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  • The alpha helix of the intermediate region in hGBP-1 acts as a coupler for enhanced GMP formation
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2020-01-16
    Sudeepa Rajan; Apurba Kumar Sau

    The interferon-gamma inducible large GTPase human guanylate binding protein-1 (hGBP-1) plays a key role in anti-pathogenic and anti-proliferative functions. This protein hydrolyzes GTP to both GDP and GMP (predominant product) through sequential phosphate cleavages, which makes it functionally distinct from other GTPases. Previous study on truncated variants of hGBP-1 suggested that the α-helix present in the intermediate region is essential for dimerization and thus for GMP formation. However, the role of this helix in the full-length protein in GMP formation is not clearly understood. Here, we present that substitution of the helix with a Gly-rich flexible (GGS)3 sequence in the full-length hGBP-1 (termed as linker protein) showed a drastic decrease in GMP formation. Unlike wild-type, the linker protein is not capable of undergoing substrate-induced dimerization and thereby transition state-induced tetramerization, suggesting the importance of the helix in oligomerization. Furthermore, we examined the effect of interactions between this helix and the α2-helix of the globular domain in GMP formation through mutational studies. The L118G mutation in the α2-helix showed a significantly reduced GMP formation. These results indicate that the interactions of the α-helix with the α2-helix are essential for enhanced GMP production. We propose that these interactions help in the oligomerization-assisted proper positioning of the catalytic machinery for efficient second phosphate cleavage. These findings thus provide a better understanding into the regulation of GMP formation in a large GTPase hGBP-1.

    更新日期:2020-01-17
  • Changes in strand 6B and helix B during neuroserpin inhibition: Implication in severity of clinical phenotype
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2020-01-16
    Mohammad Farhan Ali; Abhinav Kaushik; Dinesh Gupta; Shoyab Ansari; Mohamad Aman Jairajpuri

    Neuroserpin (NS) is predominantly expressed in brain and inhibits tissue-type plasminogen activator (tPA) with implications in brain development and memory. Nature of conformational change in pathological variants in strand 6B and helix B of NS that cause a relatively mild to severe epilepsy (and/or dementia) remains largely elusive. MD simulation with wild type (WT) NS, strand 6B and helix B variants indicated that substitution in this region affects the conformation of the strands 5B, 5A and reactive centre loop. Therefore, we designed variants of NS in strand 6B (I46D and F48S) and helix B (A54F, L55A and L55P) to investigate their role in tPA inhibition mechanism and propensity to aggregate. An interaction analysis showed disturbance of a hydrophobic patch centered at strands 5B, 6B and helix B in I46D and F48S but not in A54F, L55A, L55P and WT NS. Purified I46D, F48S and L55P variants showed decrease in fluorescence emission intensity but with similar α-helical content, results of A54F and L55A were comparable to WT NS. Analysis of tPA inhibition showed marginal effect on A54F and L55A variant with tPA-NS complex formation. In contrast, I46D, F48S and L55P variants showed massive decrease in tPA inhibition, with no tPA-NS complex formation. Analysis of native PAGE under under polymerization condition showed prompt conversion of I46D, F48S and L55P to latent conformation but not A54F and L55A variants. Identification of these novel conformational changes will aid in the understanding of variable clinical phenotype of shutter region NS variants and other serpins.

    更新日期:2020-01-17
  • Falcipain cysteine proteases of malaria parasites: An update
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2020-01-09
    Philip J. Rosenthal

    Background The malaria parasite Plasmodium falciparum expresses four related papain-family cysteine proteases known as falcipains. These proteases play critical roles in the parasite life cycle, and as such are potential targets for new modes of antimalarial chemotherapy, as discussed in this review. Scope of review This review summarizes available knowledge describing falcipain cysteine proteases of malaria parasites. Major conclusions Based on available data the falcipains can be broken into two sub-families, the falcipain-1 and the falcipain-2/3 sub-families. Falcipain-1 has been difficult to study; it appears to play its most important roles in nonerythrocytic parasites, but not the erythrocytic stage responsible for human disease. Falcipain-2 and falcipain-3 have similar biochemical features, and are expressed sequentially during the erythrocytic cycle. Inhibition of either of these enzymes blocks hemoglobin hydrolysis and completion of the parasite developmental cycle. Knockout of falcipain-2 blocks hemoglobin hydrolysis, but parasites recover, presumably due to subsequent expression of falcipain-3. Knockout of falcipain-3 has not been possible, suggesting that the protease is essential for erythrocytic parasites. Determination of structures of falcipains and extensive chemistry efforts have facilitated identification of numerous small molecule falcipain inhibitors as potential new antimalarial agents. Other malaria parasites express close homologs of falcipain-1 and falcipain-2/3 proteases, suggesting that agents that target the falcipains will also be active against other human malaria parasites. General Significance. Falcipain-2 and falcipain-3 play vital roles during the erythrocytic stage of infection with P. falciparum and thus are promising targets for new agents to treat malaria.

    更新日期:2020-01-09
  • Cysteinyl cathepsins in cardiovascular diseases
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2020-01-09
    Xian Zhang; Songyuan Luo; Minjie Wang; Guo-Ping Shi

    Cysteinyl cathepsins are lysosomal/endosomal proteases that mediate bulk protein degradation in these intracellular acidic compartments. Yet, studies indicate that these proteases also appear in the nucleus, nuclear membrane, cytosol, plasma membrane, and extracellular space. Patients with cardiovascular diseases (CVD) show increased levels of cathepsins in the heart, aorta, and plasma. Plasma cathepsins often serve as biomarkers or risk factors of CVD. In aortic diseases, such as atherosclerosis and abdominal aneurysms, cathepsins play pathogenic roles, but many of the same cathepsins are cardioprotective in hypertensive, hypertrophic, and infarcted hearts. During the development of CVD, cathepsins are regulated by inflammatory cytokines, growth factors, hypertensive stimuli, oxidative stress, and many others. Cathepsin activities in inflammatory molecule activation, immunity, cell migration, cholesterol metabolism, neovascularization, cell death, cell signaling, and tissue fibrosis all contribute to CVD and are reviewed in this article in memory of Dr. Nobuhiko Katunuma for his contribution to the field.

