Enzyme Complex Research Articles

N-terminal cleavage of cyclophilin D boosts its ability to bind F-ATP synthase.

Cyclophilin (CyP) D is a regulator of the mitochondrial F-ATP synthase. Here we report the discovery of a form of CyPD lacking the first 10 (mouse) or 13 (human) N-terminal residues (ΔN-CyPD), a protein region with species-specific features. NMR studies on recombinant human full-length CyPD (FL-CyPD) and ΔN-CyPD form revealed that the N-terminus is highly flexible, in contrast with the rigid globular part. We have studied the interactions of FL and ΔN-CyPD with F-ATP synthase at the OSCP subunit, a site where CyPD binding inhibits catalysis and favors the transition of the enzyme complex to the permeability transition pore. At variance from FL-CyPD, ΔN-CyPD binds OSCP in saline media, indicating that the N-terminus substantially decreases the binding affinity for OSCP. We also provide evidence that calpain 1 is responsible for generation of ΔN-CyPD in cells. Altogether, our work suggests the existence of a novel mechanism of modulation of CyPD through cleavage of its N-terminus that may have significant pathophysiological implications.

Screening, Gene Cloning and Expression of Cellulase-Producing Strain Bacillus subtilis Xh-16.

Cellulase is a complex enzyme system composed of multiple hydrolytic enzymes. It can degrade cellulose into glucose and improve the utilization efficiency of cellulose resources. Cellulase produced by microorganisms is the main method used in industry, offering the advantages of convenience and being environmentally friendly. A strain Xh-16 with high cellulase production was screened from the rotten rice straw. It was identified as Bacillus subtilis by morphological identification, physiological and biochemical identification, and 16S rRNA gene sequence analysis. Strain Xh-16 was used to degrade rice straw. After a 48h cellulase treatment, the complete degradation and structural breakdown of the straw were observed. We cloned the endoglucanase Cel5L gene of Bacillus subtilis Xh-16 and induced expression of the cloned gene in Escherichia coli BL21 (DE3). The results showed that the coding length of Cel5L gene was 1500bp, and the molecular weight of the encoded protein was about 55kDa. The molecular formula is C2456H3811N671O761S10 and it has 7709 atoms and 499 amino acids. Cel5L differs significantly from some the glycoside hydrolase family 5 cellulases because it has only one carbohydrate binding module family 3 at the C-terminus of its catalytic domain. The cellulase gene Cel5L in Xh-16 can encode active cellulase and can be heterologously expressed in Escherichia coli, which makes Escherichia coli have cellulase function. This study serves as a foundation for further research on cellulase diversity in Bacillus subtilis and offers insights for enhancing cellulase production.

Mitochondrial Mutations in Cardiovascular Diseases: Preliminary Findings

Background/Objectives: Mitochondria are the main organelles for ATP synthesis able to produce energy for several different cellular activities. Cardiac cells require high amounts of energy and, thus, they contain a high number of mitochondria. Consequently, mitochondrial dysfunction in these cells is a crucial factor for the development of cardiovascular diseases. Mitochondria constitute central regulators of cellular metabolism and energy production, producing approximately 90% of the cells’ energy needs in the form of ATP via oxidative phosphorylation. The mitochondria have their own circular, double-stranded DNA encoding 37 genes. Any mitochondrial DNA sequence anomaly may result in defective oxidative phosphorylation and lead to cardiac dysfunction. Methods: In this study, we investigated the potential association between mitochondrial DNA mutation and cardiovascular disease. Cardiac tissue and serum samples were collected from seven patients undergoing coronary artery bypass grafting. Total DNA was extracted from cardiac muscle tissue specimens and serum and each sample was subjected to polymerase chain reaction (PCR) to amplify the NADH dehydrogenase 1 (ND1) gene, which is part of the mitochondrial complex I enzyme complex and was screened for mutations. Results: We identified one patient with a homoplasmic A to G substitution mutation in cardiac tissue DNA and two patients with heteroplasmic A3397G mutation in serum DNA. Specifically, amplicon sequence analysis revealed a homoplasmic A3397G substitution in the ND1 gene in a tissue sample of the patient with ID number 1 and a heteroplasmic mutation in A3397G in serum samples of patients with ID numbers 3 and 6, respectively. The A to G substitution changes the amino acid from methionine (ATA) to valine (GTA) at position 31 of the ND1 gene. Conclusions: The detection of this novel mutation in patients with coronary artery disease may contribute to our understanding of the association between mitochondrial dysfunction and the disease, implying that mitochondria may be key players in the pathogenesis of cardiovascular diseases.

