Ugi bisamides based on pyrrolyl-ß-chlorovinylaldehyde and their unusual transformations.

By one-pot four- and three-component Ugi reactions involving convertible isocyanides and unexplored pyrrole-containing ß-chlorovinylaldehyde, a small library of 20 bisamides with unusual behavior in post-Ugi transformations was prepared and characterized. Surprisingly, a well-documented approach to obtain peptide-containing carboxylic acids through acid hydrolysis of the convertible isocyanide moiety in the Ugi bisamides proceeded in an unexpected manner in our case, leading to the formation of derivatives of amides of heterylidenepyruvic acid. An optimized synthetic protocol for this transformation was elaborated and a plausible sequence involving the elimination of the 2-chloroacetamide moiety and the conversion of the ß-chlorovinyl fragment into a vinyl one is provided.

Phosphorylation of (Ser 291) in the linker insert of Syk negatively regulates ITAM signaling in platelets.

Receptor-induced tyrosine phosphorylation of spleen tyrosine kinase (Syk) has been studied extensively in hematopoietic cells. Metabolic mapping and high-resolution mass spectrometry, however, indicate that one of the most frequently detected phosphorylation sites encompassed S297 (S291 in mice) located within the linker B region of Syk. It has been reported that Protein kinase C (PKC) phosphorylates Syk S297, thus influencing Syk activity. However, conflicting studies suggest that this phosphorylation enhances as well as reduces Syk activity. To clarify the function of this site, we generated Syk S291A knock-in mice. We used platelets as a model system as they possess Glycoprotein VI (GPVI), a receptor containing an immunoreceptor tyrosine-based activation motif (ITAM) which transduces signals through Syk. Our analysis of the homozygous mice indicated that the knock-in platelets express only one isoform of Syk, while the wild-type expresses two isoforms at 69 and 66 kDa. When the GPVI receptor was activated with collagen-related peptide (CRP), we observed an increase in functional responses and phosphorylations in Syk S291A platelets. This potentiation did not occur with AYPGKF or 2-MeSADP, although they also activate PKC isoforms. Although there was potentiation of platelet functional responses, there was no difference in tail bleeding times. However, the time to occlusion in the FeCl3 injury model was enhanced. These data indicate that the effects of Syk S291 phosphorylation represent a significant outcome on platelet activation and signaling in vitro but also reveals its multifaceted nature demonstrated by the differential effects on physiological responses in vivo.

What is the context Spleen tyrosine kinase (Syk) is present a number of cells and important in controlling the functions of various cells and organs.Syk is known to exist in two isoforms Syk L (long form or Syk A) and Syk S (short form or Syk B).It is known that phosphorylation events regulate Syk activation and activity.In several inflammatory disease conditions, Syk mutants are known to play a role.Phosphorylation of the Syk residue Serine 291 is known to occur, but its function in the regulation of Syk activation or activity is not known.What is new In this study, we generated a mutant mouse Syk S291A, which cannot be phosphorylated on serine residue. We evaluated the function of platelets isolated from these mice and compared them to platelets isolated from wild type littermates.We observed that the mutation in Syk L unexpectedly caused Syk S to disappear from a number of tissues.Platelet functions are enhanced in mutant mouse platelets compared to those from wild-type mice.What is the impact These studies enhance our understanding of the impact of Serine 291 phosphorylation on the function of Syk in platelets.

Role of Tula-Family Proteins in Cell Signaling and Activation: Advances and Challenges.

This Special Issue entitled "Role of Tula-Family Proteins in Cell Signaling and Activation: Advances and Challenges" is focused on a relatively novel vertebrate gene/protein family termed alternatively TULA, UBASH3, or STS [...].

The Pediatric and Young Adult Choroidal and Ciliary Body Melanoma Genetic Study, A Survey by the European Ophthalmic Oncology Group.