    更新日期:2020-01-09
  • High-resolution structure of intramolecularly proteolyzed human mucin-1 SEA domain
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2020-01-07
    Martín E. Noguera; Jean Jakoncic; Mario R. Ermácora
    更新日期:2020-01-07
  • Regulation of Herbaspirillum seropedicae NifA by the GlnK PII signal transduction protein is mediated by effectors binding to allosteric sites
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-12-19
    Adriano Alves Stefanello; Marco Aurélio Schuler de Oliveira; Emanuel Maltempi de Souza; Fábio de Oliveira Pedrosa; Leda Satie Chubatsu; Luciano Fernandes Huergo; Ray Dixon; Rose Adele Monteiro

    Herbaspirillum seropedicae is a plant growth promoting bacterium that is able to fix nitrogen and to colonize the surface and internal tissues of important crops. Nitrogen fixation in H. seropedicae is regulated at the transcriptional level by the prokaryotic enhancer binding protein NifA. The activity of NifA is negatively affected by oxygen and positively stimulated by interaction with GlnK, a PII signaling protein that monitors intracellular levels of the key metabolite 2-oxoglutarate (2-OG) and functions as an indirect sensor of the intracellular nitrogen status. GlnK is also subjected to a cycle of reversible uridylylation in response to intracellular levels of glutamine. Previous studies have established the role of the N-terminal GAF domain of NifA in intramolecular repression of NifA activity and the role of GlnK in relieving this inhibition under nitrogen-limiting conditions. However, the mechanism of this control of NifA activity is not fully understood. Here, we constructed a series of GlnK variants to elucidate the role of uridylylation and effector binding during the process of NifA activation. Our data support a model whereby GlnK uridylylation is not necessary to activate NifA. On the other hand, binding of 2-OG and MgATP to GlnK are very important for NifA activation and constitute the most important signal of cellular nitrogen status to NifA.

    更新日期:2019-12-20
  • Analysis of uracil DNA glycosylase (UNG2) stimulation by replication protein A (RPA) at ssDNA-dsDNA junctions
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-12-19
    Brian P. Weiser

    Replication Protein A (RPA) is a single-stranded DNA binding protein that interacts with DNA repair proteins including Uracil DNA Glycosylase (UNG2). Here, I report DNA binding and activity assays using purified recombinant RPA and UNG2. Using synthetic DNA substrates, RPA was found to promote UNG2's interaction with ssDNA-dsDNA junctions regardless of the DNA strand polarity surrounding the junction. RPA stimulated UNG2's removal of uracil bases paired with adenine or guanine in DNA as much as 17-fold when the uracil was positioned 21 bps from ssDNA-dsDNA junctions, with the largest degree of stimulation occurring when RPA was in molar excess compared to DNA. I found that RPA becomes sequestered on ssDNA regions surrounding junctions which promotes its spatial targeting of UNG2 near the junction. However, when RPA concentration exceeds free ssDNA, RPA promotes UNG2's activity without spatial constraints in dsDNA regions. These effects of RPA on UNG2 were found to be mediated primarily by interactions between RPA's winged-helix domain and UNG2's N-terminal domain, but when the winged-helix domain is unavailable, a secondary interaction between UNG2's N-terminal domain and RPA can occur. This work supports a widespread role for RPA in stimulating uracil base excision repair.

    更新日期:2019-12-20
  • Twenty-five years of nomenclature and classification of proteolytic enzymes
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-12-12
    Neil D. Rawlings

    Proteolytic enzymes and their homologues have been classified into clans by comparing the tertiary structures of the peptidase domains, into families by comparing the protein sequences of the peptidase domains, and into protein-species by comparing various attributes including domain architecture, substrate preference, inhibitor interactions, subcellular location, and phylogeny. The results are compared with the earlier classification by Rawlings & Barrett (Rawlings and Barrett, 1993 [1]). The numbers of sequences, protein-species, families, clans and even catalytic type have substantially increased during the intervening 26 years. The alternative classifications by catalytic type and/or activity are shown not to reflect evolutionary relationships.

    更新日期:2019-12-13
  • The structure of a prokaryotic feruloyl-CoA hydratase-lyase from a lignin-degrading consortium with high oligomerization stability under extreme pHs
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-12-10
    Marcelo Vizoná Liberato, Juscemácia N. Araújo, Victoria Sodré, Thiago Augusto Gonçalvez, Nathalia Vilela, Eduardo Cruz Moraes, Wanius Garcia, Fabio Marcio Squina

    In the context of increasing demand for renewable alternatives of fuels and chemicals, the valorization of lignin emerges as a value-adding strategy in biorefineries and an alternative to petroleum-derived molecules. One of the compounds derived from lignin is ferulic acid (FA), which can be converted into valuable molecules such as vanillin. In microorganisms, FA biotransformation into vanillin can occur via a two-step reaction catalyzed by the sequential activity of a feruloyl-CoA synthetase (FCS) and an feruloyl-CoA hydratase-lyase (FCHL), which could be exploited industrially. In this study, a prokaryotic FCHL derived from a lignin-degrading microbial consortium (named LM-FCHL) was cloned, successfully expressed in soluble form and purified. The crystal structure was solved and refined at 2.1 Å resolution. The LM-FCHL is a hexamer composed of a dimer of trimers, which showed to be quite stable under extreme pH conditions. Finally, small angle X-ray scattering corroborates the hexameric state in solution and indicates flexibility in the protein structure. The present study contributes to the field of lignin valorization to valuable molecules by establishing the biophysical and structural characterization for a novel FCHL member of unique characteristics.

    更新日期:2019-12-11
  • Small protease inhibitors in tick saliva and salivary glands and their role in tick-host-pathogen interactions
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-12-07
    Larissa Almeida Martins, Jan Kotál, Chaima Bensaoud, Jindřich Chmelař, Michail Kotsyfakis

    Ticks must durably suppress vertebrate host responses (hemostasis, inflammation, immunity) to avoid rejection and act as vectors of many pathogenic microorganisms that cause disease in humans and animals. Transcriptomics and proteomics studies have been used to study tick-host-pathogen interactions and have facilitated the systematic characterization of salivary composition and molecular dynamics throughout tick feeding. Tick saliva contains a complement of protease inhibitors that are differentially produced during feeding, many of which inhibit blood coagulation, platelet aggregation, vasodilation, and immunity. Here we focus on two major groups of protease inhibitors, the small molecular weight Kunitz inhibitors and cystatins. We discuss their role in tick-host-pathogen interactions, how they mediate the interaction between ticks and their hosts, and how they might be exploited both by pathogens to invade hosts and as candidates for the treatment of various human pathologies.

    更新日期:2019-12-07
  • Sterilization of epidermal growth factor with supercritical carbon dioxide and peracetic acid; analysis of changes at the amino acid and protein level
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-29
    David M. Bednarski, Ellen E. Lantz, Cedric E. Bobst, Anthony R. Eisenhut, Stephen J. Eyles, Julien P. Fey

    Aseptic processing and terminal sterilization become increasingly challenging as medical devices become more complex and include active biologics. Terminal sterilization is preferred for patient safety and production costs. We aimed to determine how sterilization using supercritical CO2 (scCO2) with low levels of peracetic acid (PAA) affects amino acids and human epidermal growth factor (EGF) as a model protein. In a benchtop reactivity test, the amino acids methionine, tryptophan, arginine and lysine reacted with low levels of PAA in solution. At PAA levels used for scCO2 sterilization, however, mass spectrometry only identified oxidative adducts on methionine and tryptophan. Mass spectrometry analysis of EGF exposed to scCO2/PAA identified oxidative adducts on residues Met21, Trp49 and Trp50, as well as a low level of truncations after residues Trp49 and Trp50. Importantly, processing of EGF in solution with scCO2 did not affect its native conformation, and sterilized EGF maintained its activity in cell proliferation assays. When processing samples in lyophilized form with scCO2/PAA, amino acids did not react with PAA and the presence of adducts was strongly reduced on methionine and tryptophan, both as single amino acids and in EGF. Truncation after tryptophan residues did not occur. EGF sterilized in the lyophilized form retained its activity when processing occurred with added moisture. These results have significant implications for the maintenance of biological function in sterilized decellularized scaffolds and the ability to manufacture terminally sterilized combination devices containing therapeutic peptides or proteins.

