Novel class of peptides disintegrating biological membranes to aid in the characterization of membrane proteins.

Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

nfrared thermography as a complementary method when diagnosing birth-related brachial plexus injury.

OBJECTIVES: The main objective of this study was to determine whether infrared thermography could be used as an efficient technique to evaluate the impact of a birth-related brachial plexus injury on the temperature of the injured arm and whether it could be used as a complementary method when diagnosing this injury in clinical praxis. BACKGROUND: Clinically, the brachial plexus injury is a peripheral paresis, which occurs when nerves that send signals from the spinal cord to the shoulder, arm, and hand are stretched or compressed. In principle, the brachial plexus injury, as a long-lasting injury, should be causing hypothermia of the injured arm. METHODS: The usage of contactless infrared thermography could offer a "new view" of the diagnostic process in this case. The present study, therefore, describes a process of clinical infrared thermography examination of three patients of different age and presents results from those examinations. RESULTS AND CONCLUSION: From our results, it can be confirmed that the birth-related brachial plexus injury affects the temperature of the affected arm, especially in the area of the cubital fossa, to an extent that the thermal camera is capable of detecting significant temperature differences between the healthy and injured arms (Tab. 3, Fig. 7, Ref. 13). Text in PDF www.elis.sk Keywords: birth brachial plexus injury, upper type palsy, peripheral palsy, infrared thermography.

Composition of raft-like cell membrane microdomains resistant to styrene-maleic acid copolymer (SMA) solubilization.

An advantageous alternative to the use of detergents in biochemical studies on membrane proteins are the recently developed styrene-maleic acid (SMA) amphipathic copolymers. In our recent study [1] we demonstrated that using this approach, most T cell membrane proteins were fully solubilized (presumably in small nanodiscs), while two types of raft proteins, GPI-anchored proteins and Src family kinases, were mostly present in much larger (>250 nm) membrane fragments markedly enriched in typical raft lipids, cholesterol and lipids containing saturated fatty acid residues. In the present study we demonstrate that disintegration of membranes of several other cell types by means of SMA copolymer follows a similar pattern and we provide a detailed proteomic and lipidomic characterization of these SMA-resistant membrane fragments (SRMs).

Chronic inflammation decreases HSC fitness by activating the druggable Jak/Stat3 signaling pathway.

Chronic inflammation represents a major threat to human health since long-term systemic inflammation is known to affect distinct tissues and organs. Recently, solid evidence demonstrated that chronic inflammation affects hematopoiesis; however, how chronic inflammation affects hematopoietic stem cells (HSCs) on the mechanistic level is poorly understood. Here, we employ a mouse model of chronic multifocal osteomyelitis (CMO) to assess the effects of a spontaneously developed inflammatory condition on HSCs. We demonstrate that hematopoietic and nonhematopoietic compartments in CMO BM contribute to HSC expansion and impair their function. Remarkably, our results suggest that the typical features of murine multifocal osteomyelitis and the HSC phenotype are mechanistically decoupled. We show that the CMO environment imprints a myeloid gene signature and imposes a pro-inflammatory profile on HSCs. We identify IL-6 and the Jak/Stat3 signaling pathway as critical mediators. However, while IL-6 and Stat3 blockage reduce HSC numbers in CMO mice, only inhibition of Stat3 activity significantly rescues their fitness. Our data emphasize the detrimental effects of chronic inflammation on stem cell function, opening new venues for treatment.

Molecular interactions of adaptor protein PSTPIP2 control neutrophil-mediated responses leading to autoinflammation.