Purpose: To explore the genetic background of choroidal and ciliary body melanoma among children and young adults, with special focus on BAP1 germline variants in this age group. Methods: Patients under the age of 25 and with confirmed choroidal or ciliary body melanoma were included in this retrospective, multicenter observational study. Nuclear BAP1 immunopositivity was used to evaluate the presence of functional BAP1 in the tumor. Next-generation sequencing using Ion Torrent platform was used to determine pathogenic variants of BAP1, EIF1AX, SF3B1, GNAQ and GNA11 and chromosome 3 status in the tumor or in DNA extracted from blood or saliva. Survival was analyzed using Kaplan-Meier estimates. Results: The mean age at diagnosis was 17 years (range 5.0-24.8). A germline BAP1 pathogenic variant was identified in an 18-year-old patient, and a somatic variant, based mainly on immunohistochemistry, in 13 (42%) of 31 available specimens. One tumor had a somatic SF3B1 pathogenic variant. Disomy 3 and the absence of a BAP1 pathogenic variant in the tumor predicted the longest metastasis-free survival. Males showed longer metastasis-free survival than females (P = 0.018). Conclusions: We did not find a stronger-than-average BAP1 germline predisposition for choroidal and ciliary body melanoma among children and young adults compared to adults. Males had a more favorable survival and disomy 3, and the absence of a BAP1 mutation in the tumor tissue predicted the most favorable metastasis-free survival. A BAP1 germline pathogenic variant was identified in one patient (1%), and a somatic variant based mainly on immunohistochemistry in 13 (42%).

Biochemical characterization of spleen tyrosine kinase (SYK) isoforms in platelets.

Alternate splicing is among the regulatory mechanisms imparting functional diversity in proteins. Studying protein isoforms generated through alternative splicing is therefore critical for understanding protein functions in many biological systems. Spleen tyrosine kinase (Syk) plays an essential role in ITAM/hemITAM signaling in many cell types, including platelets. However, the spectrum of Syk isoforms expressed in platelets has not been characterized. Syk has been shown to have a full-length long isoform SykL and a shorter SykS lacking 23 amino acid residues within its interdomain B. Furthermore, putative isoforms lacking another 23 amino acid-long sequence or a combination of the two deletions have been postulated to exist. In this report, we demonstrate that mouse platelets express full-length SykL and the previously described shorter isoform SykS, but lack other shorter isoforms, whereas human platelets express predominantly SykL. These results both indicate a possible role of alternative Syk splicing in the regulation of receptor signaling in mouse platelets and a difference between signaling regulation in mouse and human platelets.

Platelets express two sizes of the Syk molecule with possible alternate functions in the cell. We need to understand how these two differ in their structure so that further studies can be developed by selectively deleting one of them to evaluate their function in platelets. This study shows that platelet Syk molecules differ in their structure with and without a linker region in the molecule.

Synthesis of imidazo[1,2-a]pyridine-containing peptidomimetics by tandem of Groebke-Blackburn-Bienaymé and Ugi reactions.

Peptidomimetics with a substituted imidazo[1,2-a]pyridine fragment were synthesized by a tandem of Groebke-Blackburn-Bienaymé and Ugi reactions. The target products contain substituted imidazo[1,2-a]pyridine and peptidomimetic moieties as pharmacophores with four diversity points introduced from readily available starting materials, including scaffold diversity. A small focused compound library of 20 Ugi products was prepared and screened for antibacterial activity.

TULA Proteins in Men, Mice, Hens, and Lice: Welcome to the Family.

The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key biological functions, including immunity and hemostasis, in mammalian biological systems. Negative regulation of signaling through immune receptor tyrosine-based activation motif (ITAM)- and hemITAM-bearing receptors mediated by Syk-family protein tyrosine kinases appears to be a major molecular mechanism of the down-regulatory effect of TULA-family proteins, which possess protein tyrosine phosphatase (PTP) activity. However, these proteins are likely to carry out some PTP-independent functions as well. Whereas the effects of TULA-family proteins overlap, their characteristics and their individual contributions to cellular regulation also demonstrate clearly distinct features. Protein structure, enzymatic activity, molecular mechanisms of regulation, and biological functions of TULA-family proteins are discussed in this review. In particular, the usefulness of the comparative analysis of TULA proteins in various metazoan taxa, for identifying potential roles of TULA-family proteins outside of their functions already established in mammalian systems, is examined.

Phosphorylation of spleen tyrosine kinase at Y346 negatively regulates ITAM-mediated signaling and function in platelets.

Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor-based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVI-induced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses in vitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses.

TULA-Family Regulators of Platelet Activation.