    更新日期:2019-11-29
  • Characterization of pyranose oxidase variants for bioelectrocatalytic applications
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-27
    Annabelle T. Abrera, Hucheng Chang, Daniel Kracher, Roland Ludwig, Dietmar Haltrich

    Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H2O2. POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with improved electron acceptor turnover and reduced oxygen turnover were characterized as potential anode biocatalysts. Pre-steady-state kinetics of the oxidative half-reaction of ToPOx variants T166R, Q448H, L545C, and L547R with these alternative electron acceptors were evaluated using stopped-flow spectrophotometry. Higher kinetic constants were observed as compared to the wild-type ToPOx for some of the variants. Subsequently, the variants were immobilized on glassy carbon electrodes. Cyclic voltammetry measurements were performed to measure the electrochemical responses of these variants with glucose as substrate in the presence of 1,4-BQ, DCPIP, or ferrocene methanol as redox mediators. High catalytic efficiencies (Imaxapp/KMapp) compared to the wild-type POx proved the potential of these variants for future bioelectrocatalytic applications, in biosensors or biofuel cells. Among the variants, L545C showed the most desirable properties as determined kinetically and electrochemically.

    更新日期:2019-11-28
  • New insights into human endometrial aminopeptidases in both implantation and menstruation
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-23
    Shigehiko Mizutani, Kunio Matsumoto, Yukio Kato, Eita Mizutani, Hidesuke Mizutani, Akira Iwase, Kiyosumi Shibata

    The endometrium cycle involves proliferation of endometrial epithelial cells in preparation for implantation of fertilized ovum. With ovulation, the endometrium secretes nutrients such as peptides and amino acids into the endometrial cavity. The histological evidence of ovulation in normal menstrual cycle includes subnuclear glycogen vacuoles surrounded by placental leucine aminopeptidase (P-LAP) in endometrial epithelial cells. P-LAP is an essentially involved in intracellular trafficking of glucose transporter (GLUT) 4 which is primarily important for glucose uptake in skeletal muscles and fat tissues. On the other hand, glucose influx from blood into endometrial epithelial cells is not mainly mediated by GLUTs, but by coincident appearing progesterone just after ovulation. Progesterone increases permeability of not only plasma membranes, but also lysosomal membranes, and this may be primarily involved in glucose influx. Progesterone also expands the exocytosis in the endometrium after ovulation, and endometrial secretion after ovulation is possibly apocrine and holocrine, which is augmented and exaggerated exocytosis of the lysosomal contents. The endometrial spiral arteries/arterioles are surrounded by endometrial stromal cells which are differentiated into decidual/pre-decidual cells. Decidual cells are devoid of aminopeptidase A (APA), possibly leading to enhancement of Angiotensin-II action in decidual cell area due to loss of its degradation by APA. Angiotensin-II is thought to exert growth-factor-like effects in post-implantation embryos in decidual cells, thereby contributing to implantation. Without implantation, angiotensin-II constricts the endometrial spiral arteries/arterioles to promote menstruation. Thus, P-LAP and APA may be involved in homeostasis in uterus via regulating glucose transport and vasoconstrictive peptides.

    更新日期:2019-11-26
  • On the relationship between structure and catalytic effectiveness in solid surface-immobilized enzymes: Advances in methodology and the quest for a single-molecule perspective
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-25
    Juan M. Bolivar, Bernd Nidetzky

    The integration of enzymes with solid materials is important in many biotechnological applications, including the use of immobilized enzymes for biocatalytic synthesis. The development of functional enzyme-material composites is restrained by the lack of molecular-level insight into the behavior of enzymes in confined, surface-near environments. Here, we review recent advances in surface-sensitive spectroscopic techniques that push boundaries for the determination of enzyme structure and orientation at the solid-liquid interface. We discuss recent evidence from single-molecule studies showing that analyses sensitive to the temporal and spatial heterogeneities in immobilized enzymes can succeed in disentangling the effects of conformational stability and active-site accessibility on activity. Different immobilization methods involve distinct trade-off between these effects, thus emphasizing the need for a holistic (systems) view of immobilized enzymes for the rational development of practical biocatalysts.

    更新日期:2019-11-26
  • The substrate of the glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa provides structural stability
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-22
    Edaena Benítez-Rangel, Annia Rodríguez-Hernández, Roberto Velasco-García

    In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP+. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the Kd values determined for the PaG6PDH-G6P complex (78.0 ± 7.9 μM) and the K0.5 values determined for G6P (57.9 ± 2.5 and 104.5 ± 9.3 μM in the NADP+- and NAD+-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD+ and NADP+ from those that use NADP+ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.

    更新日期:2019-11-22
  • Metagenomics: Is it a powerful tool to obtain lipases for application in biocatalysis?
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-20
    Janaina Marques de Almeida, Robson Carlos Alnoch, Emanuel Maltempi de Souza, David Alexander Mitchell, Nadia Krieger

    In recent years, metagenomic strategies have been widely used to isolate and identify new enzymes from uncultivable components of microbial communities. Among these enzymes, various lipases have been obtained from metagenomic libraries from different environments and characterized. Although many of these lipases have characteristics that could make them interesting for application in biocatalysis, relatively little work has been done to evaluate their potential to catalyze industrially important reactions. In the present article, we highlight the latest research on lipases obtained through metagenomic tools, focusing on studies of activity and stability and investigations of application in biocatalysis. We also discuss the challenges of metagenomic approaches for the bioprospecting of new lipases.

    更新日期:2019-11-20
  • 更新日期:2019-11-20
  • Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-16
    Rhys Cutlan, Simone De Rose, Michail N. Isupov, Jennifer A. Littlechild, Nicholas J. Harmer

    Biocatalysis, the use of enzymes in chemical transformations, is an important green chemistry tool. Cascade reactions combine different enzyme activities in a sequential set of reactions. Cascades can occur within a living (usually bacterial) cell; in vitro in ‘one pot’ systems where the desired enzymes are mixed together to carry out the multi-enzyme reaction; or using microfluidic systems. Microfluidics offers particular advantages when the product of the reaction inhibits the enzyme(s). In vitro systems allow variation of different enzyme concentrations to optimise the metabolic ‘flux’, and the addition of enzyme cofactors as required. Cascades including cofactor recycling systems and modelling approaches are being developed to optimise cascades for wider industrial scale use. Two industrially important enzymes, transaminases and carboxylic acid reductases are used as examples regarding their applications in cascade reactions with other enzyme classes to obtain important synthons of pharmaceutical interest.

