Adipose Tissue-Derived Stem Cell Extracellular Vesicles Suppress Glioblastoma Proliferation, Invasiveness and Angiogenesis.

Extracellular vesicles (EVs) are attractive anticancer drug delivery candidates as they confer several fundamental properties, such as low immunogenicity and the ability to cross biological barriers. Mesenchymal stem cells (MSCs) are convenient producers for high EV yields, and patient-derived adipose tissue MSC-EVs could serve as personalised carriers. However, MSC-EV applications raise critical concerns as their natural cargo can affect tumour progression in both inducing and suppressing ways. In this study, we investigated the effect of adipose tissue-derived mesenchymal stem cell EVs (ASC-EVs) on several glioblastoma (GBM) cell lines to define their applicability for anticancer therapies. ASC-EVs were isolated from a cell-conditioned medium and characterised by size and specific markers. The internalisation of fluorescently labelled ASC-EVs by human GBM cells HROG36, U87 MG, and T98G was evaluated by fluorescent microscopy. Changes in GBM cell proliferation after ASC-EV application were determined by the metabolic PrestoBlue assay. Expression alterations in genes responsible for cell adhesion, proliferation, migration, and angiogenesis were evaluated by quantitative real-time PCR. ASC-EV effects on tumour invasiveness and neoangiogenesis in ovo were analysed on the chicken embryo chorioallantoic membrane model (CAM). ASC-EV treatment reduced GBM proliferation in vitro and significantly downregulated invasiveness-related genes ITGα5 (in T98G and HROG63) and ITGß3 (in HROG36) and the vascularisation-inducing gene KDR (in all GBM lines). Additionally, an approximate 65% reduction in the GBM invasion rate was observed in CAM after ASC-EV treatment. Our study indicates that ASC-EVs possess antitumour properties, reducing GBM cell proliferation and invasiveness, and can be applied as anticancer therapeutics and medicine carriers.

Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most aggressive CNS tumour with no efficient treatment, partly due to the retention of anticancer drugs by the blood-brain barrier (BBB) and their insufficient concentration in tumour cells. Extracellular vesicles (EVs) are attractive drug carriers because of their biocompatibility and ability to cross the BBB. Additional efficiency can be achieved by adding GBM-cell-specific ligands. GBM cells overexpress integrins; thus, one of the most straightforward targeting strategies is to modify EVs with integrin-recognising molecules. This study investigated the therapeutic potential of genetically engineered EVs with elevated membrane levels of the integrin-binding peptide RGD (RGD-EVs) against GBM cells in vitro. For RGD-EV production, stable RGD-HEK 293FT cells were generated by using a pcDNA4/TO-Lamp2b-iRGD-HA expression vector and performing antibiotic-based selection. RGD-EVs were isolated from RGD-HEK 293FT-cell-conditioned medium and characterised by size (Zetasizer), specific markers (ELISA) and RGD expression (Western Blot). Internalisation by human GBM cells HROG36 and U87 MG and BJ-5ta human fibroblasts was assessed by fluorescent EV RNA labelling. The effect of doxorubicin-loaded RGD-EVs on GBM cells was evaluated by the metabolic PrestoBlue viability assay; functional GAPDH gene knockdown by RGD-EV-encapsulated siRNA was determined by RT-qPCR. RGD-EVs had 40% higher accumulation in GBM cells (but not in fibroblasts) and induced significantly stronger toxicity by loaded doxorubicin and GAPDH silencing by loaded siRNA compared to unmodified EVs. Thus, RGD modification substantially increases the specific delivery capacity of HEK 293FT-derived EVs to GBM cells.

A SEMA3 Signaling Pathway-Based Multi-Biomarker for Prediction of Glioma Patient Survival.

