Tumor Cell-Associated IL-1α Affects Breast Cancer Progression and Metastasis in Mice through Manipulation of the Tumor Immune Microenvironment.

IL-1α is a dual function cytokine that affects inflammatory and immune responses and plays a pivotal role in cancer. The effects of intracellular IL-1α on the development of triple negative breast cancer (TNBC) in mice were assessed using the CRISPR/Cas9 system to suppress IL-1α expression in 4T1 breast cancer cells. Knockout of IL-1α in 4T1 cells modified expression of multiple genes, including downregulation of cytokines and chemokines involved in the recruitment of tumor-associated pro-inflammatory cells. Orthotopical injection of IL-1α knockout (KO) 4T1 cells into BALB/c mice led to a significant decrease in local tumor growth and lung metastases, compared to injection of wild-type 4T1 (4T1/WT) cells. Neutrophils and myeloid-derived suppressor cells were abundant in tumors developing after injection of 4T1/WT cells, whereas more antigen-presenting cells were observed in the tumor microenvironment after injection of IL-1α KO 4T1 cells. This switch correlated with increased infiltration of CD3+CD8+ and NKp46+cells. Engraftment of IL-1α knockout 4T1 cells into immunodeficient NOD.SCID mice resulted in more rapid tumor growth, with increased lung metastasis in comparison to engraftment of 4T1/WT cells. Our results suggest that tumor-associated IL-1α is involved in TNBC progression in mice by modulating the interplay between immunosuppressive pro-inflammatory cells vs. antigen-presenting and cytotoxic cells.

The Peptidyl-Prolyl cis-trans isomerase, Pin1, associates with Protein Kinase C θ via a critical Phospho-Thr-Pro motif in the V3 regulatory domain.

Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.

A Missense Variation in PHACTR2 Associates with Impaired Actin Dynamics, Dilated Cardiomyopathy, and Left Ventricular Non-Compaction in Humans.

Dilated cardiomyopathy (DCM) with left ventricular non-compaction (LVNC) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure, and excessive risk of sudden cardiac death. Using whole-exome sequencing to investigate a possible genetic cause of DCM with LVNC in a consanguineous child, a homozygous nucleotide change c.1532G>A causing p.Arg511His in PHACTR2 was found. The missense change can affect the binding of PHACTR2 to actin by eliminating the hydrogen bonds between them. The amino acid change does not change PHACTR2 localization to the cytoplasm. The patient's fibroblasts showed a decreased globular to fibrillary actin ratio compared to the control fibroblasts. The re-polymerization of fibrillary actin after treatment with cytochalasin D, which disrupts the actin filaments, was slower in the patient's fibroblasts. Finally, the patient's fibroblasts bridged a scar gap slower than the control fibroblasts because of slower and indirect movement. This is the first report of a human variation in this PHACTR family member. The knock-out mouse model presented no significant phenotype. Our data underscore the importance of PHACTR2 in regulating the monomeric actin pool, the kinetics of actin polymerization, and cell movement, emphasizing the importance of actin regulation for the normal function of the human heart.

Termination of TCR-mediated activation signals is regulated by CrkII-dependent Cbl-mediated ubiquitination and degradation of C3G.

Crk adaptor proteins are key players in signal transduction from multiple cell surface receptors, including the T cell antigen receptor (TCR). The involvement of CrkII in the early stages of T cell activation is well documented, but little is known about its role during the termination of the activation response. We substantiated findings showing that CrkII utilizes its SH3N and SH2 domains to constitutively associate with C3G and transiently with Cbl in resting and TCR/CD3-stimulated T cells, respectively. Association of CrkII with Cbl peaks within 1 min post-TCR/CD3 stimulation, and involves the formation of multiple CrkII-containing complexes of different molecular mass. Ubiquitination of C3G commences at ∼5 min post TCR/CD3 stimulation concomitantly with its degradation. This entire process conversely correlates with the levels of expression of CrkII and is dependent on the presence of the CrkII-bound Cbl protein. The data suggest that CrkII functions as a scaffold that brings Cbl into close proximity with C3G in TCR/CD3-stimulated T cells and that tyrosine phosphorylation and activation of Cbl promotes C3G ubiquitination and degradation. We suggest that this mechanism contributes to the termination of the TCR/CD3-induced activation signal and helps tune the length and intensity of T cell-mediated immune responses.

Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells.

The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of ZAP70, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here, we demonstrate that ZAP70 in TCR/CD3-activated mouse spleen and thymus cells, as well as human Jurkat T cells, is regulated by the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin A (CypA) and that this regulation is abrogated by cyclosporin A (CsA), a CypA inhibitor. We found that TCR crosslinking promoted a rapid and transient, Lck-dependent association of CypA with the interdomain B region, at the ZAP70 regulatory domain. CsA inhibited CypA binding to ZAP70 and prevented the colocalization of CypA and ZAP70 at the cell membrane. In addition, imaging analyses of antigen-specific T cells stimulated by MHC-restricted antigen-fed antigen-presenting cells revealed the recruitment of ZAP70-bound CypA to the immunological synapse. Enzymatically active CypA downregulated the catalytic activity of ZAP70 in vitro, an effect that was reversed by CsA in TCR/CD3-activated normal T cells but not in CypA-deficient T cells, and further confirmed in vivo by FRET-based studies. We suggest that CypA plays a role in determining the activity of ZAP70 in TCR-engaged T cells and impact on T cell activation by intervening with the activity of multiple downstream effector molecules.

IGF2 Mediates Resistance to Isoform-Selective-Inhibitors of the PI3K in HPV Positive Head and Neck Cancer.

Over 50% of human papilloma positive head-and-neck cancer (HNCHPV+) patients harbor genomic-alterations in PIK3CA, leading to hyperactivation of the phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) pathway. Nevertheless, despite PI3K pathway activation in HNCHPV+ tumors, the anti-tumor activities of PI3K pathway inhibitors are moderate, mostly due to the emergence of resistance. Thus, for potent and long-term tumor management, drugs blocking resistance mechanisms should be combined with PI3K inhibitors. Here, we delineate the molecular mechanisms of the acquisition of resistance to two isoform-selective inhibitors of PI3K (isiPI3K), alpelisib (BYL719) and taselisib (GDC0032), in HNCHPV+ cell lines. By comparing the transcriptional landscape of isiPI3K-sensitive tumor cells with that of their corresponding isiPI3K-acquired-resistant tumor cells, we found upregulation of insulin growth factor 2 (IGF2) in the resistant cells. Mechanistically, we show that upon isiPI3K treatment, isiPI3K-sensitive tumor cells upregulate the expression of IGF2 to induce cell proliferation via the activation of the IGF1 receptor (IGF1R). Stimulating tumor cells with recombinant IGF2 limited isiPI3K efficacy and released treated cells from S phase arrest. Knocking-down IGF2 with siRNA, or blocking IGF1R with AEW541, resulted in superior anti-tumor activity of isiPI3K in vitro and ex vivo. In vivo, the combination of isiPI3K and IGF1R inhibitor induced stable disease in mice bearing either tumors generated by the HNCHPV+ UM-SCC47 cell line or HPV+ patient-derived xenografts. These findings indicate that IGF2 and the IGF2/IGF1R pathway may constitute new targets for combination therapies to enhance the efficacy of PI3K inhibitors for the treatment of HNCHPV+.

N-Glycans Mediate the Ebola Virus-GP1 Shielding of Ligands to Immune Receptors and Immune Evasion.