Untargeted metabolomics and PacBio analysis on bioactive components and microbial community in co-fermentation of black soldier fly larva

Fermentation can enhance nutritional value and safety of insect protein, this study utilized probiotic Bacillus subtilis (B. subtilis) and complex enzyme containing chitinase and protease to ferment the paste of Black Soldier Fly larva (BSFL), decomposing anti-nutritional factor chitin and protein in paste while inhibiting the proliferation of harmful microorganisms. The result indicated a 40 % degradation of chitin after fermentation, accompanied by an increase in the variety and quantity of amino acids and peptides, functional substances such as raffinose and cucurbitacin significantly increased, while the levels of antibiotics such as erythromycin and ofloxacin had decreased; after fermentation, there is a significant difference in the microbial distribution between bacteria, co-fermentation and CK, the indigenous microbiota of BSF and pathogenic bacteria such as Klebsiella pneumoniae and Clostridiaceae bacteria were significantly inhibited, anaerobic bacteria, including Anaerosalibacter, Caldicoprobacter and Tissierella, exhibit a marked increase; significant changes are detected in the carbon sources, amino acids, and key enzymes related to other metabolic pathways of B. subtilis during the fermentation process. Overall, we have developed a method for fermenting BSFL paste, aiming at enhance its probiotic properties, nutritional value, and safety. This study provided groundwork for utilizing fermented insects as a novel protein source for food and fodder.

Engineered bacterial lipoate protein ligase A (lplA) restores lipoylation in cell models of lipoylation deficiency.

Protein lipoylation, a vital lysine posttranslational modification (PTM), plays a crucial role in the function of key mitochondrial TCA cycle enzymatic complexes. In eukaryotes, lipoyl PTM synthesis occurs exclusively through de novo pathways, relying on lipoyl synthesis/transfer enzymes, dependent upon mitochondrial fatty acid and Fe-S cluster biosynthesis. Dysregulation in any of these pathways leads to diminished cellular lipoylation. Efficient restoration of lipoylation in lipoylation deficiency cell states using either chemical or genetic approaches has been challenging due to pathway complexity and multiple upstream regulators. To address this challenge, we explored the possibility that a bacterial lipoate protein ligase (lplA) enzyme, that can salvage free lipoic acid bypassing the dependency on de novo synthesis, could be engineered to be functional in human cells. Overexpression of the engineered lplA in lipoylation null cells restored lipoylation levels, cellular respiration, and growth in low glucose conditions. Engineered lplA restored lipoylation in all tested lipoylation null cell models, mimicking defects in mitochondrial fatty acid synthesis (MECR KO), Fe-S cluster biosynthesis (BOLA3 KO), and specific lipoylation regulating enzymes (FDX1, LIAS and LIPT1 KOs). Furthermore, we describe a patient with a homozygous c.212C>T variant LIPT1 with a previously uncharacterized syndromic congenital sideroblastic anemia. K562 erythroleukemia cells engineered to harbor this missense LIPT1 allele recapitulate the lipoylation deficient phenotype and exhibit impaired proliferation in low glucose that is completely restored by engineered lplA. This synthetic approach offers a potential therapeutic strategy for treating lipoylation disorders.

Identification and Analysis of Phenolic Compounds in Vaccinium uliginosum L. and Its Lipid-Lowering Activity In Vitro.

Vaccinium uliginosum L. (VU), rich in polyphenols, is an important wild berry resource primarily distributed in extremely cold regions. However, the detailed composition of Vaccinium uliginosum L. polyphenols (VUPs) has not been reported, which limits the development and utilization of VU. In this study, VU-free polyphenols (VUFPs) and VU-bound polyphenols (VUBPs) were, respectively, extracted using an ultrasonic, complex enzyme and alkali extraction method; the compositions were identified using ultra-performance liquid chromatography-electrospray ionization mass spectrometry, and lipid-lowering activity in vitro was evaluated. The results showed that 885 polyphenols and 47 anthocyanins were detected in the VUFPs and VUBPs, and 30 anthocyanin monomers were firstly detected in VU. Compared with the model group, the accumulation of lipid droplets and the total cholesterol and triglyceride contents in the high-concentration VUP group reduced by 36.95%, 65.82%, and 62.43%, respectively, and liver damage was also alleviated. It was also found that VUP can regulate the level of Asialoglycoprotein receptor 1, a new target for lipid lowering. In summary, this study provides a detailed report on VUP for the first time, confirming that VUP has lipid-lowering potential in vitro. These findings suggest new strategies and theoretical support for the development and utilization of VU, especially in the field of functional foods.

