2M1 could affect the prognosis of DLBCL patients by affecting the immune environment, and the expression of AP2M1 affected the response of many drugs in DLBCL treatment, indicating AP2M1 as a potential therapy target in DLBCL.

Identification and selection of optimal reference genes for qPCR-based gene expression analysis in Fucus distichus under various abiotic stresses.

Quantitative gene expression analysis is an important tool in the scientist's belt. The identification of evenly expressed reference genes is necessary for accurate quantitative gene expression analysis, whether by traditional RT-PCR (reverse-transcription polymerase chain reaction) or by qRT-PCR (quantitative real-time PCR; qPCR). In the Stramenopiles (the major line of eukaryotes that includes brown algae) there is a noted lack of known reference genes for such studies, largely due to the absence of available molecular tools. Here we present a set of nine reference genes (Elongation Factor 1 alpha (EF1A), Elongation Factor 2 alpha (EF2A), Elongation Factor 1 beta (EF1B), 14-3-3 Protein, Ubiquitin Conjugating Enzyme (UBCE2), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Actin Related Protein Complex (ARP2/3), Ribosomal Protein (40s; S23), and Actin) for the brown alga Fucus distichus. These reference genes were tested on adult sporophytes across six abiotic stress conditions (desiccation, light and temperature modification, hormone addition, pollutant exposure, nutrient addition, and wounding). Suitability of these genes as reference genes was quantitatively evaluated across conditions using standard methods and the majority of the tested genes were evaluated favorably. However, we show that normalization genes should be chosen on a condition-by-condition basis. We provide a recommendation that at least two reference genes be used per experiment, a list of recommended pairs for the conditions tested here, and a procedure for identifying a suitable set for an experimenter's unique design. With the recent expansion of interest in brown algal biology and accompanied molecular tools development, the variety of experimental conditions tested here makes this study a valuable resource for future work in basic biology and understanding stress responses in the brown algal lineage.

RAB33B recruits the ATG16L1 complex to the phagophore via a noncanonical RAB binding protein

ABSTRACT Autophagosome formation is a fundamental process in macroautophagy/autophagy, a conserved self-eating mechanism in all eukaryotes, which requires the conjugating ATG (autophagy related) protein complex, ATG12–ATG5-ATG16L1 and lipidated MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). How the ATG12–ATG5-ATG16L1 complex is recruited to membranes is not fully understood. Here, we demonstrated that RAB33B plays a key role in recruiting the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 revealed the recognition mechanism between RAB33B and ATG16L1. ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation. Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. In this work, we reported a new mechanism for the recruitment of the ATG12–ATG5-ATG16L1 complex to phagophores by RAB33B, which is required for autophagosome formation. Abbreviations : ATG: autophagy-related; Cα: alpha carbon; CCD: coiled-coil domain; CLEM: correlative light and electron microscopy; DTE: dithioerythritol; EBSS: Earle’s balanced salt solution; EDTA: ethylenediaminetetraacetic acid; EGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; FLIM: fluorescence lifetime imaging microscopy; FRET: Förster resonance energy transfer; GDP: guanosine diphosphate; GOLGA2/GM130: golgin A2; GppNHp: guanosine 5ʹ-[β,γ-imido]triphosphate; GST: glutathione S-transferase; GTP: guanosine triphosphate; GTPγS: guanosine 5ʹ-O-[gamma-thio]triphosphate; HA (tag): hemagglutinin (tag); HEK: human embryonic kidney; HeLa: Henrietta Lacks; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); IgG: immunoglobulin G; Kd: dissociation constant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCF7: Michigan cancer foundation-7; MEF: mouse embryonic fibroblast; MEM: minimum essential medium Eagle; MST: microscale thermophoresis; NEAA: non-essential amino acids; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RAB: RAS-associated binding; RB1CC1/FIP200: RB1 inducible coiled-coil protein 1; RBP: RAB-binding protein; SD: standard deviation; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TBS-T: tris-buffered saline-tween 20; WD (repeat): tryptophan-aspartic acid (repeat); WIPI2B: WD repeat domain phosphoinositide interacting 2B; WT: wild type

