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Taenia solium can cause human taeniasis and/or cysticercosis. The latter can in some instances cause human neurocysticercosis which is considered a priority in disease-control strategies and the prevention of mental health problems. Glutathione transferases are crucial for the establishment and long-term survival of T. solium; therefore, we structurally analyzed the 24-kDa glutathione transferase gene (Ts24gst) of T. solium and biochemically characterized its product. The gene promoter showed potential binding sites for transcription factors and xenobiotic regulatory elements. The gene consists of a transcription start site, four exons split by three introns, and a polyadenylation site. The gene architecture is conserved in cestodes. Recombinant Ts24GST (rTs24GST) was active and dimeric. Anti-rTs24GST serum showed slight cross-reactivity with human sigma-class GST. A 3D model of Ts24GST enabled identification of putative residues involved in interactions of the G-site with GSH and of the H-site with CDNB and prostaglandin D 2 . Furthermore, rTs24GST showed optimal activity at 45 °C and pH 9, as well as high structural stability in a wide range of temperatures and pHs. These results contribute to the better understanding of this parasite and the efforts directed to fight taeniasis/cysticercosis., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Epitope tags recognized by specific antibodies have been widely used over the last few decades, notably to localize tagged proteins within cells by immunofluorescence. The diversity of tags and antibodies usually prevents a side-by-side comparison of the efficiency with which each antibody recognizes its cognate tag. We expressed chimeric proteins, each composed of an invariant domain (IL2Ra) associated with a specific epitope tag. Double immunofluorescence allowed us to quantify in parallel the reference signal generated by the anti-IL2Ra antibody and the signal generated by the anti-epitope tag antibody. Since all antibodies used in this study were recombinant antibodies fused to the same mouse Fc domain, the generated signals were directly comparable. Three groups of tags/antibodies were revealed: 'good' antibodies generated high signals even when used at a low concentration (50 ng·mL -1 ), 'fair' antibodies generated a high signal only at high concentrations (5000 ng·mL -1 ), and 'mediocre' antibodies generated positive but weak signals. Except for an anti-myc antibody, similar results were obtained when cells were fixed in paraformaldehyde or methanol. These results provide a side-by-side quantitative evaluation of different tag/antibody pairs. This information will be useful to optimize the choice of epitope tags and to choose optimal antibodies., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Filensin and phakinin are lens fiber cell-specific proteins that constitute the beaded filaments (BFs) that are critical for maintaining lens transparency. In the Shumiya cataract rat, filensin 94 kDa undergoes N- and C-terminal proteolytic processing to give a transient 50 kDa fragment and a final 38 kDa fragment, just before opacification. To characterize the effects of this processing on filensin function, recombinant proteins representing the two filensin fragments, termed Fil(30-416) and Fil(30-369), respectively, were examined. Fil(30-416) lacks the N-terminal 29 amino acids and the C-terminal 248 amino acids. Fil(30-369) lacks the N-terminal 29 residues and the C-terminal 295 residues. In cell-free assembly characterized by electron microscopy, filensin and Fil(30-416) co-polymerized with phakinin and formed rugged, entangled filaments, whereas Fil(30-369) formed only aggregates. In cultured SW-13 and MCF-7 cells expressing fluorescent fusion proteins, filensin and Fil(30-416) co-polymerized with phakinin and formed cytoplasmic sinuous filaments with different widths, while Fil(30-369) gave aggregates. Therefore, while truncation of the N-terminal 29 amino acids did not affect filament formation, truncation of the C-terminal 295 but not the 248 residues resulted in failure of filament formation. These results indicate that the tail B region (residues 370-416) of rat filensin is essential for filament formation with phakinin. Truncation of the tail B region by proteolytic processing in the cataract rat lens might interfere with BF formation and thereby contribute to opacification., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The activation of oncogenic C-terminal binding Protein (CtBP) transcriptional activity is coupled with NAD(H) binding and homo-oligomeric assembly, although the level of CtBP assembly and nucleotide binding affinity continues to be debated. Here, we apply biophysical techniques to address these fundamental issues for CtBP1 and CtBP2. Our ultracentrifugation results unambiguously demonstrate that CtBP assembles into tetramers in the presence of saturating NAD + or NADH with tetramer to dimer dissociation constants about 100 nm. Isothermal titration calorimetry measurements of NAD(H) binding to CtBP show dissociation constants between 30 and 500 nm, depending on the nucleotide and paralog. Given cellular levels of NAD + , CtBP is likely to be fully saturated with NAD under physiological concentrations suggesting that CtBP is unable to act as a sensor for NADH levels., (© 2022 Federation of European Biochemical Societies.)

Plant metacaspases type I (MCA-Is), the closest structural homologs of caspases, are key proteases in stress-induced regulated cell death processes in plants. However, no plant MCA-Is have been characterized in vitro to date. Here, we show that only plant MCA-Is contain a highly hydrophobic loop within the C terminus of their p10 domain. When removed, soluble and proteolytically active plant MCA-Is can be designed and recombinantly produced. We show that the activity of MCA-I depends on calcium ions and that removal of the hydrophobic loop does not affect cleavage and covalent binding to its inhibitor SERPIN. This novel approach will finally allow the development of tools to detect and manipulate the activity of these cysteine proteases in vivo and in planta., (© 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1-9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1-9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation-inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin-Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2., (© 2021 Federation of European Biochemical Societies.)

