Phosphotyrosine Binding Domain Research Articles

KRIT1 stabilizes endothelial adherens junctions independent of Rap1 via regulation of β1‐integrin

The junctional scaffold KRIT1 interacts with adherens junctions (AJ) proteins to stabilize endothelial cell-cell contacts. Loss of KRIT1 function leads to Cerebral Cavernous Malformations (CCM), a disease characterized by leaky vascular lesions as the result of perturbed endothelial proliferation and inflammatory signaling. As a true Rap1 effector, KRIT1 localizes to and associates with AJ after binding active Rap1. Inhibition of this interaction by mutation of the Rap1-binding site (KRIT1-R452E) prevents KRIT1 from localizing to AJ and stabilizing barrier function. However, it remains unclear whether KRIT1 can stabilize endothelial junctions independently of Rap1. In order to study the mechanisms of KRIT1-mediated junction stabilization, we targeted mCherry-tagged KRIT1-R452E to the plasma membrane using a C-terminal lipid modification (MT-R452E). Immunofluorescence studies indicated that this construct localized to cell-cell contacts similar to WT-KRIT1 in endothelial cells depleted of endogenous KRIT1 (KRIT1 shRNA). While MT-R452E partially rescued AJ staining in these cells, it was unable to rescue barrier function, suggesting that both KRIT1 and Rap1 may be necessary at AJ to stabilize the endothelial barrier. As Rap1 binding may also regulate a putative intramolecular interaction between KRIT1's first NPxY motif and its C-terminal PTB domain, we sought to evaluate whether stabilization of endothelial junctions was dependent on KRIT1 conformation. The NPxY motif of MT-R452E was mutated (N192A-Y195A) to yield a constitutively open, membrane-targeted construct (MT-R452E-ΔNPxY). Immunofluorescence confirmed that this construct localized to cell-cell contacts, and appeared to rescue AJ staining after loss of endogenous KRIT1. Interestingly, open conformation R452E lacking any targeting sequence (R452E-ΔNPxY) also rescued AJ staining despite remaining primarily cytosolic. Further, both open conformation constructs rescued barrier function independent of Rap1 binding, indicating that KRIT1's junction stabilizing effects may be exerted without binding of Rap1 or localization to cell-cell contacts. As this NPxY (192-195) motif mediates binding to ICAP1a, which has been shown to regulate β1-integrin activity, we next examined whether junctional stability was associated with changes in β1-integrin activation. Using an activation-specific antibody against β1-integrin, we observed an increase in large fibrillar adhesions in cells depleted of KRIT1 (KRIT1 shRNA), or rescued with either R452E or MT-R452E, constructs which notably failed to rescue loss of barrier function. Conversely, expression of WT, MT-R452E-ΔNPXY, and R452E-ΔNPXY constructs, which do rescue barrier function, was associated with smaller, peripheral staining of focal contacts. These results suggest that KRIT1 may stabilize endothelial cell junctions independent of Rap1 by regulating β1-integrin-containing adhesive complexes. We expect that these novel findings will further our understanding of junctional physiology while facilitating the development of novel CCM therapeutics.

Adiponectin inhibits lipoplysaccharide-induced inflammation and promotes osteogenesis in hPDLCs.