Glioma is a lethal central nervous system tumor with poor patient survival prognosis. Because of the molecular heterogeneity, it is a challenge to precisely determine the type of the tumor and to choose the most effective treatment. Therefore, novel biomarkers are essential to improve the diagnosis and prognosis of glioma tumors. Class 3 semaphorin proteins (SEMA3) play an important role in tumor biology. SEMA3 transduce their signals by using neuropilin and plexin receptors, which functionally interact with the vascular endothelial growth factor-mediated signaling pathways. Therefore, the aim of this study was to explore the potential of SEMA3 signaling molecules for prognosis of glioma patient survival. The quantitative real-time PCR method was used to evaluate mRNA expression of SEMA3(A-G), neuropilins (NRP1 and NRP2), plexins (PLXNA2 and PLXND1), cadherins (CDH1 and CDH2), integrins (ITGB1, ITGB3, ITGA5, and ITGAV), VEGFA and KDR genes in 59 II-IV grade glioma tissues. Seven genes significantly associated with patient overall survival were used for multi-biomarker construction, which showed 64%, 75%, and 68% of accuracy of predicting the survival of 1-, 2-, and 3-year glioma patients, respectively. The results suggest that the seven-gene signature could serve as a novel multi-biomarker for more accurate prognosis of a glioma patient's outcome.

The Anti-Tumorigenic Activity of Sema3C in the Chick Embryo Chorioallantoic Membrane Model.

Sema3C protein, a member of the class 3 family of secreted semaphorins, play an important role in tumor development by regulating cell proliferation, migration, invasion, and angiogenesis processes. Depending on the type and malignancy grade of the tumor, Sema3C function remains controversial. In this study, we constructed a stably overexpressing Sema3C glioblastoma cell line U87 MG and tested it on the chicken embryo chorioallantoic membrane (CAM) model with the aim to reveal Sema3C protein function on angiogenesis process in ovo. Our experiments showed that Sema3C not only affects angiogenesis of CAM by inhibiting neovascularization but also acts as an anti-tumorigenic molecule by hampering U87 MG cell invasion into mesenchyme. The effects of Sema3C on CAM were similar to the effects of anti-epileptic drug sodium valproate (NaVP). Both, anti-angiogenic and anti-tumorigenic activities of Sema3C were enhanced by the treatment of NaVP and, importantly, were not attributed to the cytotoxic effects. Our studies suggest that Sema3C could be a promising target for glioblastoma treatment.

Oncosuppressive Role of RUNX3 in Human Astrocytomas.

BACKGROUND: Gliomas are the most common and aggressive among primary malignant brain tumours with significant inter- and intratumour heterogeneity in histology, molecular profile, and patient outcome. However, molecular targets that could provide reliable diagnostic and prognostic information on this type of cancer are currently unknown. Recent studies show that certain phenotypes of gliomas such as malignancy, resistance to therapy, and relapses are associated with the epigenetic alterations of tumour-specific genes. Runt-related transcription factor 3 (RUNX3) is feasible tumour suppressor gene since its inactivation was shown to be related to carcinogenesis. AIM: The aim of the study was to elucidate RUNX3 changes in different regulation levels of molecular biology starting from epigenetics to function in particular cases of astrocytic origin tumours of different grade evaluating significance of molecular changes of RUNX3 for patient clinical characteristics as well as evaluate RUNX3 reexpression effect to GBM cells. METHODS: The methylation status and protein expression levels of RUNX3 were measured by methylation-specific PCR and Western blot in 136 and 72 different malignancy grade glioma tissues, respectively. Lipotransfection and MTT were applied for proliferation assessment in U87-MG cells. RESULTS: We found that RUNX3 was highly methylated and downregulated in GBM. RUNX3 promoter methylation was detected in 69.4% of GBM (n=49) as compared to 0 to 17.2% in I-III grade astrocytomas (n=87). Weighty lower RUNX3 protein level was observed in GMB specimens compared to grade II-III astrocytomas. Correlation test revealed a weak but significant link among Runx3 methylation and protein level. Kaplan-Meier analysis showed that increased RUNX3 methylation and low protein level were both associated with shorter patient survival (p<0.05). Reexpression of RUNX3 in U87-MG cells significantly reduced glioma cell viability compared to control transfection. CONCLUSIONS: The results demonstrate that RUNX3 gene methylation and protein expression downregulation are glioma malignancy dependent and contribute to tumour progression.