The Ebola Virus (EBOV) glycoprotein (GP) sterically shields cell-membrane ligands to immune receptors such as human leukocyte antigen class-1 (HLA-I) and MHC class I polypeptide-related sequence A (MICA), thus mediating immunity evasion. It was suggested that the abundant N-glycosylation of the EBOV-GP is involved in this steric shielding. We aimed to characterize (i) the GP N-glycosylation sites contributing to the shielding, and (ii) the effect of mutating these sites on immune subversion by the EBOV-GP. The two highly glycosylated domains of GP are the mucin-like domain (MLD) and the glycan cap domain (GCD) with three and six N-glycosylation sites, respectively. We mutated the N-glycosylation sites either in MLD or in GCD or in both domains. We showed that the glycosylation sites in both the MLD and GCD domains contribute to the steric shielding. This was shown for the steric shielding of either HLA-I or MICA. We then employed the fluorescence resonance energy transfer (FRET) method to measure the effect of N-glycosylation site removal on the distance in the cell membrane between the EBOV-GP and HLA-I (HLA.A*0201 allele). We recorded high FRET values for the interaction of CFP-fused HLA.A*0201 and YFP-fused EBOV-GP, demonstrating the very close distance (<10 nm) between these two proteins on the cell membrane of GP-expressing cells. The co-localization of HLA-I and Ebola GP was unaffected by the disruption of steric shielding, as the removal of N-glycosylation sites on Ebola GP revealed similar FRET values with HLA-I. However, these mutations directed to N-glycosylation sites had restored immune cell function otherwise impaired due to steric shielding over immune cell ligands by WT Ebola GP. Overall, we showed that the GP-mediated steric shielding aimed to impair immune function is facilitated by the N-glycans protruding from its MLD and GCD domains, but these N-glycans are not controlling the close distance between GP and its shielded proteins.

Telomerase increasing compound protects hippocampal neurons from amyloid beta toxicity by enhancing the expression of neurotrophins and plasticity related genes.

The telomerase reverse transcriptase protein, TERT, is expressed in the adult brain and its exogenic expression protects neurons from oxidative stress and from the cytotoxicity of amyloid beta (Aß). We previously showed that telomerase increasing compounds (AGS) protected neurons from oxidative stress. Therefore, we suggest that increasing TERT by AGS may protect neurons from the Aß-induced neurotoxicity by influencing genes and factors that participate in neuronal survival and plasticity. Here we used a primary hippocampal cell culture exposed to aggregated Aß and hippocampi from adult mice. AGS treatment transiently increased TERT gene expression in hippocampal primary cell cultures in the presence or absence of Aß and protected neurons from Aß induced neuronal degradation. An increase in the expression of Growth associated protein 43 (GAP43), and Feminizing locus on X-3 genes (NeuN), in the presence or absence of Aß, and Synaptophysin (SYP) in the presence of Aß was observed. GAP43, NeuN, SYP, Neurotrophic factors (NGF, BDNF), beta-catenin and cyclin-D1 expression were increased in the hippocampus of AGS treated mice. This data suggests that increasing TERT by pharmaceutical compounds partially exerts its neuroprotective effect by enhancing the expression of neurotrophic factors and neuronal plasticity genes in a mechanism that involved Wnt/beta-catenin pathway.

PICOT binding to chromatin-associated EED negatively regulates cyclin D2 expression by increasing H3K27me3 at the CCND2 gene promoter.

Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3 (Grx3; Glrx3)) is a ubiquitous protein that can interact with the embryonic ectoderm development (EED) protein via each of its two C-terminal PICOT/Grx homology domains. Since EED is a Polycomb-Group protein and a core component of the polycomb repressive complex 2 (PRC2), we tested the involvement of PICOT in the regulation of PRC2-mediated H3 lysine 27 trimethylation (H3K27me3), transcription and translation of selected PRC2 target genes. A fraction of the cellular PICOT protein was found in the nuclei of leukemia cell lines, where it was associated with the chromatin. In addition, PICOT coimmunoprecipitated with chromatin-residing EED derived from Jurkat and COS-7 cell nuclei. PICOT knockdown led to a reduced H3K27me3 mark and a decrease in EED and EZH2 at the CCND2 gene promoter. In agreement, PICOT-deficient T cells exhibited a significant increase in CCND2 mRNA and protein expression. Since elevated expression levels of PICOT were reported in several different tumors and correlated in the current studies with decreased transcription and translation of the CCND2 gene, we tested whether this opposite correlation exists in human cancers. Data from the Cancer Genome Atlas (TCGA) database indicated statistically significant negative correlation between PICOT and CCND2 in eight different human tumors where the highest correlation was in lung (p = 8.67E-10) and pancreatic (p = 1.06E-5) adenocarcinoma. Furthermore, high expression of PICOT and low expression of CCND2 correlated with poor patient survival in five different types of human tumors. The results suggest that PICOT binding to chromatin-associated EED modulates the H3K27me3 level at the CCND2 gene promoter which may be one of the potential mechanisms for regulation of cyclin D2 expression in tumors. These findings also indicate that a low PICOT/CCND2 expression ratio might serve as a good predictor of patient survival in selected human cancers.

PICOT (GLRX3) is a positive regulator of stress-induced DNA-damage response.

Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3 (Glrx3)) is a ubiquitously expressed protein that possesses an N-terminal monothiol thioredoxin (Trx) domain and two C-terminal tandem copies of a monothiol Glrx domain. It has an overall highly conserved amino acid sequence and is encoded by a unique gene, both in humans and mice, without having other functional gene homologs in the entire genome. Despite being discovered almost two decades ago, the biological function of PICOT remains largely ill-defined and its ramifications are underestimated considering the fact that PICOT-deficiency in mice results in embryonic lethality. Since classical Glrxs are important regulators of the cellular redox homeostasis, we tested whether PICOT participate in the stress-induced DNA-damage response, focusing on nuclear proteins that function as integral components of the DNA repair machinery. Using wild type versus PICOT-deficient (PICOT-KD) Jurkat T cells we found that the anti-oxidant mechanism in PICOT-deficient cells is impaired, and that these cells respond to genotoxic drugs, such as etoposide and camptothecin, by increased caspase-3 activity, a reduced survival and a slower and diminished phosphorylation of the histone protein, H2AX. Nevertheless, the effect of PICOT on the drug-induced phosphorylation of H2AX was independent of the cellular levels of reactive oxygen species. PICOT-deficient cells also demonstrated reduced and slower γH2AX foci formation in response to radiation. Furthermore, immunofluorescence staining using PICOT- and γH2AX-specific Abs followed by confocal microscopy demonstrated partial localization of PICOT at the γH2AX-containing foci at the site of the DNA double strand breaks. In addition, PICOT knockdown resulted in inhibition of phosphorylation of ATR, Chk1 and Chk2 kinases, which play an essential role in the DNA-damage response and serve as upstream regulators of γH2AX. The present data suggest that PICOT protects cells from DNA damage-inducing agents by operating as an upstream positive regulator of ATR-dependent signaling pathways. By promoting the activity of ATR, PICOT indirectly regulates the phosphorylation and activation of Chk1, Chk2, and γH2AX, which are critical components of the DNA damage repair mechanism and thereby attenuate the stress- and replication-induced genome instability.

PICOT binding to the polycomb group protein, EED, alters H3K27 methylation at the MYT1 PRC2 target gene.