Introduction: Autoinflammatory diseases are characterized by dysregulation of innate immune system leading to spontaneous sterile inflammation. One of the well-established animal models of this group of disorders is the mouse strain Pstpip2cmo . In this strain, the loss of adaptor protein PSTPIP2 leads to the autoinflammatory disease chronic multifocal osteomyelitis. It is manifested by sterile inflammation of the bones and surrounding soft tissues of the hind limbs and tail. The disease development is propelled by elevated production of IL-1ß and reactive oxygen species by neutrophil granulocytes. However, the molecular mechanisms linking PSTPIP2 and these pathways have not been established. Candidate proteins potentially involved in these mechanisms include PSTPIP2 binding partners, PEST family phosphatases (PEST-PTPs) and phosphoinositide phosphatase SHIP1. Methods: To address the role of these proteins in PSTPIP2-mediated control of inflammation, we have generated mouse strains in which PEST-PTP or SHIP1 binding sites in PSTPIP2 have been disrupted. In these mouse strains, we followed disease symptoms and various inflammation markers. Results: Our data show that mutation of the PEST-PTP binding site causes symptomatic disease, whereas mice lacking the SHIP1 interaction site remain asymptomatic. Importantly, both binding partners of PSTPIP2 contribute equally to the control of IL-1ß production, while PEST-PTPs have a dominant role in the regulation of reactive oxygen species. In addition, the interaction of PEST-PTPs with PSTPIP2 regulates the production of the chemokine CXCL2 by neutrophils. Its secretion likely creates a positive feedback loop that drives neutrophil recruitment to the affected tissues. Conclusions: We demonstrate that PSTPIP2-bound PEST-PTPs and SHIP1 together control the IL-1ß pathway. In addition, PEST-PTPs have unique roles in the control of reactive oxygen species and chemokine production, which in the absence of PEST-PTP binding to PSTPIP2 shift the balance towards symptomatic disease.

The receptor-type protein tyrosine phosphatase CD45 promotes onset and severity of IL-1ß-mediated autoinflammatory osteomyelitis.

A number of human autoinflammatory diseases manifest with severe inflammatory bone destruction. Mouse models of these diseases represent valuable tools that help us to understand molecular mechanisms triggering this bone autoinflammation. The Pstpip2cmo mouse strain is among the best characterized of these; it harbors a mutation resulting in the loss of adaptor protein PSTPIP2 and development of autoinflammatory osteomyelitis. In Pstpip2cmo mice, overproduction of interleukin-1ß (IL-1ß) and reactive oxygen species by neutrophil granulocytes leads to spontaneous inflammation of the bones and surrounding soft tissues. However, the upstream signaling events leading to this overproduction are poorly characterized. Here, we show that Pstpip2cmo mice deficient in major regulator of Src-family kinases (SFKs) receptor-type protein tyrosine phosphatase CD45 display delayed onset and lower severity of the disease, while the development of autoinflammation is not affected by deficiencies in Toll-like receptor signaling. Our data also show deregulation of pro-IL-1ß production by Pstpip2cmo neutrophils that are attenuated by CD45 deficiency. These data suggest a role for SFKs in autoinflammation. Together with previously published work on the involvement of protein tyrosine kinase spleen tyrosine kinase, they point to the role of receptors containing immunoreceptor tyrosine-based activation motifs, which after phosphorylation by SFKs recruit spleen tyrosine kinase for further signal propagation. We propose that this class of receptors triggers the events resulting in increased pro-IL-1ß synthesis and disease initiation and/or progression.

Regulation of Inflammatory Response by Transmembrane Adaptor Protein LST1.

LST1 is a small adaptor protein expressed in leukocytes of myeloid lineage. Due to the binding to protein tyrosine phosphatases SHP1 and SHP2 it was thought to have negative regulatory function in leukocyte signaling. It was also shown to be involved in cytoskeleton regulation and generation of tunneling nanotubes. LST1 gene is located in MHCIII locus close to many immunologically relevant genes. In addition, its expression increases under inflammatory conditions such as viral infection, rheumatoid arthritis and inflammatory bowel disease and its deficiency was shown to result in slightly increased sensitivity to influenza infection in mice. However, little else is known about its role in the immune system homeostasis and immune response. Here we show that similar to humans, LST1 is expressed in mice in the cells of the myeloid lineage. In vivo, its deficiency results in alterations in multiple leukocyte subset abundance in steady state and under inflammatory conditions. Moreover, LST1-deficient mice show significant level of resistance to dextran sodium sulphate (DSS) induced acute colitis, a model of inflammatory bowel disease. These data demonstrate that LST1 regulates leukocyte abundance in lymphoid organs and inflammatory response in the gut.

Dysregulated NADPH Oxidase Promotes Bone Damage in Murine Model of Autoinflammatory Osteomyelitis.

Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation. Pstpip2cmo mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1ß by neutrophil granulocytes. In this study, we show that in addition to IL-1ß, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase. Pstpip2cmo neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in Pstpip2cmo mice did not affect IL-1ß levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2cmo mice.

Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis.

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6-RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non-haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4-family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l-deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.

Investigation of flexibility of neuraminidase 150-loop using tamiflu derivatives in influenza A viruses H1N1 and H5N1.