Alantolactone inhibits cell autophagy and promotes apoptosis via AP2M1 in acute lymphoblastic leukemia

BackgroundAcute lymphoblastic leukemia (ALL) is an aggressive hematopoietic malignancy that is most commonly observed in children. Alantolactone (ALT) has been reported to exhibit anti-tumor activity in different types of cancer. The aim of the present study was to investigate the anti-tumor activity and molecular mechanism of ALT in ALL.MethodsALL cell lines were treated with 1, 5 and 10 μM ALT, and cell viability was assessed using an MTT assay and RNA sequencing. Flow cytometry, JC-1 staining and immunofluorescence staining assays were used to measure cell apoptosis and autophagy. Additionally, western blot analysis was used to detect expression of apoptosis and autophagy related proteins. Finally, the effects of ALT on tumor growth were assessed in a BV173 xenograft nude mouse model.ResultsALT inhibited the proliferation of ALL cells in a dose-dependent manner. Additionally, it was demonstrated that ALT inhibited cell proliferation, colony formation, autophagy, induced apoptosis and reduced tumor growth in vivo through upregulating the expression of adaptor related protein complex 2 subunit mu 1 (AP2M1). Moreover, the autophagy activator rapamycin, attenuated the pro-apoptotic effects of ALT on BV173 and NALM6 cell lines. Overexpression of AP2M1 decreased the expression of Beclin1 and the LC3-II/LC3-1 ratio, and increased p62 expression. Knockdown of Beclin1 increased the levels of bax, cleaved caspase 3 and cytochrome C, and decreased bcl-2 expression.ConclusionsThe present study demonstrated that ALT exerts anti-tumor activity through inducing apoptosis and inhibiting autophagy by upregulating AP2M1 in ALL, highlighting a potential therapeutic strategy for treatment of ALL.

Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence

ABSTRACT Coxiella burnetii, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a Coxiella transposon mutant library, we observed that transposon insertions in cbu0626 led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the Coxiella vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as Coxiella-containing vacuoles (CCVs). We thus renamed this effector CvpF for Coxiella vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, cvpF::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for Coxiella virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal C. burnetii vacuole biogenesis. Abbreviations: ACCM: acidified citrate cystein medium; AP: adaptor related protein complex; CCV: Coxiella-containing vacuole; Cvp: Coxiella vacuolar protein; GDI: guanosine nucleotide dissociation inhibitor; GDF: GDI dissociation factor; GEF: guanine exchange factor; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase MTOR complex 1; PBS: phosphate-buffered saline; PMA: phorbol myristate acetate; SQSTM1/p62: sequestosome 1; WT: wild-type.

Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene.

We found evidence of late-onset Alzheimer disease (LOAD)-associated genetic polymorphism within an exon of Mucin 6 (MUC6) and immediately downstream from another gene: Adaptor Related Protein Complex 2 Subunit Alpha 2 (AP2A2). PCR analyses on genomic DNA samples confirmed that the size of the MUC6 variable number tandem repeat (VNTR) region was highly polymorphic. In a cohort of autopsied subjects with quantitative digital pathology data (n = 119), the size of the polymorphic region was associated with the severity of pTau pathology in neocortex. In a separate replication cohort of autopsied subjects (n = 173), more pTau pathology was again observed in subjects with longer VNTR regions (p = 0.031). Unlike MUC6, AP2A2 is highly expressed in human brain. AP2A2 expression was lower in a subset analysis of brain samples from persons with longer versus shorter VNTR regions (p = 0.014 normalizing with AP2B1 expression). Double-label immunofluorescence studies showed that AP2A2 protein often colocalized with neurofibrillary tangles in LOAD but was not colocalized with pTau proteinopathy in progressive supranuclear palsy, or with TDP-43 proteinopathy. In summary, polymorphism in a repeat-rich region near AP2A2 was associated with neocortical pTau proteinopathy (because of the unique repeats, prior genome-wide association studies were probably unable to detect this association), and AP2A2 was often colocalized with neurofibrillary tangles in LOAD.