PICOT is a ubiquitous protein that has no functional redundant ortholog and is critical for mouse embryonic development. It is involved in the regulation of signal transduction in T lymphocytes and cardiac muscle, and in cellular iron metabolism and biogenesis of Fe/S proteins. However, very little is known about the physiological role of PICOT and its mechanism of action, and on its upstream regulators or downstream target molecules. In attempt to identify new PICOT interaction partners, we adopted the yeast two-hybrid system and screened a Jurkat T cell cDNA library using the full-length human PICOT cDNA as a bait. We found that PICOT interacts with embryonic ectoderm development (EED), a Polycomb Group (PcG) protein that serves as a core component of the Polycomb repressive complex 2 (PRC2) and contributes to the regulation of chromatin remodeling and cell differentiation. Using bead immobilized GST-PICOT and GST-EED fusion proteins in a pull-down assay and reciprocal coimmunoprecipitation studies we demonstrated that the interaction between PICOT and EED also occurs in human Jurkat T cells. In addition, immunofluorescence staining of Jurkat T cells revealed partial colocalization of PICOT and EED, predominantly in the cell nuclei. A pull-down assay using the GST-EED fusion protein and lysates of cells expressing different Myc-tagged truncation products of PICOT revealed that binding of EED is mediated by each of the two C-terminal PICOT homology domains and suggests that simultaneous interaction via both domains increases the binding affinity. Furthermore, PICOT knock-down in Jurkat T cells resulted in a reduced histone H3 lysine 27 trimethylation (H3K27me3) at the PRC2 target gene, myelin transcription factor 1 (MYT1), suggesting that PICOT binding to EED alters PRC2-regulated transcriptional repression, and potentially contributes to the epigenetic regulation of chromatin silencing and remodeling.

Interleukin 1α-Deficient Mice Have an Altered Gut Microbiota Leading to Protection from Dextran Sodium Sulfate-Induced Colitis.

Inflammatory bowel diseases (IBD) are a group of chronic inflammatory disorders of the intestine, with as-yet-unclear etiologies, affecting over a million people in the United States alone. With the emergence of microbiome research, numerous studies have shown a connection between shifts in the gut microbiota composition (dysbiosis) and patterns of IBD development. In a previous study, we showed that interleukin 1α (IL-1α) deficiency in IL-1α knockout (KO) mice results in moderate dextran sodium sulfate (DSS)-induced colitis compared to that of wild-type (WT) mice, characterized by reduced inflammation and complete healing, as shown by parameters of weight loss, disease activity index (DAI) score, histology, and cytokine expression. In this study, we tested whether the protective effects of IL-1α deficiency on DSS-induced colitis correlate with changes in the gut microbiota and whether manipulation of the microbiota by cohousing can alter patterns of colon inflammation. We analyzed the gut microbiota composition in both control (WT) and IL-1α KO mice under steady-state homeostasis, during acute DSS-induced colitis, and after recovery using 16S rRNA next-generation sequencing. Additionally, we performed cohousing of both mouse groups and tested the effects on the microbiota and clinical outcomes. We demonstrate that host-derived IL-1α has a clear influence on gut microbiota composition, as well as on severity of DSS-induced acute colon inflammation. Cohousing both successfully changed the gut microbiota composition and increased the disease severity of IL-1α-deficient mice to levels similar to those of WT mice. This study shows a strong and novel correlation between IL-1α expression, microbiota composition, and clinical outcomes of DSS-induced colitis. IMPORTANCE Here, we show a connection between IL-1α expression, microbiota composition, and clinical outcomes of DSS-induced colitis. Specifically, we show that the mild colitis symptoms seen in IL-1α-deficient mice following administration of DSS are correlated with the unique gut microbiota compositions of the mice. However, when these mice are exposed to WT microbiota by cohousing, their gut microbiota composition returns to resemble that of WT mice, and their disease severity increases significantly. As inflammatory bowel diseases are such common diseases, with limited effective treatments to date, there is a great need to better understand the interactions between microbiota composition, the immune system, and colitis. This study shows correlation between microbiota composition and DSS resistance; it may potentially lead to the development of improved probiotics for IBD treatment.

Expression of functional alternative telomerase RNA component gene in mouse brain and in motor neurons cells protects from oxidative stress.