The two members of the UBASH3/TULA/STS-protein family have been shown to critically regulate cellular processes in multiple biological systems. The regulatory function of TULA-2 (also known as UBASH3B or STS-1) in platelets is one of the best examples of the involvement of UBASH3/TULA/STS proteins in cellular regulation. TULA-2 negatively regulates platelet signaling mediated by ITAM- and hemITAM-containing membrane receptors that are dependent on the protein tyrosine kinase Syk, which currently represents the best-known dephosphorylation target of TULA-2. The biological responses of platelets to collagen and other physiological agonists are significantly downregulated as a result. The protein structure, enzymatic activity and regulatory functions of UBASH3/TULA/STS proteins in the context of platelet responses and their regulation are discussed in this review.

Phosphorylation on Syk Y342 is important for both ITAM and hemITAM signaling in platelets.

Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl3 injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal.

P2Y12 antagonism results in altered interactions between platelets and regulatory T cells during sepsis.

Sepsis is a complex clinical condition resulting from a serious bloodstream infection. With mortality rates as high as 50%, improved treatments are needed. Regulatory T cells (Tregs), a subset of T lymphocytes, promote the resolution of inflammation. Septic patients have elevated levels of circulating Tregs. Platelets influence the proliferation and activation of Tregs in vitro. However, modulating platelet-Tregs interaction during sepsis may restraing Treg proliferation, leading to the restoration of immunologic homeostasis. P2Y12 is a purinergic receptor present on platelets and T lymphocytes. Blocking P2Y12 improves the outcome of sepsis. We investigated whether blocking P2Y12 alters platelet-Treg interaction in vivo. We used the murine model of sepsis, cecal ligation, and puncture (CLP) and we blocked P2Y12 using the P2Y12 antagonist, clopidogrel. Twenty-four hours after surgery, we measured Treg population sizes in the spleens of the Sham, CLP, and CLP + clopidogrel groups. We investigated the effect of blocking P2Y12 in vitro using cocultures of human platelets and T cells with or without anti-CD3/CD28. P2Y12 was blocked using AR-C69931MX. Treg population sizes were reduced in the septic mice treated with clopidogrel compared with untreated septic mice. Aggregation of platelets and CD4+ T cells was reduced in treated CLP mice compared with untreated CLP mice. P2Y12 antagonism changes how platelets influence T cells in vitro, depending on T-cell activation. In conclusion, blockade of the P2Y12 signaling pathway restrains Treg proliferation in vivo and in vitro. Targeting platelets to control Treg proliferation and activity may be a promising strategy for treating sepsis.

Iron alters the cell wall composition and intracellular lactate to affect Candida albicans susceptibility to antifungals and host immune response.

Fungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans and an inner layer of ß-glucans and chitin. The fungal cell wall is the primary target for antifungals and is recognized by host immune cells. Environmental conditions such as carbon sources, pH, temperature, and oxygen tension can modulate the fungal cell wall architecture. Cellular signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways, are responsible for sensing environmental cues and mediating cell wall alterations. Although iron has recently been shown to affect ß-1,3-glucan exposure on the cell wall, we report here that iron changes the composition of all major C. albicans cell wall components. Specifically, high iron decreased the levels of mannans (including phosphomannans) and chitin; and increased ß-1,3-glucan levels. These changes increased the resistance of C. albicans to cell wall-perturbing antifungals. Moreover, high iron cells exhibited adequate mitochondrial functioning; leading to a reduction in accumulation of lactate that signals through the transcription factor Crz1 to induce ß-1,3-glucan masking in C. albicans We show here that iron-induced changes in ß-1,3-glucan exposure are lactate-dependent; and high iron causes ß-1,3-glucan exposure by preventing lactate-induced, Crz1-mediated inhibition of activation of the fungal MAPK Cek1. Furthermore, despite exhibiting enhanced antifungal resistance, high iron C. albicans cells had reduced survival upon phagocytosis by macrophages. Our results underscore the role of iron as an environmental signal in multiple signaling pathways that alter cell wall architecture in C. albicans, thereby affecting its survival upon exposure to antifungals and host immune response.

Multicenter External Validation of the Liverpool Uveal Melanoma Prognosticator Online: An OOG Collaborative Study.