A transporter of the multidrug and toxic compound extrusion (MATE) family, Nicotiana tabacum MATE2 (NtMATE2), is located in the vacuole membrane of the tobacco plant root and is involved in the transportation of nicotine, a secondary or specialized metabolic compound in Solanaceae. Here, we report the crystal structures of NtMATE2 in its outward-facing forms. The overall structure has a bilobate V-shape with pseudo-symmetrical assembly of the N- and C-lobes. In one crystal structure, the C-lobe cavity of NtMATE2 interacts with an unidentified molecule that may partially mimic a substrate. In addition, NtMATE2-specific conformational transitions imply that an unprecedented movement of the transmembrane α-helix 7 is related to the release of the substrate into the vacuolar lumen., (© 2021 Federation of European Biochemical Societies.)

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation., (© 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In the N-degron pathway of protein degradation of Escherichia coli, the N-recognin ClpS identifies substrates bearing N-terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N-degron pathway. The residue in second position influences recognition (especially in N-terminal ends starting with leucine). N-terminal acetylation abrogates recognition. ClpF, a ClpS1-interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate-binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N-degron pathway., (© 2021 Federation of European Biochemical Societies.)

The DEK oncoprotein regulates cellular chromatin function via a number of protein-protein interactions. However, the biological relevance of its unique pseudo-SAP/SAP-box domain, which transmits DNA modulating activities in vitro, remains largely speculative. As hypothesis-driven mutations failed to yield DNA-binding null (DBN) mutants, we combined random mutagenesis with the Bacterial Growth Inhibition Screen (BGIS) to overcome this bottleneck. Re-expression of a DEK-DBN mutant in newly established human DEK knockout cells failed to reduce the increase in nuclear size as compared to wild type, indicating roles for DEK-DNA interactions in cellular chromatin organization. Our results extend the functional roles of DEK in metazoan chromatin and highlight the predictive ability of recombinant protein toxicity in E. coli for unbiased studies of eukaryotic DNA modulating protein domains., (© 2021 Federation of European Biochemical Societies.)

The c subunits of F 0 F 1 -ATP synthase (F 0 c) assemble into a ring structure, following membrane insertion that is dependent on both glycolipid MPIase and protein YidC. We analyzed the insertion and assembly processes of Propionigenium modestum F 0 c (Pm-F 0 c), of which the ring structure is resistant to SDS. Ring assembly of Pm-F 0 c requires P. modestum UncI (Pm-UncI). Ring assembly of in vitro synthesized Pm-F 0 c was observed when both YidC and Pm-UncI were reconstituted into liposomes of Escherichia coli phospholipids. Under the physiological conditions where spontaneous insertion had been blocked by diacylglycerol, MPIase was necessary for Pm-F 0 c insertion allowing the subsequent YidC/Pm-UncI-dependent ring assembly. Thus, we have succeeded in the complete reconstitution of membrane insertion and subsequent ring assembly of Pm-F 0 c., (© 2021 Federation of European Biochemical Societies.)

Several microorganisms can utilize l-rhamnose as a carbon and energy source through the nonphosphorylative metabolic pathway, in which l-rhamnose 1-dehydrogenase (RhaDH) catalyzes the NAD(P) + -dependent oxidization of l-rhamnose to l-rhamnono-1,4-lactone. We herein investigated the crystal structures of RhaDH from Azotobacter vinelandii in ligand-free, NAD + -bound, NADP + -bound, and l-rhamnose- and NAD + -bound forms at 1.9, 2.1, 2.4, and 1.6 Å resolution, respectively. The significant interactions with the 2'-phosphate group of NADP + , but not the 2'-hydroxyl group of NAD + , were consistent with a preference for NADP + over NAD + . The C5-OH and C6-methyl groups of l-rhamnose were recognized by specific residues of RhaDH through hydrogen bonds and hydrophobic contact, respectively, which contribute to the different substrate specificities from other aldose 1-dehydrogenases in the short-chain dehydrogenase/reductase superfamily., (© 2021 Federation of European Biochemical Societies.)

ATP-binding cassette (ABC) transporter proteins include efflux pumps that confer multixenobiotic resistance to zebrafish embryos, a valuable toxico/pharmacological model. Here, we established an automated microscopy-based rhodamine B dye accumulation assay in which enhanced dye accumulation in live zebrafish embryos indicates inhibition of multixenobiotic efflux transporter activity. Twenty structurally divergent known substrates and/or inhibitors of human ABC transporters and environmentally relevant compounds were examined using this assay and the ATPase activity of recombinant zebrafish Abcb4 as readouts. These two assays confirmed that Abcb4 functions as an efflux transporter in zebrafish, whereas they gave discordant results for some of the tested substances. The dye accumulation assay in zebrafish embryos could be useful to screen environmental pollutants and other chemicals for efflux transporter interaction in a medium-throughput fashion., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Elucidating how proteins adapt from halophilic to mesophilic environments will enable a better understanding of protein evolution and folding. In this study, by directed evolution and site-directed mutagenesis of the halophilic ubiquitin-like protein (ULP) Samp2, we find that substitution of the prebiotic amino acid Asp31 by Gly is uniquely effective in the mesophilic adaptation of ULP. Sequence analysis shows that substitution of Asp/Glu in halophilic ULPs by Gly in mesophilic ULPs has higher occurrence than other substitutions, supporting the unique role of the substitution in the mesophilic adaptation of ULP. Molecular dynamic simulations indicate that the mesophilic adaptation might result from the effect of the substitution on the conformational flexibility of ULP., (© 2020 Federation of European Biochemical Societies.)