    更新日期:2019-11-18
  • Xylanases from marine microorganisms: A brief overview on scope, sources, features and potential applications
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-16
    Fatemeh Izadpanah Qeshmi, Ahmad Homaei, Pedro Fernandes, Roohullah Hemmati, Bauke W. Dijkstra, Khosro Khajeh
    更新日期:2019-11-18
  • Biochemical properties and biotechnological applications of microbial enzymes involved in the degradation of polyester-type plastics
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-16
    Aneta K. Urbanek, Aleksandra M. Mirończuk, Alberto García-Martín, Ana Saborido, Isabel de la Mata, Miguel Arroyo

    Application of polyester-degrading enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. Many hydrolases from several fungi and bacteria have been discovered and successfully evaluated for their activity towards different aliphatic polyesters (PHA, PBS, PBSA, PCL, PLA), aromatic polyesters (PET, PBT, PMT) as well as their co-polyesters (PBST, PBAT, PBSTIL). This revision gives an up-to-date overview on the main biochemical features and biotechnological applications of those reported enzymes which are able to degrade polyester-based plastics, including different microbial polyester depolymerases, esterases, cutinase-like enzymes and lipases. Summarized information includes available protein sequences with the corresponding accession numbers deposited in NCBI server, 3D resolved structures, and data about optimal conditions for enzymatic activity and stability of many of these microbial enzymes that would be helpful for researchers in this topic. Although screening and identification of new native polyester hydrolases from microbial sources is undeniable according to literature, we briefly highlight the importance of the design of improved enzymes towards recalcitrant aromatic polyesters through different approaches that include site-directed mutagenesis and surface protein engineering.

    更新日期:2019-11-18
  • Identification and application of threonine aldolase for synthesis of valuable α-amino, β-hydroxy-building blocks
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-15
    Mathieu Ligibel, Charles Moore, Robert Bruccoleri, Radka Snajdrova

    Chiral β-hydroxy α-amino acid structural motifs are interesting and common synthons present in multiple APIs and drug candidates. To access these chiral building blocks either multistep chemical syntheses are required or the application of threonine aldolases, which catalyze aldol reactions between an aldehyde and glycine. Bioinformatics tools have been utilized to identify the gene encoding threonine aldolase from Vanrija humicola and subsequent preparation of its recombinant version from E. coli fermentation. We planned to implement this enzyme as a key step to access the synthesis of our target API. Beyond this specific application, the aldolase was purified, characterized and the substrate scope of this enzyme further investigated. A number of enzymatic reactions were scaled-up and the products recovered to assess the diastereoselectivity and scalability of this asymmetric synthetic approach towards β-hydroxy α-amino acid chiral building blocks.

    更新日期:2019-11-18
  • A novel enzymatic tool for transferring GalNAc moiety onto challenging acceptors
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-15
    Pavlína Nekvasilová, Iveta Andreasová, Lucie Petrásková, Helena Pelantová, Vladimír Křen, Pavla Bojarová

    The β-N-acetylhexosaminidase from Penicillium oxalicum (PoHex; EC 3.2.1.52) is a fungal glycosidase with an outstandingly high GalNAcase/GlcNAcase activity ratio. It has a remarkable synthetic capability and can process carbohydrates functionalized at various positions. However, the production in the native fungal host is lengthy, unselective and purification from the fungal medium is complicated and low yielding. We present here a novel production method of this enzyme in the eukaryotic host of Pichia pastoris, followed by elegant one-step purification to homogeneity. The resulting recombinant enzyme has improved biochemical and catalytic properties compared to the fungal wild type. Its good production yield (11 mg/400 mL cultivation media) greatly expands the scope of synthetic applications. We further demonstrate the synthetic utility and broad acceptor specificity of recombinant PoHex in the glycosylation of a series of challenging acceptors with varying structural architectures, namely secondary and tertiary hydroxyl, aldoxime and a poly-hydroxylated compound.

    更新日期:2019-11-18
  • Energy migration captures membrane-induced oligomerization of the prion protein
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-15
    Aishwarya Agarwal, Debapriya Das, Tisya Banerjee, Samrat Mukhopadhyay
    更新日期:2019-11-18
  • Unbiased libraries in protein directed evolution
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-15
    Victor Sayous, Paul Lubrano, Yanyan Li, Carlos G. Acevedo-Rocha

    Directed evolution is a powerful approach to study the molecular basis of protein evolution and to engineer proteins for a wide range of applications in synthetic organic chemistry and biotechnology. There are many methods based on random or focused mutagenesis to engineer successfully any protein trait. Focused approaches such as site-directed and saturation mutagenesis have become methods of choice for improving protein activity, selectivity, stability and many other traits because the screening step can be practically handled (bottleneck in directed evolution). Although novel mutagenesis methods based on CRISPR or solid-phase gene synthesis can eliminate bias when creating protein libraries, traditional PCR approaches, although imperfect, remain widely used due to their ease and low cost. One of the most common approaches in focused mutagenesis relies on NNK mutagenesis, however, the primer-based 22c-trick and small-intelligent methods have emerged as key tools for constructing less biased and unbiased libraries when all 20 canonical amino acids are needed for various reasons. In this minireview, we assess studies employing such methods for library creation and their areas of application. We also discuss the advantages and disadvantages of both methods and provide a perspective for creating smarter libraries.

    更新日期:2019-11-18
  • Rat cathepsin K: Enzymatic specificity and regulation of its collagenolytic activity
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-15
    Fabien Lecaille, Thibault Chazeirat, Krzysztof K. Bojarski, Justine Renault, Ahlame Saidi, V. Gangadhara N.V. Prasad, Sergey Samsonov, Gilles Lalmanach
    更新日期:2019-11-18
  • Thermophilic nucleoside phosphorylases: Their properties, characteristics and applications
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-11-02
    Sarah Kamel, Isabel Thiele, Peter Neubauer, Anke Wagner

    Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleosides in the presence of phosphate. They are valuable catalysts in the synthesis of nucleosides and their analogues, which are often used as pharmaceuticals or their precursors. Thermostable nucleoside phosphorylases are promising biocatalysts, as they withstand harsh reaction conditions such as high pH or the addition of organic solvents. In this review, the characteristics and properties of thermostable nucleoside phosphorylases are described. Differences in amino acid content and protein structure were compared to their mesophilic homologues to identify features involved in thermostability. Substrate spectra of thermostable nucleoside phosphorylases were analyzed, and it is shown that thermostable nucleoside phosphorylases have a wider substrate spectrum than their mesophilic counterparts. Thus, thermostable nucleoside phosphorylases are interesting biocatalysts for industrial applications.