Uveal melanoma (UM) is fatal in ~50% of patients as a result of disseminated disease. This study aims to externally validate the Liverpool Uveal Melanoma Prognosticator Online V3 (LUMPO3) to determine its reliability in predicting survival after treatment for choroidal melanoma when utilizing external data from other ocular oncology centers. Anonymized data of 1836 UM patients from seven international ocular oncology centers were analyzed with LUMPO3 to predict the 10-year survival for each patient in each external dataset. The analysts were masked to the patient outcomes. Model predictions were sent to an independent statistician to evaluate LUMPO3's performance using discrimination and calibration methods. LUMPO3's ability to discriminate between UM patients who died of metastatic UM and those who were still alive was fair-to-good, with C-statistics ranging from 0.64 to 0.85 at year 1. The pooled estimate for all external centers was 0.72 (95% confidence interval: 0.68 to 0.75). Agreement between observed and predicted survival probabilities was generally good given differences in case mix and survival rates between different centers. Despite the differences between the international cohorts of patients with primary UM, LUMPO3 is a valuable tool for predicting all-cause mortality in this disease when using data from external centers.

ADP exerts P2Y12 -dependent and P2Y12 -independent effects on primary human T cell responses to stimulation.

Purinergic signaling plays a complex role in inflammation. Nucleotides released by T lymphocytes, endothelial cells, and platelets during inflammation induce cellular responses by binding to receptors that regulate intracellular signaling pathways. Previous studies have found that purinergic signaling can have both proinflammatory and anti-inflammatory effects, but the roles of specific pathways in specific cell types are poorly understood. We investigated the role of the P2Y12 signaling pathway in the activation of T lymphocytes in vitro. We isolated peripheral blood mononuclear cells (PBMCs) from healthy donors and pretreated them with ADP (a P2Y12 agonist), AR-C69931MX (a P2Y12 antagonist), or both. We then stimulated PBMC using phytohemagglutinin (PHA) or anti-CD3/CD28 antibodies. We found that ADP affects T cell responses in term of cell activity and receptor expression through both P2Y12-dependent and P2Y12-independent pathways and other responses (cytokine secretion) primarily through P2Y12 -independent pathways. The ADP-mediated effect changed over time and was stimulus-specific.

TULA proteins as signaling regulators.

Two members of the UBASH3/STS/TULA family exhibit a unique protein domain structure, which includes a histidine phosphatase domain, and play a key role in regulating cellular signaling. UBASH3A/STS-2/TULA is mostly a lymphoid protein, while UBASH3B/STS-1/TULA-2 is expressed ubiquitously. Dephosphorylation of tyrosine-phosphorylated proteins by TULA-2 and, probably to a lesser extent, by TULA critically contribute to the molecular basis of their regulatory effect. The notable differences between the effects of the two family members on cellular signaling and activation are likely to be linked to the difference between their specific enzymatic activities. However, these differences might also be related to the functions of their domains other than the phosphatase domain and independent of their phosphatase activity. The down-regulation of the Syk/Zap-70-mediated signaling, which to-date appears to be the best-studied regulatory effect of TULA family, is discussed in detail in this publication.

Doebner-type pyrazolopyridine carboxylic acids in an Ugi four-component reaction.

Substituted 1H-pyrazolo[3,4-b]pyridine-4- and 1H-pyrazolo[3,4-b]pyridine-6-carboxamides have been synthetized through a Doebner-Ugi multicomponent reaction sequence in a convergent and versatile manner using diversity generation strategies: combination of two multicomponent reactions and conditions-based divergence strategy. The target products contain as pharmacophores pyrazolopyridine and peptidomimetic moieties with four points of diversity introduced from readily available starting materials including scaffold diversity. A small focused compound library of 23 Ugi products was created and screened for antibacterial activity.

The Role of Allograft Inflammatory Factor-1 in the Effects of Experimental Diabetes on B Cell Functions in the Heart.

Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in the heart and subsequently leads to myocardial remodeling, deteriorated cardiac function and heart failure. However, the etiology of the cardiac disease is unknown. Therefore, we assessed the gene expression in the left ventricle of diabetic and non-diabetic mice using Affymetrix microarray analysis. Allograft inflammatory factor-1 (AIF-1), one of the top downregulated B cell inflammatory genes, is associated with B cell functions in inflammatory responses. Real-time reverse transcriptase-polymerase chain reaction confirmed the Affymetrix data. The expression of CD19 and AIF-1 were downregulated in diabetic hearts as compared to control hearts. Using in vitro migration assay, we showed for the first time that AIF-1 is responsible for B cell migration as B cells migrated to GFP-AIF-1-transfected H9C2 cells compared to empty vector-transfected cells. Interestingly, overexpression of AIF-1 in diabetic mice prevented streptozotocin-induced cardiac dysfunction, inflammation and promoted B cell homing into the heart. Our results suggest that AIF-1 downregulation inhibited B cell homing into diabetic hearts, thus promoting inflammation that leads to the development of diabetic cardiomyopathy, and that overexpression of AIF-1 could be a novel treatment for this condition.