Leishmania internalize hemoglobin (Hb) via a specific receptor (HbR) for their survival. To identify the Hb-binding domain of HbR, we cloned and expressed several truncated proteins of HbR and determined their ability to bind Hb. Our findings reveal that 90% of Hb-binding activity is retained in HbR 41-80 in comparison with HbR 1-471 . We synthesized a 40 amino acid peptide (SSEKMKQLTMYMIHEMVEGLEGRPSTVRMLPSFVYTSDPA) corresponding to HbR 41-80 and found that it specifically binds Hb. Subsequently, we found that the HbR 41-80 peptide completely blocks Hb uptake in both promastigote and amastigote forms of Leishmania and, thereby, inhibits the growth of the parasite. These results demonstrate that HbR 41-80 is the Hb-binding domain of HbR, which might be used as a potential therapeutic agent to inhibit the growth of Leishmania., (© 2020 Federation of European Biochemical Societies.)

Glucuronoxylans represent a significant fraction of woody biomass, and its decomposition is complicated by the presence of lignin-carbohydrate complexes (LCCs). Herein, LCCs from birchwood were used to investigate the potential coordinated action of a glucuronoyl esterase (TtCE15A) and two α-glucuronidases (SdeAgu115A and AxyAgu115A). When supplementing α-glucuronidase with equimolar quantities of TtCE15A, total MeGlcpA released after 72 h by SdeAgu115A and AxyAgu115A increased from 52% to 67%, and 61% to 95%, respectively. Based on the combined TtCE15A and AxyAgu115A activities, ~ 34% of MeGlcpA in the extracted birchwood glucuronoxylan was occupied as LCCs. Notably, insoluble LCC fractions reduced soluble α-glucuronidase concentrations by up to 70%, whereas reduction in soluble TtCE15A was less than 30%, indicating different tendencies to adsorb onto the LCC substrate., (© 2020 Federation of European Biochemical Societies.)

Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1-Gcn2 interaction by binding to Gcn1, thus preventing Gcn2-mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules., (© 2020 Federation of European Biochemical Societies.)

Pseudomonas aeruginosa is one of the most important nosocomial bacteria emerging as a highly multidrug-resistant pathogen. P. aeruginosa produces two siderophores including pyochelin (PCH) to fulfil its need for iron during infections. We know that both outer and inner membrane proteins FptA and FptX are involved in the ferri-PCH uptake, but this process requires increasing molecular and biochemical knowledge. Here, using bacterial two-hybrid and copurification assays we identified the fatty acid coenzyme-A ligase FadD1 as a novel interacting partner of the inner membrane transporter FptX and found that FadD1 may play a role in PCH production. We managed to purify the FadD1-FptX inner membrane complex and obtained low-resolution 3D models, opening the way for future high-resolution structures., (© 2020 Federation of European Biochemical Societies.)

Osteoarthritis (OA) is an age-related, chronic degenerative disease. With the increasing median age of the population, this disease has become an important public health problem. New, disease-modifying therapies are needed. A potential novel molecular target is phospholipase Cγ1 (PLCγ1), a critical enzyme with important functions including calcium signaling regulation and cell proliferation. In rat chondrocytes treated with IL-1β (20 ng·mL -1 for 36 h), inhibition of PLCγ1 with U73122 (2 μm for 12 h) increased levels and expression of the cartilage matrix components Collagen2 and Aggrecan. This beneficial effect of PLCγ1 inhibition was counteracted by increased chondrocyte apoptosis and necroptosis, increased cell death, and increase levels of ROS, all potentially negative for OA. Combined treatment of IL-1β + U73122-treated chondrocytes with inhibitors of apoptosis (Z-VAD, 10 μm) and necroptosis (Nec-1, 30 μm) enhanced the increases in levels and expression of Collagen2 and Aggrecan, and prevented the increases in cell death and ROS levels. These results suggest that PLCγ1 inhibition may be a viable approach for an OA therapy, if combined with targeted inhibition of chondrocyte apoptosis and necroptosis., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Amyotrophic lateral sclerosis/frontotemporal lobar degeneration-linked proteins, TDP-43 and fused in sarcoma (FUS), bind to G-quadruplex-containing mRNAs and transport them to distal neurites for local translation. The specificity and mechanism of G4-RNA binding, however, remain largely unsolved. Using purified full-length TDP-43 and FUS and a set of seven G4-DNA/RNA, we compared their recognition properties of G4-RNAs. Both TDP-43 and FUS recognized and bound to G4-DNA/RNAs, but the target selectivity differed between two proteins. TDP-43 recognized only parallel-stranded G4-DNA/RNAs, leading to stabilize the G4 conformation. In contrast, FUS bound to all three types, parallel, hybrid, and antiparallel, of G4-DNA/RNAs, resulting in deformation of the G4 structure. We then concluded that the target selectivity and the influence on G4 RNA structure differed between TDP-43 and FUS., (© 2020 Federation of European Biochemical Societies.)