    更新日期:2019-11-18
  • Characterization of alkaline phosphatase PhoK from Sphingomonas sp. BSAR-1 for phosphate monoester synthesis and hydrolysis
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-31
    Michael Lukesch, Gábor Tasnádi, Klaus Ditrich, Mélanie Hall, Kurt Faber

    The biocatalytic activity of a so far underexploited alkaline phosphatase, PhoK from Sphingomonas sp. BSAR-1, was extensively studied in transphosphorylation and hydrolysis reactions. The use of high-energy phosphate donors and oligophosphates as suitable phosphate donor was evaluated, as well as the hydrolytic activity on a variety of phosphate monoesters. While substrates bearing free hydroxy group displayed only moderate reactivity as acceptors for transphosphorylation by PhoK, strong hydrolytic activity on a broad variety of phosphate monoesters under alkaline conditions was observed. Site-directed mutagenesis of selected amino acid residues in the active site provided valuable insights on their involvement in enzyme catalysis. The key residue Thr89 so far postulated to engage in enzyme phosphorylation was confirmed to be crucial for catalysis and could be replaced by serine, albeit with much lower catalytic efficiency.

    更新日期:2019-11-01
  • Chemoenzymatic synthesis of ultralow and low-molecular weight heparins
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-31
    Ting Wang, Li Liu, Josef Voglmeir

    Heparin is a naturally occurring glycosaminoglycan isolated from animal tissues and is medically used as an anticoagulant drug. Adulteration attempts of isolated heparin with chondroitin sulfate in the past resulted in great safety concerns. Also, increasing demands on batch-to-batch homogeneity for better evaluation and control of its pharmacodynamic and pharmacokinetic properties kindled the development of synthetic routes for the production of heparin and its derivatives. The discovery of enzymes involved in glycosaminoglycan biosynthesis and their application in chemoenzymatic synthesis makes it feasible to generate low molecular weight heparins (LMWHs) and ultra-low molecular weight heparins (ULMWHs). Understanding the scope and limitations of these enzymes currently used in the production of synthetic heparins will help to achieve more defined heparins with controlled medicative properties. Here, we summarized the recent advances in the chemoenzymatic synthesis of LMW/ULMW heparins.

    更新日期:2019-11-01
  • Photochemical regeneration of flavoenzymes – An Old Yellow Enzyme case-study
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-31
    M.C.R. Rauch, M. Pesic, M.M.E. Huijbers, M. Pabst, C.E. Paul, M. Pesic, I.W.C.E. Arends, F. Hollmann

    Direct, NAD(P)H-independent regeneration of Old Yellow Enzymes represents an interesting approach for simplified reaction schemes for the stereoselective reduction of conjugated C=C-double bonds. Simply by illuminating the reaction mixtures with blue light in the presence of sacrificial electron donors enables to circumvent the costly and unstable nicotinamide cofactors and a corresponding regeneration system. In the present study, we characterise the parameters determining the efficiency of this approach and outline the current limitations. Particularly, the photolability of the flavin photocatalyst and the (flavin-containing) biocatalyst represent the major limitation en route to preparative application.

    更新日期:2019-11-01
  • On the catalytic mechanism of bacteriophage endolysins: Opportunities for engineering
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-31
    Michael J. Love, Gayan S. Abeysekera, Andrew C. Muscroft-Taylor, Craig Billington, Renwick C.J. Dobson

    Bacteriophage endolysins have the potential to be a long-term antibacterial replacement for antibiotics. The exogenous application of endolysins on some bacteria results in rapid cell lysis. The prospects for endolysins are furthered by the ability to engineer them; novel endolysins can be developed with optimised stability, specificity, and lytic function. But the success of endolysin engineering and application requires a comprehensive understanding of the relationship between the enzymes biochemical, biophysical and bacteriolytic properties. Here, we examine their catalytic mechanisms, opportunities for developing novel endolysins, and highlight areas where a better understanding would support their long-term success as antibacterial agents.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Molecular characterization of an aggregation-prone variant of alpha-synuclein used to model synucleinopathies
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-30
    Caterina Masaracchia, Annekatrin König, Ariel A. Valiente-Gabioud, Pablo Peralta, Filippo Favretto, Timo Strohäker, Diana F. Lázaro, Markus Zweckstetter, Claudio O. Fernandez, Tiago F. Outeiro

    The misfolding and aggregation of alpha-synuclein (aSyn) are thought to be central events in synucleinopathies. The physiological function of aSyn has been related to vesicle binding and trafficking, but the precise molecular mechanisms leading to aSyn pathogenicity are still obscure. In cell models, aSyn does not readily aggregate, even upon overexpression. Therefore, cellular models that enable the study of aSyn aggregation are essential tools for our understanding of the molecular mechanisms that govern such processes. Here, we investigated the structural features of SynT, an artificial variant of aSyn that has been widely used as a model of aggregation in mammalian cell systems, since it is more prone to aggregation than aSyn. Using Nuclear Magnetic Resonance (NMR) spectroscopy we performed a detailed structural characterization of SynT through a systematic comparison with normal, unmodified aSyn. Interestingly, we found that the conformations adopted by SynT resemble those described for the unmodified protein, demonstrating the usefulness of SynT as a model for aSyn aggregation. However, subtle differences were observed at the N-terminal region involving transient intra and/or intermolecular interactions that are known to regulate aSyn aggregation. Importantly, our results indicate that disturbances in the N-terminal region of SynT, and the consequent decrease in membrane binding of the modified protein, might contribute to the observed aggregation behavior of aSyn, and validate the use of SynT, one of the few models of aSyn aggregation in cultured cells.

    更新日期:2019-11-01
  • Ubiquitin folds via a flip-twist-lock mechanism
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-30
    Manoj Mandal, Atanu Das, Chaitali Mukhopadhyay
    更新日期:2019-11-01
  • Biochemical and biophysical characterization of the smallest pyruvate kinase from Entamoeba histolytica
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-30
    Poonam Kumari, Danish Idrees, Pragyan Parimita Rath, Ramachandran Vijayan, Samudrala Gourinath
    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Use of nucleoside phosphorylases for the preparation of 5-modified pyrimidine ribonucleosides
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-30
    Cyril S. Alexeev, Mikhail S. Drenichev, Evgeniya O. Dorinova, Roman S. Esipov, Irina V. Kulikova, Sergey N. Mikhailov
    更新日期:2019-11-01
  • Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved aryl-alcohol oxidase
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-30
    Javier Viña-Gonzalez, Angel T. Martinez, Victor Guallar, Miguel Alcalde

    Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-alcohol oxidase (AAO) and 5-hydroxymethylfurfural oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.