Significance of Amphiregulin (AREG) for the Outcome of Low and High Grade Astrocytoma Patients.

Background: Amphiregulin (AREG) is one of the ligands of the epidermal growth factor receptor which levels was shown to have a tight coherence with various types of cancer. AREG was also designated to be a promising marker for several types of cancer however precious little data about AREG role in the most frequent and generally lethal human brain tumours - astrocytomas reported up to date. The aim of the study was to investigate how AREG changes at epigenetic and expression levels reflect on astrocytoma malignancy and patient outcome. Methods: In total 205 low and high grade astrocytoma samples (15 pilocytic astrocytomas, 56 diffuse astrocytomas, 32 anaplastic astrocytomas and 102 glioblastomas) were used for target mRNA, protein expression and DNA methylation analysis applying qRT-PCR, Western-Blot and MS-PCR assays, respectively. Results: Present research revealed that AREG expression level and methylation in cancer tissue is dependent on the grade of astrocytoma. GBM tissue disclosed elevated AREG mRNA expression but reduced AREG protein level as compared to grade II and grade III astrocytomas (p<0.001). Increased methylation frequency was also more abundant in GBM (74%) than grade I, II and III astrocytomas (25%, 34%, and 36%, respectively). The survival analysis revealed relevant differences in patient overall survival between AREG methylation, mRNA and protein expression groups. Kaplan-Meier analysis encompassing only malignant tumours showed similar results indicating that AREG is associated with astrocytoma patient survival independently from astrocytoma grade. Conclusions: Current findings demonstrate that AREG appearance is associated with patient survival as well as astrocytomas malignancy indicating its influence on tumour progression and suggest its applicability as a promising marker.

Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy.

BACKGROUND: The cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/prodrug systems in the field of the suicide gene therapy. Recently, by the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of specifically converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). The aim of this study is to test the applicability of the ICD Vcz / 5-FIC pair as a potential suicide gene therapy tool. METHODS: Vcz-expressing human glioblastoma U87 and epithelial colorectal adenocarcinoma Caco-2 cells were treated with 5-FIC, and the Vcz-mediated cytotoxicity was evaluated by performing an MTT assay. In order to examine anti-tumor effects of the Vcz/5-FIC system in vivo, murine bone marrow-derived mesenchymal stem cells (MSC) were transduced with the Vcz-coding lentivirus and co-injected with 5-FIC or control reagents into subcutaneous GL261 tumors evoked in C57/BL6 mice. RESULTS: 5-FIC alone showed no significant toxic effects on U87 and Caco-2 cells at 100 µM concentration, whereas the number of cells of both cell lines that express Vcz cytosine deaminase gene decreased by approximately 60% in the presence of 5-FIC. The cytotoxic effects on cells were also induced by media collected from Vcz-expressing cells pre-treated with 5-FIC. The co-injection of the Vcz-transduced mesenchymal stem cells and 5-FIC have been shown to augment tumor necrosis and increase longevity of tumorized mice by 50% in comparison with control group animals. CONCLUSIONS: We have confirmed that the novel ICD Vcz together with the non-toxic prodrug 5-FIC has a potential of being a new enzyme/prodrug system for suicide gene therapy.

The Effect of Sodium Valproate on the Glioblastoma U87 Cell Line Tumor Development on the Chicken Embryo Chorioallantoic Membrane and on EZH2 and p53 Expression.