Telomerase, a ribonucleoprotein, is highly expressed and active in many tumor cells and types, therefore it is considered to be a target for anti-cancer agents. On the other hand, recent studies demonstrated that activation of telomerase is a potential therapeutic target for age related diseases. Telomerase mainly consists of a catalytic protein subunit with a reverse transcription activity (TERT) and an RNA component (TERC), a long non-coding RNA, which serves as a template for the re-elongation of telomeres by TERT. We previously showed that TERT is highly expressed in distinct neuronal cells of the mouse brain and its expression declined with age. To understand the role of telomerase in non-mitotic, fully differentiated cells such neurons we here examined the expression of the other component, TERC, in mouse brain. Surprisingly, by first using bioinformatics analysis, we identified an alternative TERC gene (alTERC) in the mouse genome. Using further experimental approaches we described the presence of a functional alTERC in the mouse brain and spleen, in cultures of motor neurons- like cells and neuroblastoma tumor cells. The alTERC is similar (87%) to mouse TERC (mTERC) with a deletion of 18 bp in the TERC conserved region 4 (CR4). This alTERC gene is expressed and its product interacts with the endogenous mTERT protein and with an exogenous human TERT protein (hTERT) to form an active enzyme. Overexpression of the alTERC and the mTERC genes, in mouse motor neurons like cells, increased the activity of TERT without affecting its protein level. Under oxidative stress conditions, alTERC significantly increased the survival of motor neurons cells without altering the level of TERT protein or its activity.The results suggest that the expression of the alTERC gene in the mouse brain provides an additional way for regulating telomerase activity under normal and stress conditions and confers protection to neuronal cells from oxidative stress.

Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine.

In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinß4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.

In vivo regulation of human CrkII by cyclophilin A and FK506-binding protein.

Members of the Crk family of adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII are two alternative-spliced forms of a single gene which possess an N-terminal SH2 domain and an SH3 domain that mediate interaction with other proteins. CrkII possesses an additional C-terminal linker region plus an extra SH3 domain, which does not interact with other proteins, but operates as regulatory moiety. Utilizing human Jurkat T cells, we demonstrate that CrkII-SH3N binding of C3G is inhibited by cyclosporin A (CsA) plus FK506 that inhibit the cyclophilin A (CypA) and FK506 binding protein (FKBP) peptidyl-prolyl cis-trans isomerases (PPIases; also termed immunophilins), respectively. Jurkat T cells were found to express ∼ 5-fold lower levels of CrkI protein compared to CrkII, but the efficiency of C3G binding by CrkI was ∼ 5-fold higher than that of CrkII, suggesting that the majority of cellular CrkII proteins adopt a conformation that is inaccessible for C3G. Treatment of Jurkat T cells with CsA plus FK506 led to a time-dependent conformational change in overexpressed human CrkII1-236 protein-containing FRET-based biosensor, supporting the accumulation of cis conformers of human CrkII1-236 in the presence of PPIase inhibitors. Our data suggest that the Gly(219)-Pro-Tyr motif in the human CrkII linker region serves as the recognition and isomerization site of PPIases, and raise the possibility that CsA and FK506 might interfere with selected effector T cell functions via a CrkII-, but not CrkI-dependent mechanisms.

The Role of Crk Adaptor Proteins in T-Cell Adhesion and Migration.

Crk adaptor proteins are key players in signal transduction from a variety of cell surface receptors. They are involved in early steps of lymphocyte activation through their SH2-mediated transient interaction with signal transducing effector molecules, such as Cbl, ZAP-70, CasL, and STAT5. In addition, they constitutively associate, via their SH3 domain, with effector molecules, such as C3G, that mediate cell adhesion and regulate lymphocyte extravasation and recruitment to sites of inflammation. Recent studies demonstrated that the conformation and function of CrkII is subjected to a regulation by immunophilins, which also affect CrkII-dependent T-cell adhesion to fibronectin and migration toward chemokines. This article addresses mechanisms that regulate CrkII conformation and function, in general, and emphasizes the role of Crk proteins in receptor-coupled signaling pathways that control T-lymphocyte adhesion and migration to inflammatory sites.

PLEKHM2 mutation leads to abnormal localization of lysosomes, impaired autophagy flux and associates with recessive dilated cardiomyopathy and left ventricular noncompaction.

Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient's fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function.