Periodontal diseases are infections of the structures that surround and support the teeth; they are characterized by local inflammation and alveolar bone loss. Most treatments focus on only one aspect, inhibiting inflammation, or promoting osteoblasts. We set out to develop a new method that would intervene in the two aspects simultaneously. Adiponectin (APN), secreted by adipocytes, inhibits the inflammatory response and promotes osteogenesis. However, its role in human periodontal ligament cells (hPDLCs) is unclear. Therefore, we aim to investigate whether APN could suppress lipopolysaccharide (LPS)-induced inflammation and promote osteogenesis in hPDLCs. In the present study, we stimulated hPDLCs with LPS in the presence or absence of APN. Real-time PCR and Western blotting results demonstrated that APN partially inhibited the activation of the classical nuclear factor κ-B (NF-κB) pathway. These results were confirmed by a change of expressions of NF-κB downstream inflammatory genes, such as decreased cyclooxygenase (COX)-2 and tumor necrosis factor α (TNF-α), along with increased interleukin (IL)-10. As for the role of APN in osteogenesis, Alizarin Red S staining showed that APN treatment induced more calcium deposition nodules than controls. We also found that APN enhanced the expression of osteoblast-related genes (osteopontin (OPN), collagen 1, osteocalcin, alkaline phosphatase, runt-related transcription factor 2 (RUNX2), and bone morphogenetic protein 2) in hPDLCs via the APPL1 (the adaptor protein containing PH domain, PTB domain, and leucine zipper motif 1)/p38 signal transduction pathway. Therefore, APN inhibits LPS-induced inflammation and promotes osteogenesis in hPDLCs and may have potential therapeutic value in treating periodontitis by inhibiting the inflammatory lesions and contributing to bone tissue regeneration.

Macrophage regulator of G-protein signaling 12 contributes to inflammatory pain hypersensitivity.

BackgroundPain is a predominant symptom in rheumatoid arthritis (RA) patients that results from joint inflammation and is augmented by central sensitization. Regulator of G-protein signaling 12 (RGS12) is the largest protein in the RGS protein family and plays a key role in the development of inflammation. This study investigated the regulation of RGS12 in inflammatory pain and explored the underlying mechanisms and potential RA pain targets.MethodsMacrophage-specific RGS12-deficient (LysM-Cre+;RGS12fl/fl) mice were generated by mating RGS12fl/fl mice with LysM-Cre+ transgenic mice. Collagen antibody-induced arthritis (CAIA) models were induced in LysM-Cre+;RGS12fl/fl mice by the administration of a cocktail of five monoclonal antibodies and LPS. Mouse nociception was examined using the von Frey and heat plate tests. Primary macrophages and RAW264.7 cells were used to analyze the regulatory function and mechanism of RGS12 in vitro. The expression and function of RGS12 and COX2 (cyclooxygenase 2) were determined by real-time PCR, ELISA, and luciferase assays.ResultsAblation of RGS12 in macrophages decreased pain-related phenotypes, such as paw swelling, the clinical score, and the inflammatory score, in the CAIA model. LysM-Cre+;RGS12fl/fl mice displayed increased resistance to thermal and mechanical stimulation from day 3 to day 9 during CAIA, indicating the inhibition of inflammatory pain. Overexpression of COX2 and PGE2 in macrophages enhanced RGS12 expression, and PGE2 regulated RGS12 expression through the G-protein-coupled receptors EP2 and EP4. Furthermore, RGS12 or the RGS12 PTB domain strengthened the transcriptional regulation of COX2 by NF-κB, whereas inhibiting NF-κB suppressed RGS12-mediated regulation of COX2 in macrophages.ConclusionsOur results demonstrate that the deletion of RGS12 in macrophages attenuates inflammatory pain, which is likely due to impaired regulation of the COX2/PGE2 signaling pathway.

The role of tensins in malignant neoplasms.

Tensins belong to the family of adhesion proteins which form focal adhesions serving as a bridge between the extracellular matrix and intracellular actin skeleton. The tensin family consists of four members (tensin-1 to -4) which are widely expressed in normal and cancerous tissues. The presence of Src homology 2 and phosphotyrosine binding domains is a unique feature of tensins which enables them to interact with tyrosine-phosphorylated proteins in PI3K/Akt and β-integrin/FAK signaling pathways. The tensin-mediated signaling pathway regulates physiological processes including cell motility and cytoskeleton integrity. The expression of tensins varies among cancers. Several papers report tensins as tumor suppressive proteins, whereas tensins may promote epithelial to mesenchymal transition and cancer cell metastasis. Recent findings and further research on tensins as therapeutic targets in cancers may contribute to identifying effective anti-cancer therapy. In this review we focus on the role of tensins in normal and cancer cells. We discuss potential mechanism(s) involved in carcinogenesis.

Biallelic DAB1 Variants Are Associated With Mild Lissencephaly and Cerebellar Hypoplasia.