The SH2 domain-containing protein of 76 kDa, SLP76, is an important adaptor protein that coordinates a complex protein network downstream of T-cell receptors (TCR), ultimately regulating the immune response. Upon phosphorylation on Ser376, SLP76 interacts with 14-3-3 adaptor proteins, which leads to its proteolytic degradation. This provides a negative feedback mechanism by which TCR signalling can be controlled. To gain insight into the 14-3-3/SLP76 protein-protein interaction (PPI), we have determined a high-resolution crystal structure of a SLP76 synthetic peptide containing Ser376 with 14-3-3σ. We then characterized its binding to 14-3-3 proteins biophysically by means of fluorescence polarization and isothermal titration calorimetry. Furthermore, we generated two recombinant SLP76 protein constructs and characterized their binding to 14-3-3. Our work lays the foundation for drug design efforts aimed at targeting the 14-3-3/SLP76 interaction and, thereby, TCR signalling., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen-bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high-temperature environments., (© 2020 Federation of European Biochemical Societies.)

Identifying the targetome of a microRNA is key for understanding its functions. Cross-linking and immunoprecipitation (CLIP) methods capture native miRNA-mRNA interactions in cells. Some of these methods yield small amounts of chimeric miRNA-mRNA sequences via ligation of 5'-phosphorylated RNAs produced during the protocol. Here, we introduce chemically synthesized microRNAs (miRNAs) bearing 2'-, 3'-cyclic phosphate groups, as part of a new CLIP method that does not require 5'-phosphorylation for ligation. We show in a system that models miRNAs bound to their targets, that addition of recombinant bacterial ligase RtcB increases ligation efficiency, and that the transformation proceeds via a 3'-phosphate intermediate. By optimizing the chemistry underlying ligation, we provide the basis for an improved method to identify miRNA targetomes., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Ferroportin (Fpn) is an essential mammalian iron transporter that is negatively regulated by the hormone hepcidin. Our current molecular understanding of Fpn-mediated iron efflux and regulation is limited due to a lack of biochemical, biophysical and high-resolution structural studies. A critical step towards understanding the transport mechanism of Fpn is to obtain sufficient quantities of pure and stable protein for downstream studies. As such, we detail here an expression and purification protocol for mouse Fpn yielding milligram quantities of pure protein. We have generated deletion constructs exhibiting enhanced thermal stability and which retained iron-transport activity and hepcidin responsiveness, providing a platform for further biophysical studies of Fpn., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Cytochromes P450 (CYPs or P450s) comprise a superfamily of heme-containing monooxygenases that are involved in a variety of biological processes. CYPs have broad utilities in industry, but most exhibit low thermostability, limiting their use on an industrial scale. Highly thermostable enzymes can be obtained from thermophiles in geothermal areas, including hot springs, offshore oil-producing wells and volcanoes. Here, we report the identification of a gene encoding for a thermophilic CYP from the Binh Chau hot spring metagenomic database, which was designated as P450-T2. The deduced amino acid sequence showed the highest identity of 73.15% with CYP203A1 of Rhodopseudomonas palustris, supporting that P450-T2 is a member of the CYP203A subfamily. Recombinant protein expression yielded 541 nm. The optimal temperature and pH of P450-T2 were 50 °C and 8.0, respectively. The half-life of P450-T2 was 50.2 min at 50 °C, and its melting temperature was 56.80 ± 0.08 °C. It was found to accept electrons from all tested redox partners systems, with BmCPR-Fdx2 being the most effective partner. Screening for putative substrates revealed binding of phenolic compounds, such as l-mimosine and emodin, suggesting a potential application of this new thermophilic P450 in the production of the corresponding hydroxylated products., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Irc3 is a superfamily II DNA helicase required for the maintenance of mitochondrial DNA stability in Saccharomyces cerevisiae. Here, we show that recombinant Irc3 is a monomeric protein and that it can form a binary complex with forked DNA. The catalytically active enzyme is a monomer as no positive cooperativity of ATP hydrolysis or DNA unwinding can be detected. Interestingly, we find that Irc3 prefers to unwind the nascent lagging strand at a replication fork. Using DNase I footprinting, we demonstrate that Irc3 captures DNA substrates by establishing a strong contact at the DNA branching point. Additional protections on the lagging strand template suggest a 3'-to-5' polarity for Irc3 movement., (© 2020 Federation of European Biochemical Societies.)

Rv1019, a member of an uncharacterized tetracycline resistance regulator family of transcriptional regulators of Mycobacterium tuberculosis H37Rv, was found to be differentially expressed during dormancy and reactivation in vitro. In this study, we show that this protein binds to its own promoter and acts as a negative regulator of its own expression. It forms dimers in vitro which is essential for the DNA binding activity. We also show that Rv1019 and downstream genes Rv1020 (mfd) and Rv1021 (mazG) are cotranscribed. Constitutive expression of Rv1019 in M. smegmatis downregulated MSMEG_5423 (mfd) and MSMEG_5422 (mazG), suggesting that Rv1019 negatively regulates these downstream genes which encode key proteins involved in DNA repair. M. smegmatis expressing Rv1019 was found to be sensitive to DNA-damaging environments, suggesting its role in regulating the DNA damage response in mycobacterium., (© 2020 Federation of European Biochemical Societies.)

The ability of Mycobacteria to overcome oxidative stress is of paramount importance for its survival within the host. One of the key enzymes that are involved in protecting the bacterium from reactive oxygen species is the catalase-peroxidase (KatG). However, in strains resistant to the antibiotic isoniazid, KatG is rendered ineffective, which is associated with an increased expression of alkylhydroperoxide reductase subunit C (AhpC). Mycobacterial AhpC possesses a unique helical displacement when compared to its bacterial counterparts. Here, via mutagenesis studies, we demonstrate the importance of this helix for redox modulation of the catalytic activity of AhpC. Along with structural insights from crystallographic data, the impact of critical residues on the structure and flexibility of the helix and on AhpC oligomerization is described., (© 2020 Federation of European Biochemical Societies.)

Chitooligosaccharide oxidase (ChitO) is a fungal carbohydrate oxidase containing a bicovalently bound FAD cofactor. The enzyme is known to catalyse the oxidation of chitooligosaccharides, oligomers of N-acetylated glucosamines derived from chitin degradation. In this study, the unique substrate acceptance was explored by testing a range of N-acetyl-d-glucosamine derivatives, revealing that ChitO preferentially accepts carbohydrates with a hydrophobic group attached to C2. The enzyme also accepts streptozotocin, a natural product used to treat tumours. Elucidation of the crystal structure provides an explanation for the high affinity towards C2-decorated glucosamines: the active site has a secondary binding pocket that accommodates groups attached at C2. Docking simulations are fully in line with the observed substrate preference. This work expands the knowledge on this versatile enzyme., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Heat shock protein 90 (Hsp90) is a key regulator of nitric oxide synthase (NOS) in vivo. Despite its functional importance, little is known about the underlying molecular mechanism. Here, purified dimeric human Hsp90α was used to investigate whether (and if so, how) Hsp90 affects the FMN-heme interdomain electron transfer (IET) step in NOS. Hsp90α increases the IET rate for rat neuronal NOS (nNOS) in a dose-saturable manner, and a single charge-neutralization mutation at conserved Hsp90 K585 abolishes the effect. The kinetic results with added Ficoll 70, a crowder, further indicate that Hsp90 enhances the FMN-heme IET through specific association with nNOS. The Hsp90-nNOS docking models provide hints on the putative role of Hsp90 in constraining the available conformational space for the FMN domain motions., (© 2020 Federation of European Biochemical Societies.)

Formation of clathrin-coated vesicles (CCVs) in receptor-mediated endocytosis is a mechanistically well-established process, in which clathrin, the adaptor protein complex AP-2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)-based in vitro CCV reconstitution system with chemically defined components and the full-length recombinant proteins clathrin, AP-2, epsin-1, and dynamin-2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin-1 alone does not have the propensity for scission but is required for bud formation, whereas AP-2 and clathrin are not sufficient. Thus, our study reveals that epsin-1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Xylanase B, a member of subfamily 7 of the GH30 (glycoside hydrolase family 30) from Talaromyces cellulolyticus (TcXyn30B), is a bifunctional enzyme with glucuronoxylanase and xylobiohydrolase activities. In the present study, crystal structures of the native enzyme and the enzyme-product complex of TcXyn30B expressed in Pichia pastoris were determined at resolutions of 1.60 and 1.65 Å, respectively. The enzyme complexed with 2 2 -(4-O-methyl-α-d-glucuronyl)-xylobiose (U 4m2 X) revealed that TcXyn30B strictly recognizes both the C-6 carboxyl group and the 4-O-methyl group of the 4-O-methyl-α-d-glucuronyl side chain by the conserved residues in GH30-7 endoxylanases. The crystal structure and site-directed mutagenesis indicated that Asn-93 on the β2-α2-loop interacts with the non-reducing end of the xylose residue at subsite-2 and is likely to be involved in xylobiohydrolase activity. These findings provide structural insight into the mechanisms of substrate recognition of GH30-7 glucuronoxylanase and xylobiohydrolase., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

The activation of hepatic stellate cells (HSCs) is associated with liver fibrosis, the pathological feature of most forms of chronic hepatic damage, and is accompanied by abnormal deposition of the extracellular matrix (ECM). During the pathological process, acid-sensing ion channel 1a (ASIC1a), which is responsible for Ca 2+ transportation, is involved in the activation of HSCs. It has previously been identified that ASIC1a is related to pyroptosis in articular chondrocytes. However, it remains unclear whether ASIC1a restrains pyroptosis during liver fibrosis. Here, we determined that the levels of pyroptosis-associated speck-like protein, gasdermin D, caspase-1, nucleotide-binding oligomerization domain (NOD)-like receptor 3, and apoptosis-associated speck-like protein (ASC) decreased, while the level of α-smooth muscle actin and collagen-I increased upon introduction of ASIC1a into an acid-induced model. Inhibition or silencing of ASIC1a and the use of Ca 2+ -free medium were able to promote the pyroptosis of activated HSCs, which reduced their deposition. In summary, our study indicates that ASIC1a inhibits pyroptosis of HSCs and that inhibition of ASIC1a may be able to promote pyroptosis to relieve liver fibrosis., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process., (© 2020 John Wiley & Sons Ltd.)

The mitochondrial unfolded protein response (UPR mt ) is characterized by the transcriptional induction of mitochondrial chaperone and protease genes in response to impaired mitochondrial proteostasis and is regulated by ATF5 and CHOP in mammalian cells. However, the detailed mechanisms underlying the UPR mt are currently unclear. Here, we show that HSF1 is required for activation of mitochondrial chaperone genes, including HSP60, HSP10, and mtHSP70, in mouse embryonic fibroblasts during inhibition of matrix chaperone TRAP1, protease Lon, or electron transfer complex 1 activity. HSF1 bound constitutively to mitochondrial chaperone gene promoters, and we observed that its occupancy was remarkably enhanced at different levels during the UPR mt . Furthermore, HSF1 supported the maintenance of mitochondrial function under the same conditions. These results demonstrate that HSF1 is required for induction of mitochondrial chaperones during the UPR mt , and thus, it may be one of the guardians of mitochondrial function under conditions of impaired mitochondrial proteostasis., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Transglutaminases are a family of enzymes that catalyse the cross-linking of proteins by forming covalent bonds between lysine and glutamine residues in various polypeptides. Cross-linking reactions are involved in blood clots, skin formation, embryogenesis and apoptosis. Clinically, these enzymes appear to be implicated in neurodegenerative diseases, tumours and coeliac diseases. Transglutaminases have great potential for use in the food industry because of their ability to cross-link proteins that are not normally linked. Here, a gene coding for transglutaminase from Atlantic cod was cloned into a bacterial expression vector and used to transform protein expression in a strain of Escherichia coli. The successful expression of recombinant transglutaminase protein from Atlantic cod (AcTG-1) as a soluble protein upon induction at low temperature was confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry analysis. Biochemical characterisation demonstrated that the transglutaminase was active between 0 and 65 °C, but was completely inactivated after 20-min incubation at 70 °C. Interestingly, the enzyme displayed cold-adapted features, such as temperature instability combined with high catalytic efficiency at low temperatures (8-16 °C). In addition, the enzyme had optimal activity at 50 °C, a new feature for a cold-adapted enzyme. AcTG-1 was active in the pH range from 6 to 9, with an optimum at pH 8, and required 5 mm calcium for maximum activity. Potential calcium-binding sites in the enzyme were predictable, making the enzyme an appropriate model for studying structure-function relationships in the calcium-dependent transglutaminase family. In vitro gel analysis revealed that transglutaminase cross-linked casein, collagen and gelatin. The binding of fish fillets in the presence of recombinant AcTG-1 provided further macroscopic proof for the potential application of AcTG-1 as a biological cross-linker in the food industry. Once binding occurred, fish fillets withstood further processing such as frying, boiling, freeze-thawing and chilling. The low-temperature activity and new enzymatic properties of AcTG-1 appear to offer advantages over commercially available enzymatic glues in the food industry., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Obesity during pregnancy has been shown to increase the risk of metabolic diseases in the offspring. However, the factors within the maternal milieu which affect offspring phenotypes and the underlying mechanisms remain unknown. The adipocyte hormone leptin plays a key role in regulating energy homeostasis and is known to participate in sex-specific developmental programming. To examine the action of leptin on fetal growth, placental gene expression and postnatal offspring metabolism, we injected C57BL mice with leptin or saline on gestational day 12 and then measured body weights (BWs) of offspring fed on a standard or obesogenic diet, as well as mRNA expression levels of insulin-like growth factors and glucose and amino acid transporters. Male and female offspring born to leptin-treated mothers exhibited growth retardation before and a growth surge after weaning. Mature male offspring, but not female offspring, exhibited increased BWs on a standard diet. Leptin administration prevented the development of hyperglycaemia in the obese offspring of both sexes. The placentas of the male and female foetuses differed in size and gene expression, and leptin injection decreased the fetal weights of both sexes, the placental weights of the male foetuses and placental gene expression of the GLUT1 glucose transporter in female foetuses. The data suggest that mid-pregnancy is an ontogenetic window for the sex-specific programming effects of leptin, and these effects may be exerted via fetal sex-specific placental responses to leptin administration., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Lysophosphatidylcholine acyltransferase 3 (LPCAT3) is an important enzyme in phospholipid remodeling, a process that influences the biophysical properties of cell membranes and thus cell function. Multiple lines of evidence suggest that LPCAT3 is involved in several diseases, including atherosclerosis, non-alcoholic steatohepatitis, and carcinoma. Thus, LPCAT3 may have potential as a therapeutic target for these diseases. In the present study, we devised an assay based on reversed-phase HPLC to measure LPCAT3 activity, which may facilitate the identification of LPCAT3 inhibitors and activators. We found that optimal pH and temperature of recombinant human LPCAT3 are 6.0 and 30 °C, respectively. The enzyme K m values for substrates NBD-labelled lysophosphatidylcholine and arachidonoyl CoA were 266.84 ± 3.65 and 11.03 ± 0.51 μmol·L -1 , respectively, and the V max was 39.76 ± 1.86 pmol·min -1 ·U -1 . Moreover, we used our new method to determine the IC 50 of a known LPCAT inhibitor, TSI-10. In conclusion, this novel assay can be used to measure the effects of compounds on LPCAT3 activity., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

The flaviviral heterodimeric serine protease NS2B-NS3, consisting of the NS3 protease domain and the NS2B co-factor, is essential for ZIKA virus maturation and replication in cells. For in vitro studies a 'linked' construct, where a polyglycine linker connects NS2B CF and NS3 pro , is often used. This construct undergoes autocatalytic cleavage. Here, we show that linked ZIKV NS2B CF -NS3 pro is cleaved in cis in the NS2B CF exclusively at position R95 and not at the previously proposed alternate cleavage site at residue R29 in the NS3 pro . Cleavage neither affects protease stability nor activity, despite some observed differences in spectroscopic behavior. This minimally modified construct may thus be useful for future structural and functional studies of the flaviviral protease, for example when testing new inhibitors., (© 2019 Federation of European Biochemical Societies.)

Voltage-dependent anion channel isoform 2 of the yeast Saccharomyces cerevisiae (yVDAC2) was believed for many years to be devoid of channel activity. Recently, we isolated yVDAC2 and showed that it exhibits channel-forming activity in the planar lipid bilayer system when in its so-called native form. Here, we describe an alternative strategy for yVDAC2 isolation, through heterologous expression in bacteria and refolding in vitro. Recombinant yVDAC2, like its native form, is able to form voltage-dependent channels. However, some differences between native and recombinant yVDAC2 emerged in terms of voltage dependence and ion selectivity, suggesting that, in this specific case, the recombinant protein might be depleted of post-translational modification(s) that occur in eukaryotic cells., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Mitochondrial uncoupling protein 1 (UCP1) is well known for its thermogenic function in brown adipose tissue (BAT). Since UCP1 expends energy on thermogenesis, UCP1 activation has been considered an approach to ameliorate obesity. As a tool for uncovering yet unknown mechanisms of UCP1 activation, we generated a transgenic mouse model in which UCP1 expression levels are reflected in fluorescence derived from monomeric red fluorescent protein 1 (mRFP1). In these UCP1-mRFP1 BAC transgenic mice, fluorescence intensity mimics the change in UCP1 expression levels evoked through physiological or pharmacological stimulation. This transgenic mouse model will be useful in the search for bioactive compounds with the ability to induce UCP1 and for revealing undiscovered mechanisms of BAT activation., (© 2019 Federation of European Biochemical Societies.)

Arbuscules are the central structures of the symbiotic association between terrestrial plants and arbuscular mycorrhizal (AM) fungi. However, arbuscules are also ephemeral structures, and following development, these structures are soon digested and ultimately disappear. Currently, little is known regarding the mechanism underlying the digestion of senescent arbuscules. Here, biochemical and functional analyses were integrated to test the hypothesis that a purple acid phosphatase, GmPAP33, controls the hydrolysis of phospholipids during arbuscule degeneration. The expression of GmPAP33 was enhanced by AM fungal inoculation independent of the P conditions in soybean roots. Promoter-β-glucuronidase (GUS) reporter assays revealed that the expression of GmPAP33 was mainly localized to arbuscule-containing cells during symbiosis. The recombinant GmPAP33 exhibited high hydrolytic activity towards phospholipids, phosphatidylcholine, and phosphatidic acid. Furthermore, soybean plants overexpressing GmPAP33 exhibited increased percentages of large arbuscules and improved yield and P content compared with wild-type plants when inoculated with AM fungi. Mycorrhizal RNAi plants had high phospholipid levels and a large percentage of small arbuscules. These results in combination with the subcellular localization of GmPAP33 at the plasma membrane indicate that GmPAP33 participates in arbuscule degeneration during AM symbiosis via involvement in phospholipid hydrolysis., (© 2019 John Wiley & Sons Ltd.)

Endothelin (ET)-1 is involved in the vascular system, cell proliferation and apoptosis. ET receptors consist of ET type A receptor (ET A R) and ET type B receptor (ET B R). ET A R and ET B R generally exhibit opposite responses, although many exceptions exist. In the present study, we attempted to identify ET A R- or ET B R-specific binding proteins to understand the differences in ET A R- and ET B R-mediated responses after ET-1 stimulation. The 78-kDa glucose-regulated protein (GRP78) showed a stronger binding affinity towards ET B R than towards ET A R. Moreover, GRP78 overexpression promoted ET B R-mediated ERK activation and GRP78 silencing suppressed ET B R-mediated ERK activation. Furthermore, ET B R can localize GRP78 to the cell periphery. These results suggest that the interaction of ET B R with GRP78 affects ERK activation and GRP78 localization., (© 2019 Federation of European Biochemical Societies.)