    更新日期:2019-11-01
  • Interaction between immunoglobulin G and peroxidase-like iron oxide nanoparticles: Physicochemical and structural features of the protein
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-29
    Anna V. Bychkova, Mariia V. Lopukhova, Luybov A. Wasserman, Pavel G. Pronkin, Yevgeniy N. Degtyarev, Alexander I. Shalupov, Alexandra D. Vasilyeva, Lyubov’ V. Yurina, Alexander L. Kovarski, Alexey S. Kononikhin, Evgene N. Nikolaev

    The study is devoted to the oxidative modification of immunoglobulin G (IgG) on the surface of peroxidase-like iron oxide magnetic nanoparticles (MNPs) under conditions of induced reactive oxygen species (ROS) generation and without them. A pronounced change of thermodynamic parameters of denaturation has been detected for IgG in solutions containing MNPs under hydrogen peroxide action during 24 h of incubation. Dynamic light scattering measurements and UV–Visible spectrophotometry have been used to show aggregation in these solutions. Ferromagnetic resonance (FMR) was used to compare IgG coating thickness on individual MNPs under conditions of induced ROS generation and without them. The similarity between IgG adsorption on MNPs under these conditions after 24 h of incubation has been confirmed by the fluorescence measurements. The sites of IgG oxidative modifications that take place on MNPs surface and some evidences of the influence of oxidative modification and adsorption on the chemical structure of IgG were revealed by HPLC MS/MS analysis.

    更新日期:2019-10-29
  • Purification and characterization of two forms of the homologously expressed lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-10-28
    Margarita V. Semenova, Alexander V. Gusakov, Vadim D. Telitsin, Aleksandra M. Rozhkova, Elena G. Kondratyeva, Arkady P. Sinitsyn

    Two forms of C1/C4-oxidizing lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum (Talaromyces verruculosus) homologously expressed in P. verruculosum B1-537 auxotrophic strain were isolated in a homogeneous state using two-stage chromatography. The PvLPMO9A-hm form represented a full-size enzyme encoded by the intact lpmo1 gene, while the PvLPMO9A-lm was a truncated enzyme variant consisting of a conserved catalytic core of AA9 family LPMOs and lacking a C-terminal extra peptide sequence that is present in PvLPMO9A-hm. The N-terminal histidine was partially methylated in both enzymes. Most of properties of PvLPMO9A-hm and PvLPMO9A-lm, such as specific activities determined using the 2,6-dimethoxyphenol/H2O2 assay, pH-optima of activity observed at pH 7.5, synergistic effects exhibited with purified cellobiohydrolase I (Cel7A) and/or endoglucanase II (Cel5A) from P. verruculosum in hydrolysis of Avicel and milled aspen wood, were also very similar, except for the higher PvLPMO9A-hm thermostability studied using differential scanning calorimetry (DSC). The DSC profile for the PvLPMO9A-hm holoenzyme demonstrated two overlapping peaks (with maxima at 56.3 and 59.6 °C) due to the presence of two unfolding protein domains, while the PvLPMO9A-lm DSC profile represented one peak with maximum at 48.1 °C. After removing the active site copper with EDTA, the PvLPMO9A-hm and PvLPMO9A-lm melting temperatures decreased by ~10–11 and ~1 °C, respectively. These data show that both active site copper and C-terminal domain present in the PvLPMO9A-hm protect the enzyme from thermal unfolding, while the stabilizing effect of metal is much less pronounced in the truncated PvLPMO9A-lm form.

    更新日期:2019-10-28
  • Ticket to a bubble ride: Cargo sorting into exosomes and extracellular vesicles
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-02-26
    Sushma Anand, Monisha Samuel, Sharad Kumar, Suresh Mathivanan

    Extracellular vesicles (EVs) are released by cells into the extracellular milieu to facilitate intercellular communication in both physiological and pathological condition. EVs contain selective repertoires of proteins, RNAs, lipids and metabolites that moderate signalling pathways in the recipient cells. The enrichment of a particular set of proteins or RNAs within the EVs highlights the existence of specific sorting mechanisms that orchestrate the selective packaging of the cargo. The molecular machinery of cargo sorting has remained obscure over the years and functional studies are required to understand this complex mechanism. In this article, we offer a brief overview of the molecular mechanisms that are known to regulate sorting of various molecules into EVs. We also discuss how different pathways of biogenesis alter the exosomal cargo as well and the implications of the cellular state on the content of the EVs. Understanding the sorting of exosomal cargo could further be exploited in clinical settings for targeted drug delivery and to block disease progression.

    更新日期:2019-10-25
  • Exosomal proteins constitute an essential part of the human adipose tissue secretome
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2018-11-28
    Sonja Hartwig, Elisabetta De Filippo, Simon Göddeke, Birgit Knebel, Jorg Kotzka, Hadi Al-Hasani, Michael Roden, Stefan Lehr, Henrike Sell

    Adipose tissue is an endocrine organ, secreting various adipokines, either directly or via extracellular vesicles, including exosomes. Exosomes are vesicles of 40–150 nm size that represent a novel concept of biomolecule release. We purified exosomes from isolated primary human preadipocytes differentiated to mature adipocytes. The analyses of these exosomal preparations by LC-MS identified 884 proteins, so called exoadipokines. The comparison of exoadipokines with previously identified human exosome-associated proteins in ExoCarta database show an overlap of 817 proteins, but also revealed 67 proteins not assigned to human exosomes, yet. We further compared all exoadipokines to our previously reported reference secretome of human adipose tissue (http://diabesityprot.org/), finding 212 common proteins, whereas 672 proteins were specific for the exosomal fraction. Bioinformatic analyses revealed that the 212 common proteins can be assigned to all major functions of adipose tissue secreted proteins e.g. molecules involved in fibrotic processes or inflammation. In contrast, the exosome-specific proteins were rather assigned to signaling pathways and membrane-mediated processes. In conclusion, the isolation of exosomes allows to further specify the functionality of adipokines and exoadipokines as part of the adipocyte secretome in signaling and interorgan crosstalk.

    更新日期:2019-10-25
  • Proteomic profiling reveals key cancer progression modulators in shed microvesicles released from isogenic human primary and metastatic colorectal cancer cell lines
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2018-11-29
    Wittaya Suwakulsiri, Alin Rai, Rong Xu, Maoshan Chen, David W. Greening, Richard J. Simpson

    Extracellular vesicles comprise two main classes - exosomes and shed microvesicles (sMVs). Whilst much is known about exosome cargo content and functionality, sMVs are poorly understood. Here, we describe the large-scale purification of sMVs released from primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines using a combination of differential ultracentrifugation and isopycnic iodixanol density centrifugation. The yield of SW480-sMVs and SW620-sMVs was 0.75 mg and 0.80 mg, respectively. Both SW480-/SW620-sMVs are heterogeneous in size (100–600 nm diameter) and exhibit identical buoyant densities (1.10 g/mL). In contrast to exosomes, sMVs are ALIX−, TSG101−, CD63− and CD9−. Quantitative mass spectrometry identified 1295 and 1300 proteins in SW480-sMVs and SW620-sMVs, respectively. Gene Ontology enrichment analysis identified ‘cell adhesion’ (CDH1, OCLN, CTN families), ‘signalling pathway’ (KRAS, NRAS, MAPK1, MAP2K1), and ‘translation/RNA related’ processes (EIF, RPL, HNRNP families) in both sMV types. Strikingly, SW480- and SW620-sMVs exhibit distinct protein signatures - SW480-sMVs being enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, while SW620-sMVs are enriched in PRKCA, MACC1, FGFR4 and MTOR/MARCKS signalling networks. Both SW480- and SW620-sMVs are taken up by NIH3T3 fibroblasts resulting in similar cell invasion capability. This study provides, for the first time, molecular insights into sMVs and CRC biology.

    更新日期:2019-10-25
  • The germinal centre kinase Don3 is crucial for unconventional secretion of chitinase Cts1 in Ustilago maydis
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2018-10-11
    Jörn Aschenbroich, Kai P. Hussnaetter, Peter Stoffels, Thorsten Langner, Sabrina Zander, Björn Sandrock, Michael Bölker, Michael Feldbrügge, Kerstin Schipper

    Unconventional secretion has emerged as an increasingly important cellular process in eukaryotic cells. The underlying translocation mechanisms are diverse and often little understood. We study unconventional secretion of chitinase Cts1 in the corn smut fungus Ustilago maydis. This protein participates in the cytokinesis of yeast cells. During budding it localizes to the septated fragmentation zone where it presumably functions in the degradation of remnant chitin to allow separation of mother and daughter cell. However, the mechanistic details of Cts1 export remain unclear. Here we investigated the mechanism of unconventional Cts1 secretion with a focus on cytokinesis. Cell-cycle inhibition experiments supported the hypothesis that Cts1 export is connected to cytokinesis. To substantiate this finding we analysed gene deletion mutants impaired in cell separation and discovered that strains defective in secondary septum formation were affected in Cts1 export. The germinal centre kinase Don3 had a particularly strong influence on unconventional secretion. Using a synthetic switch, we unambiguously verified an essential role of Don3 for cytokinesis-dependent Cts1 export via the fragmentation zone. Thus, we gained novel insights into the mechanism of unconventional secretion and discovered the first regulatory component of this process.

    更新日期:2019-10-25
  • 更新日期:2019-10-25
  • Pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-06-12
    Jessica Schira-Heinen, Leonie Grube, Daniel M. Waldera-Lupa, Falk Baberg, Maike Langini, Omid Etemad-Parishanzadeh, Gereon Poschmann, Kai Stühler

    Proteins are released from cells by different secretory pathways. The secretory pathway via the ER-Golgi route - realized by a signal sequence - is referred to as “classical secretion”. In contrast, alternative secretory pathways were summarized as “unconventional protein secretion”. Until now, unconventional protein secretion was lacking attention due to the absence of detailed mechanistic insight and limited experimental access. However, there is a growing number of experimental data showing that a large proportion of secreted proteins is released by these alternative routes. Secretomics - the analysis of all secreted proteins of a cell population - offers the opportunity to gain more functional insight into unconventional protein secretion. Several pitfalls in secretome analysis starting with the analyzed cell model and sample preparation to data analysis have to be considered for detailed characterization of the secretome. Here, we highlight the investigation of secretomes by quantitative LC-MS/MS analysis and discuss pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis.

    更新日期:2019-10-25
  • Predicting eukaryotic protein secretion without signals
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2018-12-04
    Henrik Nielsen, Eirini I. Petsalaki, Linlin Zhao, Kai Stühler

    Predicting unconventional protein secretion is a much harder problem than predicting signal peptide-based protein secretion, both due to the small number of examples and due to the heterogeneity and the limited knowledge of the pathways involved, especially in eukaryotes. However, the idea that secreted proteins share certain properties regardless of the secretion pathway used made it possible to construct the prediction method SecretomeP in 2004. Here, we take a critical look at SecretomeP and its successors, and we also discuss whether multi-category subcellular location predictors can be used to predict unconventional protein secretion in eukaryotes. A new benchmark shows SecretomeP to perform much worse than initially estimated, casting doubt on the underlying hypothesis. On a more positive note, recent developments in machine learning may have the potential to construct new methods which can not only predict unconventional protein secretion but also point out which parts of a sequence are important for secretion.

    更新日期:2019-10-25
  • Effects of lithospermic acid on hIAPP aggregation and amyloid-induced cytotoxicity by multiple analytical methods
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-09-14
    Bo Pang, Xinyu Bian, Junpeng Xing, Shu Liu, Zhiqiang Liu, Fengrui Song

    The abnormal aggregation of human islet amyloid polypeptide (hIAPP) is a crucial pathogenic factor associated with type 2 diabetes (T2D). The development of effective inhibitors to prevent hIAPP aggregation is a common therapeutic strategy against T2D. Lithospermic acid (LA) is a natural compound with diversified biological activities. In this study, electrospray ionization coupled with ion mobility–mass spectrometry, thioflavin T fluorescence assay, Congo red binding assay, Nile red fluorescence assay, circular dichroism spectroscopy, transmission electron microscopy, cell toxicity, lactate dehydrogenase assay (LDH) assay and molecular docking were combined to explore the influence of LA on hIAPP aggregation. Results showed that LA had favorable binding affinity to hIAPP and formed hIAPP–LA complexes, which could alter the relative abundance of the compact and extended conformers and promoted the transition of extended structures to compact conformers. LA also displayed strong inhibitory actions on fibrillation and potential protective effects against hIAPP-induced cell toxicity. Therefore, the obtained results were useful to understand the possible inhibitory mechanism of LA on hIAPP aggregation and provided valuable reference for the screening of potent amyloid inhibitors.

    更新日期:2019-10-25
  • Structural studies of the Hsp70/Hsp90 organizing protein of Plasmodium falciparum and its modulation of Hsp70 and Hsp90 ATPase activities
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-09-13
    Noeli S.M. Silva, Dayane E. Bertolino-Reis, Paulo R. Dores-Silva, Fátima B. Anneta, Thiago V. Seraphim, Leandro R.S. Barbosa, Júlio C. Borges
    更新日期:2019-10-25
  • The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-09-13
    Ina Buchholz, Felix Nagel, Annelie Klein, Preshit R. Wagh, Ujjwal M. Mahajan, Andreas Greinacher, Markus M. Lerch, Julia Mayerle, Mihaela Delcea

    One of the most common mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene is the N34S variant which is strongly associated with chronic pancreatitis. Although it is assumed that N34S mutation constitutes a high-risk factor, the underlying pathologic mechanism is still unknown. In the present study, we investigated the impact of physiological stress factors on SPINK1 protein structure and trypsin inhibitor function using biophysical methods. Our circular dichroism spectroscopy data revealed differences in the secondary structure of SPINK1 and N34S mutant suggesting protein structural changes induced by the mutation as an impairment that could be disease-relevant. We further confirmed that both SPINK1 (KD of 0.15 ± 0.06 nM) and its N34S variant (KD of 0.08 ± 0.02 nM) have similar binding affinity and inhibitory effect towards trypsin as shown by surface plasmon resonance and trypsin inhibition assay studies, respectively. We found that stress conditions such as altered ion concentrations (i.e. potassium, calcium), temperature shifts, as well as environmental pH lead to insignificant differences in trypsin inhibition between SPINK1 and N34S mutant. However, we have shown that the environmental pH induces structural changes in both SPINK1 constructs in a different manner. Our findings suggest protein structural changes in the N34S variant as an impairment of SPINK1 and environmental pH shift as a trigger that could play a role in disease progression of pancreatitis.