Literature data support evidences that glioblastoma (GBM) patients experience prolonged survival due to sodium valproate (NaVP) treatment. The study assessed the human GBM cell U87 xenograft studied in the chicken embryo chorioallantoic membrane (CAM) model evaluating NaVP effect on tumor. Three groups of tumors (each n = 10) were studied: nontreated, treated with 4 mM, and treated with 8 mM of NaVP. The majority of tumors without NaVP treatment during tumor growth destroyed the chorionic epithelium, invaded the mesenchyme, and induced angiogenesis. Incidence of tumor formation on CAM without invasion into the mesenchyme was higher when U87 cells were treated with NaVP; the effect significantly increased with NaVP concentration. Treatment with 8 mM of NaVP did not show clear dynamics of tumor growth during 5 days; at the same time, the angiogenesis failed. With a strong staining of EZH2, p53 in tumors without NaVP treatment was found, and NaVP significantly decreased the expression of EZH2- and p53-positive cells; the effect was significantly higher at its 8 mM concentration. NaVP has a function in blocking the growth, invasion, and angiogenesis of tumor in the CAM model; tumor growth interferes with EZH2 and p53 molecular pathways, supporting the NaVP potential in GBM therapy.

Testin (TES) as a candidate tumour suppressor and prognostic marker in human astrocytoma.

Astrocytomas are one of the most common brain tumours; however, the current methods used to characterize these tumours are inadequate. The establishment of molecular markers may identify variables required to improve tumour characterization and subtyping, and may aid to specify targets for improved treatment with essential prognostic value for patient survival. One such candidate is testin (TES), which was reported to have prognostic value for glioblastoma patients. However, the role of TES protein in gliomagenesis is currently unknown. In the present study, the methylation status of the TES promoter was investigated in post-operative astrocytoma tumours of different malignancy grade, and its association with the survival of astrocytoma patients was evaluated. In addition, the expression of TES protein was investigated in the same set of astrocytoma tumours tissue, and the association of protein expression with glioma patients survival was evaluated. The methylation status of TES was assessed by methylation-specific polymerase chain reaction in 138 different grade astrocytoma samples. Western blot analysis was used to characterize the expression pattern of TES in 86 different grade astrocytoma specimens: 13 of pathological grade I, 31 of pathological grade II, 17 of pathological grade III and 25 of pathological grade IV (glioblastoma). Statistical analyses were conducted to investigate the association between tumour molecular pattern, patient clinical variables and overall survival. The methylation analysis of the TES promoter exhibited a distinct profile between astrocytomas of different malignancy grade (P<0.001). Furthermore, gene promoter methylation was significantly associated with patients' age, survival and pathological grade (P<0.001). The protein expression level of TES was significantly lower in glioblastoma (grade IV astrocytoma) than in lower grade (II-III) astrocytoma tissue (P=0.028 and P=0.04, respectively). Additionally, short overall survival of patients was markedly associated with low TES protein expression (P=0.007). However, no association between TES methylation and TES protein expression was noticed. The present study demonstrated that decreased expression of TES may be important in tumour progression and prognosis in human astrocytomas. TES may be a useful marker for predicting the clinical outcome of astrocytoma patients.

Large-Scale Screening of Preferred Interactions of Human Src Homology-3 (SH3) Domains Using Native Target Proteins as Affinity Ligands.

The Src Homology-3 (SH3) domains are ubiquitous protein modules that mediate important intracellular protein interactions via binding to short proline-rich consensus motifs in their target proteins. The affinity and specificity of such core SH3 - ligand contacts are typically modest, but additional binding interfaces can give rise to stronger and more specific SH3-mediated interactions. To understand how commonly such robust SH3 interactions occur in the human protein interactome, and to identify these in an unbiased manner we have expressed 324 predicted human SH3 ligands as full-length proteins in mammalian cells, and screened for their preferred SH3 partners using a phage display-based approach. This discovery platform contains an essentially complete repertoire of the ∼300 human SH3 domains, and involves an inherent binding threshold that ensures selective identification of only SH3 interactions with relatively high affinity. Such strong and selective SH3 partners could be identified for only 19 of these 324 predicted ligand proteins, suggesting that the majority of human SH3 interactions are relatively weak, and thereby have capacity for only modest inherent selectivity. The panel of exceptionally robust SH3 interactions identified here provides a rich source of leads and hypotheses for further studies. However, a truly comprehensive characterization of the human SH3 interactome will require novel high-throughput methods based on function instead of absolute binding affinity.

High CHI3L1 expression is associated with glioma patient survival.

BACKGROUND: Survival of glioma patients with the same tumor histology and grade can vary significantly, and some low-grade gliomas transform to a more malignant phenotype. There is a need of molecular signatures, which are better predictors of the patient diagnosis, outcome of treatment, and prognosis than the diagnosis provided by histopathology. We propose CHI3L1 mRNA expression as a prognostic biomarker for patients with glioma. METHODS: We measured CHI3L1 expression with quantitative real time-polymerase chain reaction (qRT-PCR) in the cohort of 98 patients with different grade glioma: 10 grade I pylocytic astrocytomas, 30 grade II diffuse astrocytomas, 20 grade III anaplastic astrocytomas, and 38 grade IV astrocytomas (glioblastomas). Statistical analyses were conducted to investigate the association between CHI3L1 mRNA expression levels and patient clinical variables. RESULTS: We demonstrated that mRNA expression of CHI3L1 was evidently higher in glioblastoma than in lower grade glioma tissues. We evaluated correlations between CHI3L1 expression, clinicopathological characteristics, and the outcomes of the patients. Patients with high CHI3L1 expression had a shorter overall survival (p < 0.001). CONCLUSIONS: Findings presented in our study showed that increased mRNA level of CHI3L1 could be associated with the progression of astrocytoma and poor patient survival not only for glioblastoma, but for lower grade astrocytoma tumors as well. Further investigation will be required to evaluate CHI3L1 value as a molecular marker for astrocytoma prognoses and for novel treatment strategies against all grade astrocytomas.

High level of Sema3C is associated with glioma malignancy.

BACKGROUND: Malignant gliomas are characterized by the tendency of cancerous glial cells to infiltrate into normal brain tissue, thereby complicating targeted treatment of this type of cancer. Recent studies suggested involvement of Sema3C (semaphorin 3C) protein in tumorigenesis and metastasis in a number of cancers. The role of Sema3C in gliomagenesis is currently unclear. In this study, we investigated how expression levels of Sema3C in post-operative glioma tumors are associated with the malignancy grade and the survival of the patient. FINDINGS: Western blot analysis was used for detection of Sema3C protein levels in 84 different grade glioma samples: 12 grade I astrocytomas, 30 grade II astrocytomas, 17 grade III astrocytomas, and 25 grade IV astrocytomas (glioblastomas). Sema3C mRNA levels in gliomas were analysed by real-time PCR. Several statistical methods have been used to investigate associations between Sema3C protein and mRNA levels and clinical variables and survival outcome. The results demonstrated that protein levels of Sema3C were markedly increased in glioblastomas compared to grade I-III astrocytoma tissues and were significantly associated with the shorter overall survival of patients. High accumulation of Sema3C positively associated with the age of patients and pathological grade, but did not correlate with patient's gender. Sema3C mRNA levels showed no association with either grade of glioma or patient survival. CONCLUSIONS: The data presented in this work suggest that the increased levels of Sema3C protein may be associated with the progression of glioma tumor and has a potential as a prognostic marker for outcome of glioma patients. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1564066714158642.

Hepatitis C virus NS5A protein blocks epidermal growth factor receptor degradation via a proline motif- dependent interaction.

