Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects

The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.

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TAMI-50. CHITINASE-3-LIKE-1 PROTEIN BINDING COMPLEXES REGULATE IMMUNE SUPPRESSION IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.937 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii224-ii224

Author(s):

Apeng Chen ◽

Yinan Jiang ◽

Zhengwei Li ◽

Han Zou ◽

Ulises Santiago ◽

...

Keyword(s):

Protein Binding ◽

Immune Evasion ◽

Immune Suppression ◽

Tumor Infiltrating Lymphocytes ◽

Cell Mediated Immunity ◽

Binding Motif ◽

Loss Of Function ◽

Immunodeficient Mice ◽

Galectin 3

Abstract Glioblastoma (GBM), the most common and lethal brain tumor, remains incurable despite intensive multimodal treatment. While immunotherapies have been highly effective in some types of cancer, the disappointing results from clinical trials for GBM immunotherapy represent continued challenges. GBM is highly immunosuppressive and resistant to immunotherapy because of glioma cells escaping from immune surveillance by reprograming the tumor microenvironment (TME). However, understanding the mechanisms of immune evasion by GBM remains elusive. Here, we found that Chitinase-3-like-1 (CHI3L1) is highly expressed in GBM and associated with a poor clinical outcome. CHI3L1, also known as human homolog YKL-40, plays a role in tissue remodeling, inflammation and cancer. Interestingly, we found that genetic knockdown (KD) of Chi3l1 in syngeneic immunocompetent mouse GBM models resulted in increased tumor-infiltrating lymphocytes, tumor size reduction, and improved animal survival. Surprisingly, the parallel loss-of-function experiment revealed that Chi3l1 KD did not repress tumor progression in the orthotopic immunodeficient mice with deficient T and B cells. These results suggest the predominant role of CHI3L1 in regulating the GBM immune TME, rather than in tumor cells per se. Mechanistically, we discovered that Galectin-3 (Gal-3) and Galectin-3 binding protein (Gal-3BP) interact competitively with the same binding motif on CHI3L1, leading to selective migration of protumor M2-like versus antitumor M1-like bone marrow-derived macrophages (BMDMs) and resident microglia (MG). Transcriptomic analysis revealed that pro-inflammatory signature and T cell mediated immunity and cytotoxicity signaling are significantly enriched in tumor associated macrophages/microglia (TAMs) composed of BMDMs and MG, which were isolated from tumors with Chi3l1 KD versus wild type. In vitro validations suggest that CHI3L1-Gal-3, but not CHI3L1-Gal-3BP protein binding complex, activates PI3K/AKT/mTOR signaling to control the TAM switch of immune suppression and immune stimulation. Together, these results shed light on molecular mechanism of GBM immune evasion and potential new immunotherapeutic strategies for GBM treatment.

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The tumour-suppressor function of PTEN requires an N-terminal lipid-binding motif

Biochemical Journal ◽

10.1042/bj20031839 ◽

2004 ◽

Vol 379 (2) ◽

pp. 301-307 ◽

Cited By ~ 103

Author(s):

Steven M. WALKER ◽

Nick R. LESLIE ◽

Nevin M. PERERA ◽

Ian H. BATTY ◽

C. Peter DOWNES

Keyword(s):

Catalytic Activity ◽

Lipid Binding ◽

Tumour Suppressor ◽

Binding Motif ◽

Loss Of Function ◽

Wild Type ◽

Suppressor Function ◽

Tumour Suppressor Function ◽

Anionic Lipids

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour-suppressor protein is a phosphoinositide 3-phosphatase which antagonizes phosphoinositide 3-kinase-dependent signalling by dephosphorylating PtdIns(3,4,5)P3. Most tumour-derived point mutations of PTEN induce a loss of function, which correlates with profoundly reduced catalytic activity. However, here we characterize a point mutation at the N-terminus of PTEN, K13E from a human glioblastoma, which displayed wild-type activity when assayed in vitro. This mutation occurs within a conserved polybasic motif, a putative PtdIns(4,5)P2-binding site that may participate in membrane targeting of PTEN. We found that catalytic activity against lipid substrates and vesicle binding of wild-type PTEN, but not of PTEN K13E, were greatly stimulated by anionic lipids, especially PtdIns(4,5)P2. The K13E mutation also greatly reduces the efficiency with which anionic lipids inhibit PTEN activity against soluble substrates, supporting the hypothesis that non-catalytic membrane binding orientates the active site to favour lipid substrates. Significantly, in contrast to the wild-type enzyme, PTEN K13E failed either to prevent protein kinase B/Akt phosphorylation, or inhibit cell proliferation when expressed in PTEN-null U87MG cells. The cellular functioning of K13E PTEN was recovered by targeting to the plasma membrane through inclusion of a myristoylation site. Our results establish a requirement for the conserved N-terminal motif of PTEN for correct membrane orientation, cellular activity and tumour-suppressor function.

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The JAK Inhibitor Tofacitinib Rescues Intestinal Barrier Defects Caused by Disrupted Epithelial-macrophage Interactions

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjaa182 ◽

2020 ◽

Author(s):

Marianne R Spalinger ◽

Anica Sayoc-Becerra ◽

Christ Ordookhanian ◽

Vinicius Canale ◽

Alina N Santos ◽

...

Keyword(s):

Barrier Function ◽

Tyrosine Phosphatase ◽

Janus Kinase ◽

Epithelial Barrier ◽

Intestinal Epithelial ◽

Jak Inhibitor ◽

Loss Of Function ◽

Junction Protein

Abstract Background and Aims Loss-of-function variants in protein tyrosine phosphatase non-receptor type-2 [PTPN2] promote susceptibility to inflammatory bowel diseases [IBD]. PTPN2 regulates Janus-kinase [JAK] and signal transducer and activator of transcription [STAT] signalling, while protecting the intestinal epithelium from inflammation-induced barrier disruption. The pan-JAK inhibitor tofacitinib is approved to treat ulcerative colitis, but its effects on intestinal epithelial cell-macrophage interactions and on barrier properties are unknown. We aimed to determine if tofacitinib can rescue disrupted epithelial-macrophage interaction and barrier function upon loss of PTPN2. Methods Human Caco-2BBe intestinal epithelial cells [IECs] and THP-1 macrophages expressing control or PTPN2-specific shRNA were co-cultured with tofacitinib or vehicle. Transepithelial electrical resistance and 4 kDa fluorescein-dextran flux were measured to assess barrier function. Ptpn2fl/fl and Ptpn2-LysMCre mice, which lack Ptpn2 in myeloid cells, were treated orally with tofacitinib citrate twice daily to assess the in vivo effect on the intestinal epithelial barrier. Colitis was induced via administration of 1.5% dextran sulphate sodium [DSS] in drinking water. Results Tofacitinib corrected compromised barrier function upon PTPN2 loss in macrophages and/or IECs via normalisation of: [i] tight junction protein expression; [ii] excessive STAT3 signalling; and [iii] IL-6 and IL-22 secretion. In Ptpn2-LysMCre mice, tofacitinib reduced colonic pro-inflammatory macrophages, corrected underlying permeability defects, and prevented the increased susceptibility to DSS colitis. Conclusions PTPN2 loss in IECs or macrophages compromises IEC-macrophage interactions and reduces epithelial barrier integrity. Both of these events were corrected by tofacitinib in vitro and in vivo. Tofacitinib may have greater therapeutic efficacy in IBD patients harbouring PTPN2 loss-of-function mutations.

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RBM10 Interacts with CTBNNBIP1 and Represses Lung Adenocarcinoma Progression Through the Wnt/β- catenin Pathway

10.21203/rs.3.rs-480093/v1 ◽

2021 ◽

Author(s):

Yingyue Cao ◽

Xin Wang ◽

Qingwei Meng ◽

Jianxiong Geng ◽

Shanqi Xu ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Molecular Mechanism ◽

Rna Binding ◽

Bioinformatics Analysis ◽

Protein Extraction ◽

Binding Motif ◽

Loss Of Function ◽

Mesenchymal Transition

Abstract Background: RNA-binding motif protein 10 (RBM10), one of the RNA-binding protein (RBP) family, has a tumor suppressor role in various tumors. However, the functional role of RBM10 in lung adenocarcinoma (LUAD) and the molecular mechanism remain unclear. The aim of this study was to explore the effect of RBM10 on LUAD growth and metastasis and its molecular mechanism.Methods: Bioinformatics analysis was used to predict RBM10 expression and its associations with clinicopathological features and prognosis in LUAD. Gain- and loss- of function experiments were conducted to investigate the biological functions of RBM10 both in vitro and in vivo. RNA-seq, bioinformatics programs, western blot, qRT-PCR, TOP/FOP flash reporter, co-immunoprecipitation (co-IP), nuclear and cytoplasmic protein extraction and rescue experiments were used to reveal the underlying mechanisms.Results: Bioinformatics analysis showed that RBM10 was significantly downregulated and closely correlated with poor prognosis in LUAD patients. RBM10 silencing significantly promoted the LUAD proliferation, migration, invasion ability, while RBM10 overexpression had the opposite effects. Furthermore, upregulation of RBM10 inhibited growth and metastasis of LUAD in vivo. Additionally, RBM10 suppressed tumor progression through inhibiting epithelial to mesenchymal transition (EMT) in LUAD cells. Mechanistically, RBM10 interacts with β-catenin interacting protein 1 (CTNNBIP1) and positively regulates its expression, thus inactivating the Wnt/β-catenin pathway. Conclusions: This is the first study that reported how RBM10 suppresses cell proliferation and metastasis of LUAD by negatively regulating the Wnt/β-catenin pathway through interaction with CTNNBIP1. These data suggest that RBM10 may be a promising new target or clinical biomarker for LUAD therapy.

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Leydig cell function in mice lacking connexin43

Reproduction ◽

10.1530/rep.1.01234 ◽

2006 ◽

Vol 132 (4) ◽

pp. 607-616 ◽

Cited By ~ 21

Author(s):

Caroline N Kahiri ◽

M Wahid Khalil ◽

Francis Tekpetey ◽

Gerald M Kidder

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Leydig Cell ◽

Leydig Cells ◽

Cell Function ◽

Hydroxysteroid Dehydrogenase ◽

Wild Type ◽

Dye Transfer ◽

Junction Protein

Connexin43 (Cx43) is the most abundantly expressed member of the connexin (gap junction protein) family and the only one so far identified in mouse Leydig cell gap junctions. Mice lacking Cx43 were used to investigate its role in testicular androgen production and regulation. Testes from term fetuses were grafted under the kidney capsules of castrated adult males. After 3 weeks, serum from host mice was analyzed for androgens. In order to test their response to stimulation, the grafted testes were incubated in vitro with varying concentrations of LH and their androgen end products analyzed. Incubation with radiolabeled progesterone was followed by high performance liquid chromatography to quantify the androgen-intermediate metabolites. Radiolabeled testosterone in the presence of NADPH was used to determine the activity of testosterone-metabolizing enzymes 17β-hydroxysteroid dehydrogenase (17βHSD), 5α-reductase (5αR), and 3α-hydroxysteroid dehydrogenase (3α HSD). Serum androgen levels did not differ between hosts carrying wild-type versus null mutant grafts although Cx43-deficient testes had more 17βHSD and 5αR activity than wild-type controls. Furthermore, the genotype of grafted testes did not influence LH-stimulated androgen production in vitro. These results indicate that the steroidogenic function of Leydig cells is not compromised by the absence of Cx43, perhaps because other gap junction proteins are present. Dye transfer experiments demonstrated that Cx43-deficient Leydig cells retain intercellular coupling, indicating that Cx43 is not the only protein contributing to their gap junctions. Thus, despite their prominence in Leydig cells, Cx43 gap junctions are not essential for androgen production.

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A transient amphipathic helix in PCSK9’s prodomain facilitates low-density lipoprotein binding

10.1101/743195 ◽

2019 ◽

Author(s):

Samantha K. Sarkar ◽

Alexander C.Y. Foo ◽

Angela Matyas ◽

Tanja Kosenko ◽

Natalie K. Goto ◽

...

Keyword(s):

Conformational Changes ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Helical Structure ◽

Low Density ◽

Loss Of Function ◽

Ldl Particles ◽

Ldl Binding ◽

Α Helix

SUMMARYProprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30-40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9’s prodomain. Here we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N-terminus adopted helical structure in a membrane-mimetic environment; this effect was greatly enhanced by an R46L substitution representing an athero-protective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested the transient α-helix aligns multiple polar residues to interact with positive-charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, F515L) inhibited LDL binding, which was abolished for the R496W variant. These studies inform on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, we report the initial identification of FH-associated mutations that diminish the ability of PCSK9 to bind LDL, supporting that LDL association in the circulation inhibits PCSK9 activity.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013379117 ◽

2020 ◽

Vol 117 (41) ◽

pp. 25486-25493 ◽

Cited By ~ 1

Author(s):

Jun Xu ◽

Wei Wang ◽

Liang Xu ◽

Jia-Yu Chen ◽

Jenny Chong ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Neurological Diseases ◽

Transcription Elongation ◽

Cockayne Syndrome ◽

Stable Complex ◽

Loss Of Function ◽

Pol Ii ◽

Chromatin Remodelers ◽

Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

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Pro-Inflammatory Effect of Gliadins and Glutenins Extracted from Different Wheat Cultivars on an In Vitro 3D Intestinal Epithelium Model

International Journal of Molecular Sciences ◽

10.3390/ijms22010172 ◽

2020 ◽

Vol 22 (1) ◽

pp. 172

Author(s):

Francesca Truzzi ◽

Camilla Tibaldi ◽

Anne Whittaker ◽

Silvia Dilloo ◽

Enzo Spisni ◽

...

Keyword(s):

Three Dimensional ◽

Innate Immune Responses ◽

Wheat Proteins ◽

Monocyte Migration ◽

Junction Protein ◽

Modern Wheat ◽

In Vitro Effects ◽

Landrace Wheat ◽

Cluster Of Differentiation

There is a need to assess the relationship between improved rheological properties and the immunogenic potential of wheat proteins. The present study aimed to investigate the in vitro effects of total protein extracts from three modern and two landrace Triticum aestivum commercial flour mixes, with significant differences in gluten strength (GS), on cell lines. Cytotoxicity and innate immune responses induced by wheat proteins were investigated using Caco-2 monocultures, two dimensional (2D) Caco-2/U937 co-cultures, and three dimensional (3D) co-cultures simulating the intestinal mucosa with Caco-2 epithelial cells situated above an extra-cellular matrix containing U937 monocytes and L929 fibroblasts. Modern wheat proteins, with increased GS, significantly reduced Caco-2 cell proliferation and vitality in monoculture and 2D co-cultures than landrace proteins. Modern wheat proteins also augmented Caco-2 monolayer disruption and tight junction protein, occludin, redistribution in 3D co-cultures. Release of interleukin-8 into the cell medium and increased U937 monocyte migration in both 2D and 3D co-cultures were similarly apparent. Immuno-activation of migrating U937 cells was evidenced from cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. The modern wheat proteins, with gluten polymorphism relatedness and increased GS, were shown to be more cytotoxic and immunogenic than the landrace wheat proteins.

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TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

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