Long noncoding RNA LINC00518 acts as a competing endogenous RNA to promote the metastasis of malignant melanoma via miR-204-5p/AP1S2 axis

Long intergenic nonprotein coding RNA 518 (LINC00518) has been shown to promote cancer cell growth and metastasis in some human tumors. Although it has been reported that LINC00518 is dysregulated in melanoma, its exact role and molecular mechanism in melanoma remain unclear. RNA-seq analysis and qRT-PCR was used to detect the expression of LINC00518 in melanoma tissues. Melanoma cases from The Cancer Genome Atlas (TCGA), GEO#GSE15605 and GEO#GSE24469 were included in this study. 3D migration, transwell and scratch wound assay were used to explore the role of LINC00518 in melanoma cells. Bioinformatics, luciferase reporter assays, MS2-RIP assay, RNA pull-down assay and RNA-ChIP assay were used to demonstrate the mechanism of LINC00518 in melanoma. We found that LICN00518 was significantly upregulated in melanoma tissue, and high LICN00518 level was an independent risk factor for melanoma patients. LICN00518 promoted the invasion and migration of melanoma cells. LICN00518 exerted its role by decoying miR-204-5p to upregulate Adaptor Related Protein Complex 1 Sigma 2 Subunit (AP1S2) expression. We also demonstrated that LICN00518 promoted melanoma metastasis in vivo through pulmonary metastasis assay. This result elucidates a new mechanism for LICN00518 in the metastasis of melanoma. LICN00518 may serve as a survival indicator and potential therapeutic target in melanoma patients.

Co-expression Network Analysis Identified Key Proteins in Association With Hepatic Metastatic Colorectal Cancer.

Intense efforts have been made in colorectal cancer (CRC) treatment in recent decades. However, the mechanism of development and metastasis of CRC has not been fully cleared. This study is designed to identify key proteins involved in stage III and hepatic metastatic CRC. Protein expression profiles of paired tumor and benign tissue samples from stage III and hepatic metastatic CRC patients are characterized by using a label-free proteomics approach. Key proteins relevant to hepatic metastatic CRC are revealed by weighted gene correlation network analysis (WGCNA) and other bioinformatics tools. WGCNA reveals three hub modules: CRC without specific stage (turquoise), stage III CRC (blue), and hepatic metastatic CRC (green). Nine key proteins (heat shock protein family D member 1 (HSPD1), eukaryotic translation elongation factor 1 gamma, heterogeneous nuclear ribonucleoprotein A2/B1, fibrinogen beta chain (FGB), Talin 1, adaptor related protein complex 2 subunit alpha 2, serrate RNA effector molecule homolog, apolipoprotein C3, phosphoglucomutase 5) are identified. Moreover, upregulation of HSPD1 is validated in CRC tissue by the immunohistochemistry. Upregulation of fibrinogen is validated in metastatic CRC by plasma fibrinogen assay. This study provides the proteomic analysis of stage III and hepatic metastatic CRC to identify key proteins of CRC. FGB plays a key role to serve as diagnostic and therapeutic biomarkers for hepatic metastatic CRC.

Identification of Novel Genes in Osteoarthritic Fibroblast-Like Synoviocytes Using Next-Generation Sequencing and Bioinformatics Approaches.