Elucidating the metabolic roles of isoflavone synthase-mediated protein-protein interactions in yeast.

Transient plant enzyme complexes formed via protein-protein interactions (PPIs) play crucial regulatory roles in secondary metabolism. Complexes assembled on cytochrome P450s (CYPs) are challenging to characterize metabolically due to difficulties in decoupling the PPIs' metabolic impacts from the CYPs' catalytic activities. Here, we developed a yeast-based synthetic biology approach to elucidate the metabolic roles of PPIs between a soybean-derived CYP, isoflavone synthase (GmIFS2), and other enzymes in isoflavonoid metabolism. By reconstructing multiple complex variants with an inactive GmIFS2 in yeast, we found that GmIFS2-mediated PPIs can regulate metabolic flux between two competing pathways producing deoxyisoflavonoids and isoflavonoids. Specifically, GmIFS2 can recruit chalcone synthase (GmCHS7) and chalcone reductase (GmCHR5) to enhance deoxyisoflavonoid production or GmCHS7 and chalcone isomerase (GmCHI1B1) to enhance isoflavonoid production. Additionally, we identified and characterized two novel isoflavone O -methyltransferases interacting with GmIFS2. This study highlights the potential of yeast synthetic biology for characterizing CYP-mediated complexes.

The effects of early feeding with different pre-starter diets on growth performance, morphological development and microorganism content of the small intestine of broiler chicks

The present study was conducted to compare the effects of feeding 2 different pre-starter diets to broiler chicks at 2 different times on growth performance, slaughtering parameters, morphological development of the small intestine segments and the microorganism content of the ileum of broilers. A total of 600 day-old Ross 308 male broiler chicks were randomly distributed into 5 treatment groups with 5 replicates of 24 chicks each. The experimental treatments included: SD10: broiler chicks were fed a corn-soybean meal-based starter diet for 0-10 days; 1PSD5: chicks were fed a first pre-starter diet based on corn-soybean meal for 0-5 days, afterwards they were fed a starter diet based on corn-soybean meal for 5-10 days; 1PSD10: chicks were fed the first pre-starter diet for 0-10 days; 2PSD5: chicks were fed a second pre-starter diet based on rice-soybean meal for 0-5 days, afterwards they were fed a starter diet based on corn-soybean meal for 5-10 days; 2PSD10: chicks were fed the second pre-starter diet for 0-10 days. Broilers were fed a basal diet based on corn and soybean meal until the end of the experimental period. The 1PSD5, 1PSD10, 2PSD5 and 2PSD10 treatments significantly increased final body weight (BW) and BW gain, decreased feed intake (FI) and improved feed conversion ratio (FCR) of broilers from 0 to 42 day compared to the SD10 treatment. The lowest FI and the best FCR of broilers during the period of 0 to 42 day were obtained by the 2PSD5 treatment. The 1PSD5, 1PSD10, 2PSD5 and 2PSD10 treatments significantly enhanced the hot- and cold-carcass yields and villus heights (VHs) and decreased crypt depths of small intestine segments of broilers on day 42. However, the experimental treatments did not significantly affect the the E.coli and Lactobacillus contents of ileum of broilers on day 42. In conclusion, feeding of chicks with the pre-starter diet based on rice-soybean meal supplemented with enzyme complex and synbiotic for 0-5 days was a more effective treatment in terms of performance especially FI and FCR, carcass yields and VHs of small intestine segments of broilers.

Distinct Regions within SAP25 Recruit O-Linked Glycosylation, DNA Demethylation, and Ubiquitin Ligase and Hydrolase Activities to the Sin3/HDAC Complex.

Sin3 is an evolutionarily conserved repressor protein complex mainly associated with histone deacetylase (HDAC) activity. Many proteins are part of Sin3/HDAC complexes, and the function of most of these members remains poorly understood. SAP25, a previously identified Sin3A associated protein of 25 kDa, has been proposed to participate in regulating gene expression programs involved in the immune response but the exact mechanism of this regulation is unclear. SAP25 is not expressed in HEK293 cells, which hence serve as a natural knockout system to decipher the molecular functions uniquely carried out by this Sin3/HDAC subunit. Using molecular, proteomic, protein engineering, and interaction network approaches, we show that SAP25 interacts with distinct enzymatic and regulatory protein complexes in addition to Sin3/HDAC. Additional proteins uniquely recovered from the Halo-SAP25 pull-downs included the SCF E3 ubiquitin ligase complex SKP1/FBXO3/CUL1 and the ubiquitin carboxyl-terminal hydrolase 11 (USP11). Furthermore, mutational analysis demonstrates that distinct regions of SAP25 participate in its interaction with USP11, OGT/TETs, and SCF(FBXO3). These results suggest that SAP25 may function as an adaptor protein to coordinate the assembly of different enzymatic complexes to control Sin3/HDAC-mediated gene expression. The data were deposited with the MASSIVE repository with the identifiers MSV000093576 and MSV000093553.