TULA-family proteins: Jacks of many trades and then some.

UBASH3/STS/TULA is a novel two-member family, which exerts several key regulatory effects in multiple cell types. UBASH3B/STS-1/TULA-2 is a highly active protein tyrosine phosphatase; its major target appears to be a specific regulatory site of protein tyrosine kinases of the Syk family, dephosphorylation of which inhibits Syk and Zap-70 kinases and suppresses receptor signaling mediated by these kinases. UBASH3A/STS-2/TULA exhibits substantial homology to UBASH3B/STS-1/TULA-2, but possesses only a small fraction of phosphatase activity of UBASH3B/STS-1/TULA-2, and thus, its regulatory effect may be based also on the phosphatase-independent mechanisms. Critical physiologic effects of these proteins have been demonstrated in T lymphocytes, platelets, stem cells, and other important cell types. These proteins have also been shown to play a key role in such pathologic conditions as autoimmunity, cancer, and thrombosis. The review focuses on the recent studies of this important family of cellular regulators.

A study of enterocyte membranes during activation of apoptotic processes in chronic carrageenan-induced gastroenterocolitis.

Aim To investigate the lipid membranes of rat enterocytes in chronic carrageenan-induced gastroenterocolitis accompanied by the activation of apoptotic processes. Methods Steady-state fluorescence spectroscopy: a study by fluorescent probes - by ortho-hydroxy derivatives of 2,5-diaryl-1,3- oxazole. Activity of apoptosis signal-regulating kinase 1 and poly (ADP-ribose) polymerase in small intestinal homogenates, blood serum levels of matrix metalloproteinase-2 and caspase-3 and the level of DNA fragmentation in small intestinal homogenates were determined. Results Biochemical analysis revealed that an activation of apoptotic processes occurred in the intestinal epithelium of rats during chronic carrageenan-induced gastroenterocolitis. The fluorescence probes showed that activation of apoptotic processes in carrageenan-induced gastroenterocolitis was accompanied by changes in polar regions of rat enterocyte membranes, while no changes were revealed in more hydrophobic regions of the membranes. Conclusion The increase in hydration of membranes was attributed to the activation of the apoptosis of enterocytes. It has been shown that a fluorescent probe (2-(2'-hydroxyphenyl)-5-phenyl-1,3-oxazole) can be used for the detection of apoptosis of enterocytes.

TULA-2 Deficiency Enhances Platelet Functional Responses to CLEC-2 Agonists.

Platelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors such as glycoprotein VI, the protease-activated receptors, and C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a HemITAM (hem-immunoreceptor tyrosine activation motif)-bearing receptor that binds podoplanin and signals through spleen tyrosine kinase (Syk). T-cell ubiquitin ligand-2 (TULA-2) is a protein tyrosine phosphatase that is highly expressed in platelets and targets phosphorylated Y352 of Syk. We wanted to determine whether TULA-2 regulates Syk phosphorylation and activity downstream of CLEC-2. To that end, we used TULA-2 knockout mice and wild-type (WT) littermate controls. We found that TULA-2 deficiency enhances the aggregation and secretion response following stimulation with an excitatory CLEC-2 antibody or the CLEC-2 agonist rhodocytin. Consistently, Syk phosphorylation of Y346 is enhanced, as well as phosphorylation of the downstream signaling molecule PLCγ2, in TULA-2 knockout platelets treated with either CLEC-2 antibody or rhodocytin, compared with WT control platelets. Furthermore, the kinetics of Syk phosphorylation, as well as that of PLCγ2 and SLP-76, is enhanced in TULA-2 knockout platelets treated with 2.5-µg/mL CLEC-2 antibody compared with WT platelets. Similarly, thromboxane production was enhanced, in both amount and kinetics, in TULA-2 -/- platelets treated with 2.5-µg/mL CLEC-2 antibody. TULA-2 acts as a negative regulator of CLEC-2 signaling by dephosphorylating Syk on Y346 and restraining subsequent Syk-mediated signaling.