This study focuses on design, synthesis and in vitro evaluation of inhibitory potency of two series of sialylmimetic that target an exosite ("150-cavity") adjacent to the active site of influenza neuraminidases from A/California/07/2009 (H1N1) pandemic strain and A/chicken/Nakorn-Patom/Thailand/CU-K2-2004 (H5N1). The structure-activity analysis as well as 3-D structure of the complex of parental compound with the pandemic neuraminidase p09N1 revealed high flexibility of the 150-cavity towards various modification of the neuraminidase inhibitors. Furthermore, our comparison of two methods for inhibition constant determination performed at slightly different pH values suggest that the experimental conditions of the measurement could dramatically influence the outcome of the analysis in the compound-dependent manner. Therefore, previously reported Ki values determined at non-physiological pH should be carefully scrutinized.

The use of styrene-maleic acid copolymer (SMA) for studies on T cell membrane rafts.

An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.

DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors.

Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.

Kinetic, Thermodynamic, and Structural Analysis of Drug Resistance Mutations in Neuraminidase from the 2009 Pandemic Influenza Virus.

Neuraminidase is the main target for current influenza drugs. Reduced susceptibility to oseltamivir, the most widely prescribed neuraminidase inhibitor, has been repeatedly reported. The resistance substitutions I223V and S247N, alone or in combination with the major oseltamivir-resistance mutation H275Y, have been observed in 2009 pandemic H1N1 viruses. We overexpressed and purified the ectodomain of wild-type neuraminidase from the A/California/07/2009 (H1N1) influenza virus, as well as variants containing H275Y, I223V, and S247N single mutations and H275Y/I223V and H275Y/S247N double mutations. We performed enzymological and thermodynamic analyses and structurally examined the resistance mechanism. Our results reveal that the I223V or S247N substitution alone confers only a moderate reduction in oseltamivir affinity. In contrast, the major oseltamivir resistance mutation H275Y causes a significant decrease in the enzyme's ability to bind this drug. Combination of H275Y with an I223V or S247N mutation results in extreme impairment of oseltamivir's inhibition potency. Our structural analyses revealed that the H275Y substitution has a major effect on the oseltamivir binding pose within the active site while the influence of other studied mutations is much less prominent. Our crystal structures also helped explain the augmenting effect on resistance of combining H275Y with both substitutions.

[Autoantibodies in systemic connective tissue disease and ANCA-associated vasculitis, their relationship to interstitial lung diseases and prognosis]. / Autoprotilátky u systémových onemocnení pojiva a ANCA asociovaných vaskulitid, jejich vztah k intersticiálním plicním procesum a prognóze.

Lung disease, including interstitial lung disease (ILD), is a frequent complication of systemic connective tissue disorders (CTD) and ANCA (anti-neutrophil cytoplasmic antibody) associated vasculitis (AAV). Pulmonary manifestations are prognostic factor of CTDs and vasculitis. Autoantibodies assessment is a part of differential diagnosis algorithm of lung diseases. Autoantibodies importance is mainly clinical-diagnostic. Using detection of some autoantibodies it is possible to determine prognosis of lung involvement, especially in CTDs.Key words: autoantibodies - connective tissue disease - interstitial lung diseases - prognosis - vasculitis.

The Transmembrane Adaptor Protein SCIMP Facilitates Sustained Dectin-1 Signaling in Dendritic Cells.

Transmembrane adaptor proteins are molecules specialized in recruiting cytoplasmic proteins to the proximity of the cell membrane as part of the signal transduction process. A member of this family, SLP65/SLP76, Csk-interacting membrane protein (SCIMP), recruits a complex of SLP65/SLP76 and Grb2 adaptor proteins, known to be involved in the activation of PLCγ1/2, Ras, and other pathways. SCIMP expression is restricted to antigen-presenting cells. In a previous cell line-based study, it was shown that, in B cells, SCIMP contributes to the reverse signaling in the immunological synapse, downstream of MHCII glycoproteins. There it mainly facilitates the activation of ERK MAP kinases. However, its importance for MHCII glycoprotein-dependent ERK signaling in primary B cells has not been analyzed. Moreover, its role in macrophages and dendritic cells has remained largely unknown. Here we present the results of our analysis of SCIMP-deficient mice. In these mice, we did not observe any defects in B cell signaling and B cell-dependent responses. On the other hand, we found that, in dendritic cells and macrophages, SCIMP expression is up-regulated after exposure to GM-CSF or the Dectin-1 agonist zymosan. Moreover, we found that SCIMP is strongly phosphorylated after Dectin-1 stimulation and that it participates in signal transduction downstream of this important pattern recognition receptor. Our analysis of SCIMP-deficient dendritic cells revealed that SCIMP specifically contributes to sustaining long-term MAP kinase signaling and cytokine production downstream of Dectin-1 because of an increased expression and sustained phosphorylation lasting at least 24 h after signal initiation.