Synovitis in osteoarthritis (OA) the consequence of low grade inflammatory process caused by cartilage breakdown products that stimulated the production of pro-inflammatory mediators by fibroblast-like synoviocytes (FLS). FLS participate in joint homeostasis and low grade inflammation in the joint microenvironment triggers FLS transformation. In the current study, we aimed to identify differentially expressed genes and potential miRNA regulations in human OA FLS through deep sequencing and bioinformatics approaches. The 245 differentially expressed genes in OA FLS were identified, and pathway analysis using various bioinformatics databases indicated their enrichment in functions related to altered extracellular matrix organization, cell adhesion and cellular movement. Moreover, among the 14 dysregulated genes with potential miRNA regulations identified, src kinase associated phosphoprotein 2 (SKAP2), adaptor related protein complex 1 sigma 2 subunit (AP1S2), PHD finger protein 21A (PHF21A), lipoma preferred partner (LPP), and transcription factor AP-2 alpha (TFAP2A) showed similar expression patterns in OA FLS and OA synovial tissue datasets in Gene Expression Omnibus database. Ingenuity Pathway Analysis identified the dysregulated LPP participated in cell migration and cell spreading of OA FLS, which was potentially regulated by miR-141-3p. The current findings suggested new perspectives into understanding the novel molecular signatures of FLS involved in the pathogenesis of OA, which may be potential therapeutic targets.

Regulation of gastric smooth muscle contraction via Ca2+-dependent and Ca2+-independent actin polymerization.

In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.

ZFYVE26/SPASTIZIN and SPG11/SPATACSIN mutations in hereditary spastic paraplegia types AR-SPG15 and AR-SPG11 have different effects on autophagy and endocytosis

ABSTRACTZFYVE26/Spastizin and SPG11/Spatacsin encode 2 large proteins that are mutated in hereditary autosomal-recessive spastic paraplegia/paraparesis (HSP) type 15 (AR-SPG15) and type 11 (AR-SPG11), respectively. We previously have reported that AR-SPG15-related ZFYVE26 mutations lead to autophagy defects with accumulation of immature autophagosomes. ZFYVE26 and SPG11 were found to be part of a complex including the AP5 (adaptor related protein complex 5) and to have a critical role in autophagic lysosomal reformation with identification of autophagic and lysosomal defects in cells with both AR-SPG15- and AR-SPG11-related mutations. In spite of these similarities between the 2 proteins, here we report that ZFYVE26 and SPG11 are differently involved in autophagy and endocytosis. We found that both ZFYVE26 and SPG11 interact with RAB5A and RAB11, 2 proteins regulating endosome trafficking and maturation, but only ZFYVE26 mutations affected RAB protein interactions and activation. ZFYVE26 mutations lead to defects in the fusion between autophagosomes and endosomes, while SPG11 mutations do not affect this step and lead to a milder autophagy defect. We thus demonstrate that ZFYVE26 and SPG11 affect the same cellular physiological processes, albeit at different levels: both proteins have a role in autophagic lysosome reformation, but only ZFYVE26 acts at the intersection between endocytosis and autophagy, thus representing a key player in these 2 processes. Indeed expression of the constitutively active form of RAB5A in cells with AR-SPG15-related mutations partially rescues the autophagy defect. Finally the model we propose demonstrates that autophagy and the endolysosomal pathway are central processes in the pathogenesis of these complicated forms of hereditary spastic paraparesis.Abbreviations: ALR, autophagic lysosome reformation; AP5, adaptor related protein complex 5; AR, autosomal-recessive; HSP, hereditary spastic paraplegia/paraparesis; ATG14, autophagy related 14; BafA, bafilomycin A1; BECN1, beclin 1; EBSS, Earle balanced salt solution; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; HSP, hereditary spastic paraplegias; LBPA, lysobisphosphatidic acid; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MVBs, multivesicular bodies; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4, phosphoinositide-3-kinase regulatory subunit 4; PtdIns3P, phosphatidylinositol-3-phosphate; RFP, red fluorescent protein; RUBCN, RUN and cysteine rich domain containing beclin 1 interacting protein; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; TCC: thin corpus callosum; TF, transferrin; UVRAG, UV radiation resistance associated.