The enzyme 5,10-methylenetetrahydrofolate dehydrogenase (MTHFD) is essential for the production of certain amino acids (glycine, serine, and methionine) and nucleic acids (thymidylate and purine). Here, we identified a cDNA encoding this enzyme from the silkworm Bombyx mori . The recombinant B. mori MTHFD (bmMTHFD) expressed in Escherichia coli recognized 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate as substrate in the presence of NADP + as well as NAD + . The bmMTHFD structure was determined at a resolution of 1.75 Å by X-ray crystallography. Site-directed mutagenesis indicated that the amino acid residue Tyr49 contributed to its catalytic activity. Our findings provide insight into the mechanism underlying the activity of MTHFD from B. mori and potentially other insects and may therefore facilitate the development of inhibitors specific to MTHFD as insecticides., Competing Interests: The authors declare no conflict of interest.

A glutathione S -transferase (GST) with a potential dehalogenation function against various organochlorine substrates was identified from a polychlorobiphenyl (PCB)-degrading organism, Acidovorax sp. KKS102. A homolog of the gene BphK (biphenyl upper pathway K), named BphK-KKS , was cloned, purified and biochemically characterized. Bioinformatic analysis indicated several conserved amino acids that participated in the catalytic activity of the enzyme, and site-directed mutagenesis of these conserved amino acids revealed their importance in the enzyme's catalytic activity. The wild-type and mutant (C10F, K107T and A180P) recombinant proteins displayed wider substrate specificity. The wild-type recombinant GST reacted towards 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid, hydrogen peroxide and cumene hydroperoxide. The mutated recombinant proteins, however, showed significant variation in specific activities towards the substrates. A combination of a molecular docking study and a chloride ion detection assay showed potential interaction with and a dechlorination function against 2-, 3- and 4-chlorobenzoates (metabolites generated during PCB biodegradation) in addition to some organochlorine pesticides (dichlorodiphenyltrichloroethane, endosulfan and permethrin). It was demonstrated that the behavior of the dechlorinating activities varied among the wild-type and mutant recombinant proteins. Kinetic studies (using CDNB and glutathione) showed that the kinetic parameters K m , V max , K cat and K m / K cat were all affected by the mutations. While C10F and A180P mutants displayed an increase in GST activity and the dechlorination function of the enzyme, the K107T mutant displayed variable results, suggesting a functional role of Lys107 in determining substrate specificity of the enzyme. These results demonstrated that the enzyme should be valuable in the bioremediation of metabolites generated during PCB biodegradation., Competing Interests: The authors declare no conflict of interest.

Xyloglucan is one of the major polysaccharides found in the plant cell wall and seeds. Owing to its complex branched structure, several different hydrolases are required to degrade it. Isoprimeverose-producing enzymes (IPase) are unique among the glycoside hydrolase 3 family in that they recognize and release a disaccharide from the nonreducing end of xyloglucan oligosaccharides. Only two IPases have been previously isolated and characterized. A novel IPase from Phaeoacremonium minimum (PmIPase) was expressed and characterized. The xylopyranosyl residue at the nonreducing end of xyloglucan oligosaccharides was essential for hydrolytic activity, and PmIPase was unable to hydrolyze cellobiose into d-glucose. PmIPase had a K m for xyloglucan oligosaccharide substrate that was much lower than that of the reported IPase isolated from Aspergillus oryzae . This indicates that PmIPase was able to produce isoprimeverose efficiently from low concentrations of xyloglucan oligosaccharides. PmIPase also exhibited transglycosylation activity and was able to transfer isoprimeverose units to its substrates.

Protein phosphatase-1 (PP1) drives a large amount of phosphoSer/Thr protein dephosphorylations in eukaryotes to counteract multiple kinases in signaling pathways. The phosphatase requires divalent metal cations for catalytic activity and contains iron naturally. Iron has been suggested to have an influence on PP1 activity through Fe 2+ and Fe 3+ oxidation states. However, much biochemical and all structural data have been obtained with recombinant PP1 containing Mn 2+ ions. Purifying iron-containing PP1 from Escherichia coli has thus far not been possible. Here, we present the preparation, characterization, and structure of iron-bound PP1α in inactive and active states. We establish a key role for the electronic/redox properties of iron in PP1 activity and shed light on the difference in substrate specificity between iron- and manganese-containing PP1., (© 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The MEKK1 kinase is a key regulator of stress signaling in Arabidopsis; however, little is known about the regulation of its kinase activity. Here, we found that recombinant MEKK1, expressed in both mammalian HEK293 cells and Escherichia coli, shows a mobility shift in SDS-PAGE, and immunoblotting detected phosphorylation of serine, threonine, and tyrosine residues. N-terminal deletions, site-directed mutagenesis, and protein phosphatase treatment revealed that the mobility shift results from autophosphorylation of the kinase domain. We identified the tyrosine autophosphorylation sites in the N-terminal region of MEKK1. Tyrosine to phenylalanine mutations decrease phosphorylation of the substrate MKK1, suggesting the important role of this residue in the regulation of MEKK1 kinase activity. The present study is the first to show that plant MAPKKKs are regulated by tyrosine phosphorylation., (© 2018 Federation of European Biochemical Societies.)