Ion specific effects of alkali cations on the catalytic activity of HIV-1 protease.

Human immunodeficiency virus 1 protease (HIV-1 PR), an important therapeutic target for the treatment of AIDS, is one of the most well-studied enzymes. However, there is still much to learn about the regulation of the activity and inhibition of this key viral enzyme. Specifically, the mechanism of activation of HIV-1 PR from the viral polyprotein upon HIV maturation is still not understood. It has been suggested that external factors like pH or salt concentration might contribute to regulation of this crucial step in the viral life cycle. Recently, we analyzed the activity of HIV-1 PR in aqueous solutions of sodium and potassium chloride by experimental determination of enzyme kinetics and molecular dynamics simulations. We showed that the effect of salt concentration is cation-specific [Heyda et al., Phys. Chem. Chem. Phys., 2009 (11), 7599]. In this study, we extended this analysis for other alkali cations and found that the dependence of the initial velocity of peptide substrate hydrolysis on the nature of the cation follows the Hofmeister series, with the exception of caesium. Significantly higher catalytic efficiencies both in terms of substrate binding (K(M)) and turnover number (kcat) are observed in the presence of K+ compared to Na+ or Li+ at corresponding salt concentrations. Molecular dynamics simulations suggest that both lithium and sodium are attracted more strongly than potassium and caesium to the protein surface, mostly due to stronger interactions with carboxylate side chain groups of aspartates and glutamates. Furthermore, we observed a surprising decrease in the K(M) value for a specific substrate at very low salt concentration. The molecular mechanism of this phenomenon will be further analyzed.

HIV-1 protease inhibitor mutations affect the development of HIV-1 resistance to the maturation inhibitor bevirimat.

BACKGROUND: Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance. RESULTS: Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different proteases. CONCLUSIONS: These findings highlight the complicated interactions between the viral protease and its substrate. By providing a better understanding of these interactions, we aim to help guide the development of second generation maturation inhibitors.

Ion specific effects of sodium and potassium on the catalytic activity of HIV-1 protease.

The behavior of HIV-1 protease in aqueous NaCl and KCl solutions is investigated by kinetic measurements and molecular dynamics simulations. Experiments show cation-specific effects on the enzymatic activity. The initial velocity of peptide substrate hydrolysis increases with salt concentration more dramatically in potassium than in sodium chloride solutions. Furthermore, significantly higher catalytic efficiencies (k(cat)/K(M)) are observed in the presence of K+ compared to Na+ at comparable salt concentrations. Molecular dynamics simulations provide insight into this ion-specific behavior. Sodium is attracted more strongly than potassium to the protein surface primarily due to stronger interactions with carboxylate side chain groups of aspartates and glutamates. These effects are of particular importance for acidic amino acid residues at or near the active site of the enzyme, including a pair of aspartates at the entrance to the reaction cavity. We infer that the presence of more Na+ than K+ at the active site leads to a lower increase in enzymatic activity with increasing salt concentration in the presence of Na+, likely due to the ability of the alkali cations at the active site to lower the efficiency of substrate binding.

Design of HIV protease inhibitors based on inorganic polyhedral metallacarboranes.

HIV protease (HIV PR) is a primary target for anti-HIV drug design. We have previously identified and characterized substituted metallacarboranes as a new class of HIV protease inhibitors. In a structure-guided drug design effort, we connected the two cobalt bis(dicarbollide) clusters with a linker to substituted ammonium group and obtained a set of compounds based on a lead formula [H(2)N-(8-(C(2)H(4)O)(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))-3,3'-Co)(2)]Na. We explored inhibition properties of these compounds with various substitutions, determined the HIV PR:inhibitor crystal structure, and computationally explored the conformational space of the linker. Our results prove the capacity of linker-substituted dual-cage cobalt bis(dicarbollides) as lead compounds for design of more potent inhibitors of HIV PR.

Current and Novel Inhibitors of HIV Protease.

The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed.