Immunogenic cell death (ICD), a functionally peculiar type of apoptosis, represents a unique way to deliver danger-associated molecular patterns (DAMPs) to the tumor microenvironment. Once emitted by dying cancer cells, DAMPs orchestrate antigen-specific immune responses by acting on both innate and adaptive components of the immune system. Accumulating preclinical and clinical evidence indicates that one of these DAMPs, calreticulin (CALR) represents a novel powerful prognostic biomarker, reflecting the activation of a clinically relevant anticancer immune response in different cancer malignancies. Therefore, the assessment of CALR emission can provide a therapeutic tool for the stratification of cancer patients and the identification of individuals that are intrinsically capable to respond to a particular treatment. Here we describe methods for the quantification of CALR exposure in the tumor microenvironment of cancer patients by flow cytometry and immunohistochemistry.
- MeSH
- imunogenní buněčná smrt * MeSH
- imunohistochemie metody MeSH
- kalretikulin analýza imunologie MeSH
- lidé MeSH
- nádorové biomarkery analýza imunologie MeSH
- nádorové mikroprostředí MeSH
- nádory imunologie patologie MeSH
- průtoková cytometrie metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Dendritic cells have been widely investigated in cancer immunotherapy clinical trials for the last two decades mainly due to their robust ability to elicit an adaptive anticancer immune response of the cellular and humoral types. Immature DCs can be easily loaded with desired antigens. However, to become efficient antigen-presenting cells, DCs must first undergo a process of maturation. Protocols for the generation of DCs for use in cancer immunotherapy, including the generation of a large number of immature DCs for antigen pulsing and the selection of a well-defined immunostimulatory agent to achieve complete and reproducible maturation, which is a crucial step for further stimulation of T cell activation, must carefully consider the characteristics of DC physiology. In this report, we provided a detailed protocol for DC generation, pulsation and activation with the subsequent induction of T cell-specific immune responses.
- MeSH
- aktivace lymfocytů * MeSH
- apoptóza MeSH
- dendritické buňky imunologie MeSH
- fagocytóza MeSH
- kultivované buňky MeSH
- lidé MeSH
- nádory imunologie MeSH
- průtoková cytometrie metody MeSH
- T-lymfocyty imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Natural killer (NK) cells constitute the predominant innate lymphocyte subset that mediates the anti-viral and anti-tumor immune responses. NK cells use an array of innate receptors to sense their environment and to respond to infections, cellular stress and transformation. The resulting NK cell activation, including cytotoxicity and cytokine production, is a fundamental component of the early immune response. The most recent discoveries in NK cell biology have stimulated the translational research that has led to remarkable results for the treatment of human malignancies. Therefore, the rapid isolation of NK cells from the peripheral blood or tumor microenvironment and the subsequent assessment of cytolytic function are crucial to the study of their potency and NK cell-mediated immunosurveillance. Here, we provide protocols for NK cell isolation and the assessment of NK cell cytotoxicity using flow cytometry.
Enzymes are being increasingly utilized for acceleration of industrially and pharmaceutically critical chemical reactions. The strong demand for finding robust and efficient biocatalysts for these applications can be satisfied via the exploration of enzyme diversity. The first strategy is to mine the natural diversity, represented by millions of sequences available in the public genomic databases, by using computational approaches. Alternatively, metagenomic libraries can be targeted experimentally or computationally to explore the natural diversity of a specific environment. The second strategy, known as directed evolution, is to generate man-made diversity in the laboratory using gene mutagenesis and screen the constructed library of mutants. The selected hits must be experimentally characterized in both strategies, which currently represent the rate-limiting step in the process of diversity exploration. The traditional techniques used for biochemical characterization are time-demanding, cost, and sample volume ineffective, and low-throughput. Therefore, the development and implementation of high-throughput experimental methods are essential for discovering novel enzymes. This chapter describes the experimental protocols employing the combination of robust production and high-throughput microscale biochemical characterization of enzyme variants. We validated its applicability against the model enzyme family of haloalkane dehalogenases. These protocols can be adapted to other enzyme families, paving the way towards the functional characterization and quick identification of novel biocatalysts.
- MeSH
- genová knihovna MeSH
- lidé MeSH
- metagenomika * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Understanding the RNA binding specificity of protein is of primary interest to decipher their function in the cell. Here, we review the methodology used to solve the structures of protein-RNA complexes using solution-state NMR spectroscopy: from sample preparation to structure calculation procedures. We also describe how molecular dynamics simulations can help providing additional information on the role of key amino acid side chains and of water molecules in protein-RNA recognition.
- MeSH
- CELF proteiny chemie metabolismus MeSH
- interakční proteinové domény a motivy MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie metody MeSH
- RNA chemie genetika metabolismus MeSH
- sestřihové faktory chemie metabolismus MeSH
- simulace molekulární dynamiky * MeSH
- termodynamika MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.
- MeSH
- bakteriální proteiny chemie genetika izolace a purifikace metabolismus MeSH
- buněčná membrána metabolismus MeSH
- enzymatické testy přístrojové vybavení metody MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční mikroskopie přístrojové vybavení metody MeSH
- mutageneze MeSH
- protozoální proteiny chemie genetika izolace a purifikace metabolismus MeSH
- pyrofosfatasy chemie genetika izolace a purifikace metabolismus MeSH
- racionální návrh léčiv MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- rezonanční přenos fluorescenční energie přístrojové vybavení metody MeSH
- Saccharomyces cerevisiae MeSH
- sekvenční seřazení MeSH
- simulace molekulární dynamiky * MeSH
- software MeSH
- zobrazení jednotlivé molekuly přístrojové vybavení metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Haloalkane dehalogenases degrade halogenated compounds to corresponding alcohols by a hydrolytic mechanism. These enzymes are being intensively investigated as model systems in experimental and in silico studies of enzyme mechanism and evolution, but also hold importance as useful biocatalysts for a number of biotechnological applications. Haloalkane dehalogenases originate from various organisms including bacteria (degraders, symbionts, or pathogens), eukaryotes, and archaea. Several members of this enzyme family have been found in marine organisms. The marine environment represents a good source of enzymes with novel properties, because of its diverse living conditions. A number of novel dehalogenases isolated from marine environments show interesting characteristics such as high activity, unusually broad substrate specificity, stability, or selectivity. In this chapter, the overview of haloalkane dehalogenases from marine organisms is presented and their characteristics are summarized together with an overview of the methods for their identification and biochemical characterization.
- MeSH
- aldehydy metabolismus MeSH
- alkany metabolismus MeSH
- biokatalýza MeSH
- biotechnologie metody MeSH
- enzymatické testy metody MeSH
- halogeny metabolismus MeSH
- hydrolasy chemie izolace a purifikace metabolismus MeSH
- substrátová specifita MeSH
- technologie zelené chemie metody MeSH
- vodní organismy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.
- MeSH
- DNA vazebné proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- endopeptidasy biosyntéza chemie genetika izolace a purifikace MeSH
- Escherichia coli enzymologie MeSH
- Haemophilus influenzae enzymologie MeSH
- kinetika MeSH
- lipidové dvojvrstvy chemie MeSH
- membránové proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- molekulární biologie metody MeSH
- proteiny z Escherichia coli biosyntéza chemie genetika izolace a purifikace MeSH
- Providencia enzymologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KM and Vmax to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein.
- MeSH
- buněčná membrána enzymologie MeSH
- DNA vazebné proteiny chemie izolace a purifikace metabolismus MeSH
- endopeptidasy chemie izolace a purifikace metabolismus MeSH
- enzymatické testy metody MeSH
- Escherichia coli enzymologie MeSH
- katalytická doména MeSH
- membránové proteiny chemie izolace a purifikace metabolismus MeSH
- proteiny z Escherichia coli chemie izolace a purifikace metabolismus MeSH
- proteolýza * MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Intramembrane serine proteases of the rhomboid family are widespread, and their gradually uncovered functions in different organisms already suggest medical relevance for infectious diseases and cancer. However, selective inhibitors that could serve as research tools for rhomboids, for validation of their disease relevance, or as templates for drug development are lacking. Here I summarize the current knowledge about rhomboid protease mechanism and specificity, overview the currently used inhibitors, and conclude by proposing avenues for future development of rhomboid protease inhibitors.
- MeSH
- endopeptidasy chemie metabolismus MeSH
- inhibitory proteas chemie MeSH
- lidé MeSH
- membránové proteiny antagonisté a inhibitory chemie MeSH
- substrátová specifita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