ObjectiveWe aimed to identify pathogenic variants in a girl with epilepsy, developmental delay, cerebellar ataxia, oral motor difficulty, and structural brain abnormalities with the use of whole-exome sequencing.MethodsWhole-exome trio analysis and molecular functional studies were performed in addition to the clinical findings and neuroimaging studies.ResultsBrain MRI showed mild pachygyria, hypoplasia of the cerebellar vermis, and abnormal foliation of the cerebellar vermis, suspected for a variant in one of the genes of the Reelin pathway. Trio whole-exome sequencing and additional functional studies were performed to identify the pathogenic variants. Trio whole-exome sequencing revealed compound heterozygous splice variants in DAB1, both affecting the highly conserved functional phosphotyrosine-binding domain. Expression studies in patient-derived cells showed loss of normal transcripts, confirming pathogenicity.ConclusionsWe conclude that these variants are very likely causally related to the cerebral phenotype and propose to consider loss-of-function DAB1 variants in patients with RELN-like cortical malformations.

Aberrant oligodendroglial LDL receptor orchestrates demyelination in chronic cerebral ischemia.

Oligodendrocytes express low-density lipoprotein receptor (LDLR) to endocytose cholesterol for the maintenance of adulthood myelination. However, the potential role of LDLR in chronic cerebral ischemia-related demyelination remains unclear. We used bilateral carotid artery stenosis (BCAS) to induce sustained cerebral ischemia in mice. This hypoxic-ischemic injury caused a remarkable decrease in oligodendroglial LDLR, with impaired oligodendroglial differentiation and survival. Oligodendroglial cholesterol levels, however, remained unchanged. Mouse miR-344e-3p and the human homolog miR-410-3p, 2 miRNAs directly targeting Ldlr, were identified in experimental and clinical leukoaraiosis and were thus implicated in the LDLR reduction. Lentiviral delivery of LDLR ameliorated demyelination following chronic cerebral ischemia. By contrast, Ldlr-/- mice displayed inadequate myelination in the corpus callosum. Ldlr-/- oligodendrocyte progenitor cells (OPCs) exhibited reduced ability to differentiate and myelinate axons in vitro. Transplantation with Ldlr-/- OPCs could not rescue the BCAS-induced demyelination. Such LDLR-dependent myelin restoration might involve a physical interaction of the Asn-Pro-Val-Tyr (NPVY) motif with the phosphotyrosine binding domain of Shc, which subsequently activated the MEK/ERK pathway. Together, our findings demonstrate that the aberrant oligodendroglial LDLR in chronic cerebral ischemia impairs myelination through intracellular signal transduction. Preservation of oligodendroglial LDLR may provide a promising approach to treat ischemic demyelination.

PTB domain and leucine zipper motif 1 (APPL1) inhibits myocardial ischemia/hypoxia-reperfusion injury via inactivation of apoptotic protease activating factor-1 (APAF-1)/Caspase9 signaling pathway

ABSTRACT Myocardial ischemia/hypoxia-reperfusion injury mediates the progression of multiple cardiovascular diseases. It has been reported that knockdown of adaptor protein containing a PH domain, PTB domain and leucine zipper motif 1 (APPL1) is a significant factor for the progression of myocardial injury. However, the role of APPL1 in myocardial ischemia remains unclear. Hence, the aim of the present study was to investigate the specific mechanism underlying the role of APPL1 in myocardial ischemia. In our study, the mRNA level of APPL1 was detected by quantitative real-time PCR (RT-qPCR). The expressions of APPL1, Apoptotic protease activating factor-1 (APAF-1), cleaved caspase9 and other inflammation- and apoptosis-related proteins were determined by western blotting. The secretion of inflammatory cytokines and lactate dehydrogenase (LDH) levels were measured by commercial assay kits. The H9C2 cell viability was analyzed by cell counting kit-8 (CCK-8) assay. The apoptosis rate of H9C2 cells was analyzed by TUNEL assay. The interaction between APPL1 and APAF-1/caspase9 was determined by Immunoprecipitation (IP). Our findings demonstrated that APPL1 was low expressed in myocardial ischemia tissues and cells. APPL1 knockdown suppressed the viability of myocardial ischemia cells and aggravated hypoxia/reperfusion-induced LDH hypersecretion, inflammation and apoptosis. In addition, the overexpression of APPL1 induced inactivation of APAF-1/Caspase9 signaling pathway. Significantly, APAF1 inhibitor reversed the effect of APPL1 knockdown on viability, LDH secretion, inflammation and apoptosis. We conclude that APPL1 inhibits myocardial ischemia/hypoxia-reperfusion injury via inactivation of APAF-1/Caspase9 signaling pathway. Hence, APPL1 may be a novel and effective target for the treatment of myocardial ischemia.

Crystal Structure of Human APPL BAR-PH Heterodimer Reveals a Flexible Dimeric BAR curve: Implication in Mutual Regulation of Endosomal Targeting.

The APPL (adaptor proteins containing pleckstrin homology domain, phosphotyrosine binding domain and a leucine zipper motif) family consists of two isoforms, APPL1 and APPL2. By binding to curved plasma membrane, these adaptor proteins associate with multiple transmembrane receptors and recruit various downstream signaling components. They are involved in the regulation of signaling pathways evoked by a variety of extracellular stimuli, such as adiponectin, insulin, FSH (follicle stimulating hormone), EGF (epidermal growth factor). And they play important roles in cell proliferation, apoptosis, glucose uptake, insulin secretion and sensitivity. However, emerging evidence suggests that APPL1 and APPL2 perform different or even opposite functions and the underlying mechanism remains unclear. As APPL proteins can either homodimerize or heterodimerize in vivo, we hypothesized that heterodimerization of APPL proteins might account for the mechanism. By solving the crystal structure of APPL1-APPL2 BAR-PH heterodimer, we find that the overall structure is crescent-shaped with a longer curvature radius of 76 Å, compared to 55 Å of the APPL1 BAR-PH homodimer. However, there is no significant difference of the curvature between APPL BAR-PH heterodimer and APPL2 homodimer. The data suggest that the APPL1 BAR-PH homodimer, APPL2 BAR-PH homodimer and APPL1/APPL2 BAR-PH heterodimer may bind to endosomes of different sizes. Different positive charge distribution is observed on the concave surface of APPL BAR-PH heterodimer than the homodimers, which may change the affinity of membrane association and subcellular localization. Collectively, APPL2 may regulate APPL1 function through altering the preference of endosome binding by heterodimerization.

TBC1D1 interacting proteins, VPS13A and VPS13C, regulate GLUT4 homeostasis in C2C12 myotubes

Proteins involved in the spaciotemporal regulation of GLUT4 trafficking represent potential therapeutic targets for the treatment of insulin resistance and type 2 diabetes. A key regulator of insulin- and exercise-stimulated glucose uptake and GLUT4 trafficking is TBC1D1. This study aimed to identify proteins that regulate GLUT4 trafficking and homeostasis via TBC1D1. Using an unbiased quantitative proteomics approach, we identified proteins that interact with TBC1D1 in C2C12 myotubes including VPS13A and VPS13C, the Rab binding proteins EHBP1L1 and MICAL1, and the calcium pump SERCA1. These proteins associate with TBC1D1 via its phosphotyrosine binding (PTB) domains and their interactions with TBC1D1 were unaffected by AMPK activation, distinguishing them from the AMPK regulated interaction between TBC1D1 and AMPKα1 complexes. Depletion of VPS13A or VPS13C caused a post-transcriptional increase in cellular GLUT4 protein and enhanced cell surface GLUT4 levels in response to AMPK activation. The phenomenon was specific to GLUT4 because other recycling proteins were unaffected. Our results provide further support for a role of the TBC1D1 PTB domains as a scaffold for a range of Rab regulators, and also the VPS13 family of proteins which have been previously linked to fasting glycaemic traits and insulin resistance in genome wide association studies.

Nonphagocytic Activation of NOX2 Is Implicated in Progressive Nonalcoholic Steatohepatitis During Aging.

Older patients with obesity/type II diabetes mellitus frequently present with advanced NASH. Whether this is due to specific molecular pathways that accelerate fibrosis during aging is unknown. Activation of the Src homology 2 domain-containing collagen-related (Shc) proteins and redox stress have been recognized in aging; however, their link to NASH has not been explored. Shc expression increased in livers of older patients with NASH, as assessed by real time quantitative PCR (RT-qPCR) or western blots. Fibrosis, Shc expression, markers of senescence, and nicotinamide adenine dinucleotide phosphate, reduced form oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice, lentiviral (LV)-short hairpin Shc versus control-LV were used during FFD. For hepatocyte-specific effects, floxed (fl/fl) Shc mice on FFD were injected with adeno-associated virus 8-thyroxine-binding globulin-Cre-recombinase versus control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation, and fibrosis. To study NOX2 activation, the interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation and coimmunoprecipitations. Palmitate-induced p52Shc binding to p47phox , activating the NOX2 complex, more so at an older age. Kinetics of binding were assessed in Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) domain deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47phox interaction using RosettaDock was generated. Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit that results in redox stress and accelerated fibrosis in the aged.

RGS12 Is a Novel Critical NF-κB Activator in Inflammatory Arthritis.

SummaryRheumatoid arthritis (RA) is the most common inflammatory disease, which currently lacks effective treatment. Here, we discovered that the Regulator of G Protein Signaling 12 (RGS12) plays a key role in regulating inflammation. Transcriptional and protein analysis revealed that RGS12 was upregulated in human and mouse RA macrophages. Deletion of RGS12 in myeloid lineage or globally inhibits the development of collagen-induced arthritis including joint swelling and bone destruction. Mechanistically, RGS12 associates with NF-κB(p65) to activate its phosphorylation and nuclear translocation through PTB domain, and NF-κB(p65) regulates RGS12 expression in a transcriptional manner. The nuclear translocation ability of NF-κB(p65) and RGS12 can both be enhanced by cyclooxygenase-2 (COX2). Furthermore, ablation of RGS12 via RNA interference significantly blocks the inflammatory process in vivo and in vitro. These results demonstrate that RGS12 plays a critical role in the pathogenesis of inflammatory arthritis.

SUN-687 MODY-14: A Rare Cause of Inherited Diabetes

Case: A 29-year-old physically active, lean man presented with a three-year history of poorly controlled diabetes diagnosed during a flu-like illness and no history diabetic ketoacidosis. Despite treatment with various oral hypoglycemic medications his glycated hemoglobin was consistently above target (never falling below 9.3%) and correlated with home blood glucose readings. His mother had gestational diabetes with all three of her pregnancies. His maternal grandmother was also known to have diabetes. Pancreatic islet antibodies were negative. C-peptide levels were low. Monogenic diabetes (MODY) was suspected. Discussion: Genetic testing revealed a mutation in the Adaptor Protein, Phosphotyrosine Interaction, PH domain, and leucine zipper containing 1 (APPL1) gene. APPL1 is involved in insulin secretion and insulin signaling via the Akt pathway [1,2,3]. Other mutations of APPL1 causing MODY-14 have been previously described [4]. In our case, the substitution of glutamate for aspartate at the 265 position occurs within the unique fourth alpha helix of the BAR domain of APPL1 [5]. Generally, BAR domains contribute to APPL1 dimerization and plasma membrane association [5]. Substitution of glutamate could disrupt protein folding, impairing dimerization and phosphorylation of Akt, thereby decreased insulin secretion as seen in other mutations of the BAR domain of APPL1 [4]. Although this remains a hypothesis, loss-of-function of APPL1 would explain the lack of insulin secretion without evidence of pancreatic autoimmunity. If the hypothesis is correct, it would be a novel mutation attributable to this MODY subtype. 1. Cheng KKY, Lam KSL, Wu D, et al. APPL1 potentiates insulin secretion in pancreatic β cells by enhancing protein kinase Akt-dependent expression of SNARE proteins in mice. Proc Natl Acad Sci U S A. 2012;109(23):8919–8924. doi:10.1073/pnas.1202435109 2. Ryu J, Galan AK, Xin X, et al. APPL1 potentiates insulin sensitivity by facilitating the binding of IRS1/2 to the insulin receptor. Cell Rep. 2014;7(4):1227–1238. doi:10.1016/j.celrep.2014.04.006 3. Saito T, Jones CC, Huang S, Czech MP, Pilch PF. The interaction of Akt with APPL1 is required for insulin-stimulated Glut4 translocation. J Biol Chem. 2007;282(44):32280–32287. doi:10.1074/jbc.M704150200 4. Prudente S, Jungtrakoon P, Marucci A, et al. Loss-of-Function Mutations in APPL1 in Familial Diabetes Mellitus. Am J Hum Genet. 2015;97(1):177–185. doi:10.1016/j.ajhg.2015.05.011 5. Li J, Mao X, Dong LQ, Liu F, Tong L. Crystal Structures of the BAR-PH and PTB Domains of Human APPL1. Structure. 2007;15(5):525–533. doi:10.1016/j.str.2007.03.011

APPL1 as an important regulator of insulin and adiponectin-signaling pathways in the PCOS: A narrative review.

Adaptor protein containing a PH domain, PTB domain and leucine zipper motif 1 (APPL1) plays a central role as the main contributing factor in the adiponectin and insulin signaling. This review aims to discuss previous and recent findings concerning the role of APPL1 in the polycystic ovary syndrome (PCOS) patients with conclusions regarding more efficient therapeutic approaches. A literature review was performed in PubMed, Web of Science, ScienceDirect, Scopus, and Google Scholar from August 1999 to May 2020. This study reveals that APPL1 has a key role in adiponectin, insulin, and follicle-stimulating hormone signaling pathways occurring within the ovaries. Recent studies in mouse model systems have indicated that APPL1 can prevent diabetes, endothelial disorders, and insulin resistance. In contrast, APPL1 deficiency can lead to the metabolic and vascular disorders. APPL1 due to its potential roles in different signaling pathways might be suggested as a novel diagnostic and therapeutic option for prediction of ovarian dysfunctions and treatment of reproductive disorders, especially PCOS.

Redefining PTB domain into independently functional dual cores

Current understanding of phosphotyrosine binding (PTB) domain is limited. Recently, we revealed a novel atypical phosphotyrosine binding (aPTB) domain in CCM2, making it a dual PTB domain-containing protein. Since aPTB domain is only 1/3 of the size of typical PTB domain, we explored the possibility to decrease the size of PTB domain and demonstrate that the typical PTB domain can be divided into two similarly structural and functional cores that can independently bind to NPXY motif. Further, we reduced each PTB core into a minimum core motif (mCore) which is the functional unit of PTB domains and structurally similar to the novel aPTB domain. Based on structural data, we developed several cis- and trans-inhibitors for NPXY binding scheme, with potential applications for therapeutic strategies in human health conditions.

A SYK/SHC1 pathway regulates the amount of CFTR in the plasma membrane.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the recessive genetic disease cystic fibrosis, where the chloride transport across the apical membrane of epithelial cells mediated by the CFTR protein is impaired. CFTR protein trafficking to the plasma membrane (PM) is the result of a complex interplay between the secretory and membrane recycling pathways that control the number of channels present at the membrane. In addition, the ion transport activity of CFTR at the PM is modulated through post-translational protein modifications. Previously we described that spleen tyrosine kinase (SYK) phosphorylates a specific tyrosine residue in the nucleotide-binding domain 1 domain and this modification can regulate the PM abundance of CFTR. Here we identified the underlying biochemical mechanism using peptide pull-down assays followed by mass spectrometry. We identified in bronchial epithelial cells that the adaptor protein SHC1 recognizes tyrosine-phosphorylated CFTR through its phosphotyrosine-binding domain and that the formation of a complex between SHC1 and CFTR is induced at the PM in the presence of activated SYK. The depletion of endogenous SHC1 expression was sufficient to promote an increase in CFTR at the PM of these cells. The results identify a SYK/SHC1 pathway that regulates the PM levels of CFTR channels, contributing to a better understanding of how CFTR-mediated chloride secretion is regulated.

Biochemical characterization of TRIM72 E3 ligase and its interaction with the insulin receptor substrate 1

TRIM family of E3 ubiquitin ligases have an amino-terminal conserved tripartite motif consisting of RING, B-Box, coiled-coil domain and different C-terminal domain leading it to classification into 11 subclasses. TRIM72 is an E3 ligase of class IV and subclass 1 with its role in a multitude of cellular processes. Despite being crucial in multiple cellular processes, TRIM72 still hasn't been biochemically characterized. In the present study, we have characterized the oligomeric status of TRIM72 and found that it forms both monomers, dimers, and tetramers. We have screened a set of 12 E2s and identified two novel E2 enzymes (Ubch5c and Ubch10) that work in cooperation with TRIM72. Nevertheless, E3 ligase activity is minimal and we propose that additional regulation is required to enhance its E3 ligase activity. We have also used surface plasmon resonance to study interaction with one of its substrate proteins, IRS1, and identified the PH domain of IRS1 is mediating interaction with the TRIM72 E3 ligase while the PTB domain of IRS1, does not show any interaction.

Synthesis of high-vinyl isoprene and styrene triblock copolymers via anionic polymerization with difunctional t-BuLi initiator

Triblock copolymers poly(styrene)-block-poly(isoprene)-block-poly(styrene) (PS-b-PI-b-PS, SIS) and poly(isoprene)-block-poly(styrene)-block-poly(isoprene) (PI-b-PS-b-PI, ISI) with different vinyl structure contents were successfully synthesized by anionic polymerization using a difunctional t-BuLi initiator. The difunctional t-BuLi initiator was prepared from tert-butyllithium (t-BuLi) and 1,3-diisopropenylbenzene, and characterized by 1H NMR, 13C NMR, DEPT135, 7Li NMR, and GC–MS analysis. According to the results of 1H NMR, 13C NMR, and 2D HSQC analysis, the contents of 1,2- and 3,4-addition (vinyl group) structures for PI in SIS and ISI were in the range of 3.7%–94.6%, and depended on the reaction temperature and amount of tetrahydrofuran (THF) added. The Mn and PDI of SIS and ISI obtained by GPC were in the ranges of 13,400–281,000 Da and 1.09–1.24, respectively. The presence of two glass transition temperatures (Tg) on the DSC curve indicated microphase separation of the PI and PS domains. The low Tg was attributed to the PI domain, which was correlated with the vinyl group content in the block copolymer. For vinyl group contents of 6.9% and 94.6% in the SIS triblock copolymer, and 6.6% and 94.2% in the ISI triblock copolymer, the corresponding Tg values of the PI block were −64 °C and 12 °C, and −62 °C and 17 °C, respectively.

Rabgap1 promotes recycling of active β1 integrins to support effective cell migration.

Integrin function depends on the continuous internalization of integrins and their subsequent endosomal recycling to the plasma membrane to drive adhesion dynamics, cell migration and invasion. Here we assign a pivotal role for Rabgap1 (GAPCenA) in the recycling of endocytosed active β1 integrins to the plasma membrane. The phosphotyrosine-binding (PTB) domain of Rabgap1 binds to the membrane-proximal NPxY motif in the cytoplasmic domain of β1 integrin subunits on endosomes. Silencing Rabgap1 in mouse fibroblasts leads to the intracellular accumulation of active β1 integrins, alters focal adhesion formation, and decreases cell migration and cancer cell invasion. Functionally, Rabgap1 facilitates active β1 integrin recycling to the plasma membrane through attenuation of Rab11 activity. Taken together, our results identify Rabgap1 as an important factor for conformation-specific integrin trafficking and define the role of Rabgap1 in β1-integrin-mediated cell migration in mouse fibroblasts and breast cancer cells.

Elucidating the expression and function of Numbl during cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma (MM)

BackgroundCell adhesion-mediated drug resistance (CAM-DR) is a major clinical problem that prevents successful treatment of multiple myeloma (MM). In particular, the expression levels of integrin β1 and its sub-cellular distribution (internalization and trafficking) are strongly associated with CAM-DR development.MethodsDevelopment of an adhesion model of established MM cell lines and detection of Numbl and Integrinβ1 expression by Western Blot analysis. The interaction between Numbl and Integrinβ1 was assessed by a co-immunoprecipitation (CO-IP) method. Calcein AM assay was performed to investigate the levels of cell adhesion. Finally, the extent of CAM-DR in myeloma cells was measured using cell viability assay and flow cytometry analysis.ResultsOur preliminary date suggest that Numbl is differentially expressed in a cell adhesion model of MM cell lines. In addition to binding to the phosphotyrosine-binding (PTB) domain, the carboxyl terminal of Numbl can also interact with integrin β1 to regulate the cell cycle by activating the pro-survival PI3K/AKT signaling pathway. This study intends to verify and elucidate the interaction between Numbl and integrin β1 and its functional outcome on CAM-DR. We have designed and developed a CAM-DR model using MM cells coated with either fibronectin or bone marrow stromal cells. We assessed whether Numbl influences cell-cycle progression and whether it, in turn, contributes to activation of PI3K/AKT signal pathway through the adjustment of its carboxyl end. Finally, we showed that the interaction of Numbl with integrin β1 promotes the formation of CAM-DR in MM cells.ConclusionsOur findings elucidated the specific molecular mechanisms of CAM-DR induction and confirmed that Numbl is crucial for the development of CAM-DR in MM cells.

Multiple Origins and Specific Evolution of CRISPR/Cas9 Systems in Minimal Bacteria (Mollicutes).

CRISPR/Cas systems provide adaptive defense mechanisms against invading nucleic acids in prokaryotes. Because of its interest as a genetic tool, the Type II CRISPR/Cas9 system from Streptococcus pyogenes has been extensively studied. It includes the Cas9 endonuclease that is dependent on a dual-guide RNA made of a tracrRNA and a crRNA. Target recognition relies on crRNA annealing and the presence of a protospacer adjacent motif (PAM). Mollicutes are currently the bacteria with the smallest genome in which CRISPR/Cas systems have been reported. Many of them are pathogenic to humans and animals (mycoplasmas and ureaplasmas) or plants (phytoplasmas and some spiroplasmas). A global survey was conducted to identify and compare CRISPR/Cas systems found in the genome of these minimal bacteria. Complete or degraded systems classified as Type II-A and less frequently as Type II-C were found in the genome of 21 out of 52 representative mollicutes species. Phylogenetic reconstructions predicted a common origin of all CRISPR/Cas systems of mycoplasmas and at least two origins were suggested for spiroplasmas systems. Cas9 in mollicutes were structurally related to the S. aureus Cas9 except the PI domain involved in the interaction with the PAM, suggesting various PAM might be recognized by Cas9 of different mollicutes. Structure of the predicted crRNA/tracrRNA hybrids was conserved and showed typical stem-loop structures pairing the Direct Repeat part of crRNAs with the 5′ region of tracrRNAs. Most mollicutes crRNA/tracrRNAs showed G + C% significantly higher than the genome, suggesting a selective pressure for maintaining stability of these secondary structures. Examples of CRISPR spacers matching with mollicutes phages were found, including the textbook case of Mycoplasma cynos strain C142 having no prophage sequence but a CRISPR/Cas system with spacers targeting prophage sequences that were found in the genome of another M. cynos strain that is devoid of a CRISPR system. Despite their small genome size, mollicutes have maintained protective means against invading DNAs, including restriction/modification and CRISPR/Cas systems. The apparent lack of CRISPR/Cas systems in several groups of species including main pathogens of humans, ruminants, and plants suggests different evolutionary routes or a lower risk of phage infection in specific ecological niches.