    更新日期:2019-10-25
  • A new sensitive spectrofluorimetric method for measurement of activity and kinetic study of cholinesterases
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-09-10
    Aliya R. Mukhametgalieva, Irina V. Zueva, Aliya R. Aglyamova, Sofya V. Lushchekina, Patrick Masson

    A new spectrofluorimetric method more sensitive than the Ellman method was developed for determination of both acetylcholinesterase and butyrylcholinesterase activity and for kinetic analysis of these enzymes and their mutants. Two selected mutants of human butyrylcholinesterase (E197Q and E197G) were included in this work. As for the Ellman's method, substrates are thiocholine esters, but the chromogenic reagent, DTNB (dithio-bisnitro benzoic acid) is replaced by a fluorogenic probe, “Calbiochem Probe IV”, (3-(7-Hydroxy-2-oxo-2H-chromen-3-ylcarbamoyl)acrylic acid methylester). Compared to the classical Ellman's method, the sensitivity of this new spectrofluorimetric assay is 2 orders of magnitude higher. The method allows measurement of activity in media containing <10−11 M of cholinesterase active sites at low substrate concentrations, either under first order conditions, [S] << Km, or under conditions where kinetics obeys the Michaelis-Menten model, i.e. at [S] < 1 mM for wild-type enzymes. The method adapted to titration plate reader assays is suitable for clinical and toxicological routine analyses, for high throughput screening of novel cholinesterase mutants and screening of inhibitor libraries of pharmacological interest.

    更新日期:2019-10-25
  • Conventional and non-conventional applications of β-galactosidases
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-09-05
    Carlos Vera, Cecilia Guerrero, Carla Aburto, Andrés Cordova, Andrés Illanes
    更新日期:2019-10-25
  • 更新日期:2019-10-25
  • Expression and role of CYP505A1 in pathogenicity of Fusarium oxysporum f. sp. lactucae
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-09-03
    Daniela Minerdi, Sheila J. Sadeghi, Lara Pautasso, Simone Morra, Riccardo Aigotti, Claudio Medana, Giovanna Gilardi, Maria Lodovica Gullino, Gianfranco Gilardi
    更新日期:2019-10-25
  • Mapping of the binding site for FcμR in human IgM-Fc
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-08-23
    Rosemary A. Nyamboya, Brian J. Sutton, Rosaleen A. Calvert
    更新日期:2019-10-25
  • Sustainable synthesis of uridine-5′-monophosphate analogues by immobilized uracil phosphoribosyltransferase from Thermus thermophilus
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-07-09
    Jon del Arco, Javier Galindo, Vicente Javier Clemente-Suárez, Amaira Corrales, Jesús Fernández-Lucas

    Nowadays enzymatic synthesis of nucleic acid derivatives is gaining momentum over traditional chemical synthetic processes. Biotransformations catalyzed by whole cells or enzymes offer an ecofriendly and efficient alternative to the traditional multistep chemical methods, avoiding the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Herein we report for the first time the covalent immobilization a uracil phosphoribosyltransferase (UPRT). In this sense, UPRT from Thermus thermophilus HB8 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtUPRT). According to the catalyst load experiments, MTtUPRT3 was selected as optimal biocatalyst for further studies. MTtUPRT3 was active and stable in a broad range of temperature (70–100 °C) and in the pH interval 6–8, displaying maximum activity at 100 °C and pH 7 (activity 968 IU/gsupport, retained activity 100%). In addition, MTtUPRT3 could be reused up to 8 times in the synthesis of uridine-5′-monophosphate (UMP). Finally, MTtUPRT3 was successfully applied in the sustainable synthesis of different 5-modified uridine-5′-monophosphates at short times. Taking into account these results, MTtUPRT3 would emerge as a valuable biocatalyst for the synthesis of nucleoside monophosphates through an efficient and environmentally friendly methodology.

    更新日期:2019-10-25
  • Monomeric and homotrimeric solution structures of truncated human peroxidasin 1 variants
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-07-08
    Martina Paumann-Page, Rupert Tscheliessnig, Benjamin Sevcnikar, Romy-Sophie Katz, Irene Schwartz, Stefan Hofbauer, Vera Pfanzagl, Paul G. Furtmüller, Christian Obinger
    更新日期:2019-10-25
  • Constant domain-exchanged Fab enables specific light chain pairing in heterodimeric bispecific SEED-antibodies
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-07-08
    Sylvia Dietrich, Alec W. Gross, Stefan Becker, Björn Hock, Gerhard Stadlmayr, Florian Rüker, Gordana Wozniak-Knopp
    更新日期:2019-10-25
  • Glycosylation effects on the structure and dynamics of a full-length Cel7A cellulase
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-07-04
    Carlos Eduardo Pena, Mauricio G.S. Costa, Paulo Ricardo Batista
    更新日期:2019-10-25
  • A comprehensive comparison of the metazoan tryptophan degrading enzymes
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-07-02
    Hajime Julie Yuasa

    Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) have an independent origin; however, they have distinctly evolved to catalyze the same reaction. In general, TDO is a single-copy gene in each metazoan species, and TDO enzymes demonstrate similar enzyme activity regardless of their biological origin. In contrast, multiple IDO paralogues are observed in many species, and they display various enzymatic properties. Similar to vertebrate IDO2, invertebrate IDOs generally show low affinity/catalytic efficiency for L-Trp. Meanwhile, two IDO isoforms from scallop (IDO-I and -III) and sponge IDOs show high L-Trp catalytic activity, which is comparable to vertebrate IDO1. Site-directed mutagenesis experiments have revealed that primarily two residues, Tyr located at the 2nd residue on the F-helix (F2nd) and His located at the 9th residue on the G-helix (G9th), are crucial for the high affinity/catalytic efficiency of these ‘high performance’ invertebrate IDOs. Conversely, those two amino acid substitutions (F2nd/Tyr and G9th/His) resulted in high affinity and catalytic activity in other molluscan ‘low performance’ IDOs. In human IDO1, G9th is Ser167, whereas the counterpart residue of G9th in human TDO is His76. Previous studies have shown that Ser167 could not be substituted by His because the human IDO1 Ser167His variant showed significantly low catalytic activity. However, this may be specific for human IDO1 because G9th/His was demonstrated to be very effective in increasing the L-Trp affinity even in vertebrate IDOs. Therefore, these findings indicate that the active sites of TDO and IDO are more similar to each other than previously expected.

    更新日期:2019-10-25
  • Fungal Lanosterol 14α-demethylase: A target for next-generation antifungal design
    BBA Proteins Proteom. (IF 2.54) Pub Date : 2019-03-06
    Brian C. Monk, Alia A. Sagatova, Parham Hosseini, Yasmeen N. Ruma, Rajni K. Wilson, Mikhail V. Keniya
    更新日期:2019-10-25
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