Hepatitis C virus (HCV) establishes a persistent infection that in many cases leads to cirrhosis and hepatocellular carcinoma. The non-structural 5A protein (NS5A) has been implicated in this process as it contains a C-terminal polyproline motif (termed P2) that binds to Src homology 3 (SH3) domains to regulate cellular signalling and trafficking pathways. We have shown previously that NS5A impaired epidermal growth factor (EGF) receptor (EGFR) endocytosis, thereby inhibiting EGF-stimulated EGFR degradation by a mechanism that remained unclear. As EGFR has been implicated in HCV cell entry and trafficking of the receptor involves several SH3-domain containing proteins, we investigated in more detail the mechanisms by which NS5A perturbs EGFR trafficking. We demonstrated that the P2 motif was required for the NS5A-mediated disruption to EGFR trafficking. We further demonstrated that the P2 motif was required for an interaction between NS5A and CMS, a homologue of CIN85 that has previously been implicated in EGFR endocytosis. We provided evidence that CMS was involved in the NS5A-mediated perturbation of EGFR trafficking. We also showed that NS5A effected a loss of EGFR ubiquitination in a P2-motif-dependent fashion. These data provide clues to the mechanism by which NS5A regulates the trafficking of a key cellular receptor and demonstrate for the first time the ability of NS5A to regulate host cell ubiquitination pathways.

Glioma Malignancy-Dependent NDRG2 Gene Methylation and Downregulation Correlates with Poor Patient Outcome.

AIMS: NDRG2 (N-myc downstream regulated gene 2) gene is involved in important biological processes: cell differentiation, growth and apoptosis. Several molecular studies have shown NDRG2 as a promising diagnostic marker involved in brain tumor pathology. The aim of the study was to investigate how changes in epigenetic modification and activity of NDRG2 reflect on glioma malignancy and patient outcome. METHODS: 137 different malignancy grade gliomas were used as the study material: 14 pilocytic astrocytomas grade I, 45 diffuse astrocytomas grade II, 29 anaplastic astrocytomas grade III, and 49 grade IV astrocytomas (glioblastomas). Promoter methylation analysis has been carried out by using methylation-specific PCR, whereas RT-PCR and Western-blot analyses were used to measure NDRG2 expression levels. RESULTS: We demonstrated that NDRG2 gene methylation frequency increased whereas expression at both mRNA and protein levels markedly decreased in glioblastoma specimens compared to the lower grade astrocytomas. NDRG2 transcript and protein levels did not correlate with the promoter methylation state, suggesting the presence of alternative regulatory gene expression mechanisms that may operate in a tissue-specific manner in gliomas. Kaplan-Meier analyses revealed significant differences in survival time in gliomas stratified by NDRG2 methylation status and mRNA and protein expression levels. CONCLUSIONS: Our findings highlight the usefulness of combining epigenetic data to gene expression patterns at mRNA and protein level in tumor biomarker studies, and suggest that NDRG2 downregulation might bear influence on glioma tumor progression while being associated with higher malignancy grade.

Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.

Recognition of tandem PxxP motifs as a unique Src homology 3-binding mode triggers pathogen-driven actin assembly.

Src homology 3 (SH3) domains are globular protein interaction modules that regulate cell behavior. The classic SH3 ligand-binding site accommodates a hydrophobic PxxP motif and a positively charged specificity-determining residue. We have determined the NMR structure of insulin receptor tyrosine kinase substrate (IRTKS) SH3 domain in complex with a repeat from Escherichia coli-secreted protein F-like protein encoded on prophage U (EspF(U)), a translocated effector of enterohemorrhagic E. coli that commandeers the mammalian actin assembly machinery. EspF(U)-IRTKS interaction is among the highest affinity natural SH3 ligands. Our complex structure reveals a unique type of SH3 interaction based on recognition of tandem PxxP motifs in the ligand. Strikingly, the specificity pocket of IRTKS SH3 has evolved to accommodate a polyproline type II helical peptide analogously to docking of the canonical PxxP by the conserved IRTKS SH3 proline-binding pockets. This cooperative binding explains the high-affinity SH3 interaction and is required for EspF(U)-IRTKS interaction in mammalian cells as well as the formation of localized actin "pedestals" beneath bound bacteria. Importantly, tandem PxxP motifs are also found in mammalian ligands and have been shown to contribute to IRTKS SH3 recognition similarly.