Structure and Energetics of PET-Hydrolyzing Enzyme Complexes: A Systematic Comparison from Molecular Dynamics Simulations.

Discovered in 2016, the enzyme PETase, secreted by bacterial Ideonella Sakaiensis 201-F6, has an excellent hydrolytic activity toward poly(ethylene terephthalate) (PET) at room temperature, while it decreases at higher temperatures due to the low thermostability. Many variants have been engineered to overcome this limitation, which hinders industrial application. In this work, we systematically compare PETase wild-type (WT) and four mutants (DuraPETase, ThermoPETase, FastPETase, and HotPETase) using standard molecular dynamics (MD) simulations and unbinding free energy calculations. In particular, we analyze the enzymes' structural characteristics and binding to a tetrameric PET chain (PET4) under two temperature conditions: T1─300 K and T2─350 K. Our results indicate that (i) PET4 forms stable complexes with the five enzymes at room temperature (∼300 K) and (ii) most of the interactions are localized close to the active site of the protein, where the W185 and Y87 residues interact with the aromatic rings of the substrate. Specifically, (iii) the W185 side-chain explores different conformations in each variant (a phenomenon known in the literature as "W185 wobbling"). This suggests that the binding pocket retains structural plasticity and flexibility among the variants, facilitating substrate recognition and localization events at moderate temperatures. Moreover, (iv) PET4 establishes aromatic interactions with the catalytic H237 residue, stabilizing the catalytic triad composed of residues S160-H237-D206, and helping the system achieve an effective configuration for the hydrolysis reaction. Conversely, (v) the binding affinity decreases at a higher temperature (∼350 K), retaining moderate interactions only for HotPETase. Finally, (vi) MD simulations of complexes formed with poly(ethylene-2,5-furan dicarboxylate) (PEF) show no persistent interactions, suggesting that these enzymes are not yet optimized for binding this alternative semiaromatic plastic polymer. Our study offers valuable insights into the structural stability of these enzymes and the molecular determinants driving PET binding onto their surfaces, sheds light on the mechanistic steps that precede the onset of hydrolysis, and provides a foundation for future enzyme optimization.

Epigenetic therapies targeting histone lysine methylation: complex mechanisms and clinical challenges.

As epigenetic therapies continue to gain ground as potential treatment strategies for cancer and other diseases, compounds that target histone lysine methylation and the enzyme complexes represent a major frontier for therapeutic development. Clinically viable therapies targeting the activities of histone lysine methyltransferases (HKMT) and demethylases (HKDMs) have only recently begun to emerge following FDA approval of the EZH2 inhibitor tazemetostat in 2020 and remain limited to compounds targeting the well-studied SET domain-containing HKMTs and their opposing HKDMs. These include the H3K27 methyltransferases EZH2/EZH1, the singular H3K79 methyltransferase DOT1L, and the H3K4 methyltransferase MLL1/COMPASS as well as H3K9 and H3K36 methyltransferases. They additionally include the H3K4/9-preferential demethylase LSD1 and the H3K4-, H3K27-, and H3K36-preferential KDM5, KDM6, and KDM2 demethylase subfamilies, respectively. This Review discusses the results of recent clinical and preclinical studies relevant to all of these existing and potential therapies. It provides an update on advancements in therapeutic development, as well as more basic molecular understanding, within the past 5 years approximately. It also offers a perspective on histone lysine methylation that departs from the long-predominant "histone code" metaphor, emphasizing complex-disrupting inhibitors and proximity-based approaches rather than catalytic domain inhibitors in the outlook for future therapeutic development.