Cellular senescence-like features of lung fibroblasts derived from idiopathic pulmonary fibrosis patients.

Idiopathic pulmonary fibrosis (IPF) is an age-related fatal disease with unknown etiology and no effective treatment. In this study, we show that primary cultures of fibroblasts derived from lung biopsies of IPF patients exhibited (i) accelerated replicative cellular senescence (CS); (ii) high resistance to oxidative-stress-induced cytotoxicity or CS; (iii) a CS-like morphology (even at the proliferative phase); and (iv) rapid accumulation of senescent cells expressing the myofibroblast marker α-SMA. Our findings suggest that CS could serve as a bridge connecting lung aging and its quite frequent outcome -- pulmonary fibrosis, and be an important player in the disease progression. Consequently, targeting senescent cells offers the potential of being a promising therapeutic approach.

Association of p75(NTR) and α9ß1 integrin modulates NGF-dependent cellular responses.

Direct interaction of α9ß1 integrin with nerve growth factor (NGF) has been previously reported to induce pro-proliferative and pro-survival activities of non-neuronal cells. We investigated participation of p75(NTR) in α9ß1 integrin-dependent cellular response to NGF stimulation. Using selective transfection of glioma cell lines with these receptors, we showed a strong, cation-independent association of α9 integrin subunit with p75(NTR) on the cellular membrane by selective immunoprecipitation experiments. The presence of the α9/p75(NTR) complex increases NGF-dependent cell adhesion, proliferation and migration. Other integrin subunits including ß1 were not found in complex with p75(NTR). FRET analysis indicated that p75(NTR) and α9 integrin subunit are not closely associated through their cytoplasmic domains, most probably because of the molecular interference with other cytoplasmic proteins such as paxillin. Interaction of α9ß1 integrin with another ligand, VCAM-1 was not modulated by the p75(NTR). α9/p75(NTR) complex elevated NGF-dependent activation of MAPK Erk1/2 arty for integrin that may create active complexes with other types of receptors belonging to the TNF superfamily.

The role of cytosolic phospholipase A2 α in amyloid precursor protein induction by amyloid beta1-42 : implication for neurodegeneration.

Amyloid-ß peptides generated by proteolysis of the ß-amyloid precursor protein (APP) play an important role in the pathogenesis of Alzheimer's disease. The present study aimed to determine whether cytosolic phospholipase A2 α (cPLA2 α) plays a role in elevated APP protein expression induced by aggregated amyloid-ß1-42 (Aß) in cortical neurons and to elucidate its specific role in signal events leading to APP induction. Elevated cPLA2 α and its activity determined by phosphorylation on serine 505 as well as elevated APP protein expression, were detected in primary rat cortical neuronal cultures exposed to Aß for 24 h and in cortical neuron of human amyloid-ß1-42 brain infused mice. Prevention of cPLA2 α up-regulation and its activity by oligonucleotide antisense against cPLA2 α (AS) prevented the elevation of APP protein in cortical neuronal cultures and in mouse neuronal cortex. To determine the role of cPLA2 α in the signals leading to APP induction, increased cPLA2 α expression and activity induced by Aß was prevented by means of AS in neuronal cortical cultures. Under these conditions, the elevated cyclooxygenase-2 and the production of prostaglandin E2 (PGE2 ) were prevented. Addition of PGE2 or cyclic AMP analogue (dbcAMP) to neuronal cultures significantly increased the expression of APP protein, while the presence protein kinase A inhibitor (H-89) attenuated the elevation of APP induced by Aß. Inhibition of elevated cPLA2 α by AS prevented the activation of cAMP response element binding protein (CREB) as detected by its phosphorylated form, its translocation to the nucleus and its DNA binding induced by Aß which coincided with cPLA2 α dependent activation of CREB in the cortex of Aß brain infused mice. Our results show that accumulation of Aß induced elevation of APP protein expression mediated by cPLA2 α, PGE2 release, and CREB activation via protein kinase A pathway.