Molecular priming of Lyn by GPVI enables an immune receptor to adopt a hemostatic role.

The immune receptor signaling pathway is used by nonimmune cells, but the molecular adaptations that underlie its functional diversification are not known. Circulating platelets use the immune receptor homologue glycoprotein VI (GPVI) to respond to collagen exposed at sites of vessel injury. In contrast to immune cell responses, platelet activation must take place within seconds to successfully form thrombi in flowing blood. Here, we show that the GPVI receptor utilizes a unique intracellular proline-rich domain (PRD) to accelerate platelet activation, a requirement for efficient platelet adhesion to collagen under flow. The GPVI PRD specifically binds the Src-family kinase Lyn and directly activates it, presumably through SH3 displacement. In resting platelets, Lyn is constitutively bound to GPVI in an activated state and platelets lacking Lyn exhibit defective collagen adhesion like that of platelets with GPVI receptors lacking the PRD. These findings define a molecular priming mechanism that enables an immune-type receptor to adopt a hemostatic function. These studies also demonstrate that active kinases can constitutively associate with immune-type receptors without initiating signal transduction before receptor ligation, consistent with a recent molecular model of immune receptor signaling in which receptor ligation is required to bring active kinases to their receptor substrates.

Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.

Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effectors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin "pedestals." One effector, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich effector EspF(U) (aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment by a mechanism dependent on an Asn-Pro-Tyr (NPY(458)) sequence in the Tir C-terminal cytoplasmic domain. Tir, EspF(U), and N-WASP form a complex, but neither EspF(U) nor N-WASP bind Tir directly, suggesting involvement of another protein in complex formation. Screening of the mammalian SH3 proteome for the ability to bind EspF(U) identified the SH3 domain of insulin receptor tyrosine kinase substrate (IRTKS), a factor known to regulate the cytoskeleton. Derivatives of WASP, EspF(U), and the IRTKS SH3 domain were capable of forming a ternary complex in vitro, and replacement of the C terminus of Tir with the IRTKS SH3 domain resulted in a fusion protein competent for actin assembly in vivo. A second domain of IRTKS, the IRSp53/MIM homology domain (IMD), bound to Tir in a manner dependent on the C-terminal NPY(458) sequence, thereby recruiting IRTKS to sites of bacterial attachment. Ectopic expression of either the IRTKS SH3 domain or the IMD, or genetic depletion of IRTKS, blocked pedestal formation. Thus, enterohemorrhagic E. coli translocates 2 effectors that bind to distinct domains of a common host factor to promote the formation of a complex that triggers robust actin assembly at the plasma membrane.

Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling.

NS1 (nonstructural protein 1) is an important virulence factor of the influenza A virus. We observed that NS1 proteins of the 1918 pandemic virus (A/Brevig Mission/1/18) and many avian influenza A viruses contain a consensus Src homology 3 (SH3) domain-binding motif. Screening of a comprehensive human SH3 phage library revealed the N-terminal SH3 of Crk and CrkL as the preferred binding partners. Studies with recombinant proteins confirmed avid binding of NS1 proteins of the 1918 virus and a representative avian H7N3 strain to Crk/CrkL SH3 but not to other SH3 domains tested, including p85alpha and p85beta. Endogenous CrkL readily co-precipitated NS1 from cells infected with the H7N3 virus. In transfected cells association with CrkL was observed for NS1 of the 1918 and H7N3 viruses but not A/Udorn/72 or A/WSN/33 NS1 lacking this sequence motif. SH3 binding was dispensable for suppression of interferon-induced gene expression by NS1 but was associated with enhanced phosphatidylinositol 3-kinase signaling, as evidenced by increased Akt phosphorylation. Thus, the Spanish Flu virus resembles avian influenza A viruses in its ability to recruit Crk/CrkL to modulate host cell signaling.