Food as a factor determining the physiological state of populations of the phytopagous pests of agricultural crops

Relevance. Trophic relationships along with competition and mutualism are the most basic and significant interactions in ecosystems. To develop, survive, and multiply, insects need to consume a certain amount of nutrients at a certain ratio. Food products are complex mixes of nutrients and non-nutritive substances (sometimes toxic ones): macronutrients (proteins, carbohydrates, and lipids), micronutrients (vitamins and minerals), and water. Some nutrients are essential; insects lack the ability to synthesize them in their bodies and must obtain them from their diet or through symbiosis with beneficial organisms. Identification mechanisms being well developed in the system “phytophagous insect – plant” allow insects to successfully spread, multiply, and feed on certain plant species. The complex of hydrolytic enzymes in the insect intestine is one of the main targets for plant defense responses because these enzymes determine the availability of structural compounds for phytophagous insects. For this reason, enzymes in the insect intestine play a key role in the adaptation of insects to the pest resistance of fodder plants. For instance, when proteinase inhibitors are synthesized in a fodder plant as the result of induced insect resistance the complex of enzymes in an insect intestine might change negating the effect of these inhibitors. The development of co-adaptations due to interactions among species in food chains depends on a complicated constellation of conflicting relationships between consumers and food objects. The mechanisms of this influence may be rooted in the allelochemical interactions in the system “phytophagous insect – plant recipient”. Allelopathic interactions are among the most complex interactions because they are constituted by direct and indirect effects. Plants when damaged by phytophagous insects activate defense responses, which incorporate several mechanisms, including an increase in the concentration of secondary metabolites, many of which are phenolic compounds.The aim of the work is to describe the mechanisms of relationships in the system “phytophage-plant”. Conclusion. Management of processes of intra-water divergence of insect-phytophages in agrobiocoenoses in order to prevent the emergence of races and populations of pests adapted to live on initially resistant to them plant forms is possible in compliance with the transition to a targeted selection of agricultural crops for resistance to a complex of pests.

Biallelic variants in MRPL49 cause variable clinical presentations, including sensorineural hearing loss, leukodystrophy, and ovarian insufficiency.

Combined oxidative phosphorylation deficiency (COXPD) is a rare multisystem disorder which is clinically and genetically heterogeneous. Genome sequencing identified biallelic MRPL49 variants in individuals from five unrelated families with presentations ranging from Perrault syndrome (primary ovarian insufficiency and sensorineural hearing loss) to severe childhood onset of leukodystrophy, learning disability, microcephaly and retinal dystrophy. Complexome profiling of fibroblasts from affected individuals revealed reduced levels of the small and, a more pronounced reduction of, the large mitochondrial ribosomal subunits. There was no evidence of altered mitoribosomal assembly. The reductions in levels of OXPHOS enzyme complexes I and IV are consistent with a form of COXPD associated with biallelic MRPL49 variants, expanding the understanding of how disruption of the mitochondrial ribosomal large subunit results in multi-system phenotypes.

A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System

AbstractIn this study, a versatile method is developed to create an antibody/split‐enzyme complex for use in electrochemical immunosensors. SpyCatchers are genetically fused at each terminus of flavin adenine dinucleotide‐dependent glucose dehydrogenase (FAD‐GDH) and a protease recognition sequence is inserted into a loop region to prepare a soluble protein and the split GDH. SpyTag‐fused single‐chain variable fragments (scFvs) are employed, which leads to the simple preparation of antibody‐split enzyme complexes (split AECs). The addition of pentameric C‐reactive protein (CRP), as a representative multimeric protein, restored the GDH activity of the split AECs, as the two split AEC fragments formed active GDH when in close proximity. CRP in human serum is electrochemically detected within 5 min without any washing steps with a clinically relevant CRP detection range (0.03–1 mg dL−1). The detection system is expanded to detect hemoglobin, SARS‐CoV‐2 spike protein, and inactivated SARS‐CoV‐2 by exchanging the scFvs. These results show that the convenient preparation method of the split GDH and the split AEC by the insertion of the protease recognition sequence and the use of SpyCatcher/SpyTag can realize a rapid, homogeneous, and universal electrochemical detection system that can be integrated into a commercially available electrochemical glucose sensor.

Connecting the Dots: Telomere Shortening and Rheumatic Diseases.

Telomeres, repetitive sequences located at the extremities of chromosomes, play a pivotal role in sustaining chromosomal stability. Telomerase is a complex enzyme that can elongate telomeres by appending telomeric repeats to chromosome ends and acts as a critical factor in telomere dynamics. The gradual shortening of telomeres over time is a hallmark of cellular senescence and cellular death. Notably, telomere shortening appears to result from the complex interplay of two primary mechanisms: telomere shelterin complexes and telomerase activity. The intricate interplay of genetic, environmental, and lifestyle influences can perturb telomere replication, incite oxidative stress damage, and modulate telomerase activity, collectively resulting in shifts in telomere length. This age-related process of telomere shortening plays a considerable role in various chronic inflammatory and oxidative stress conditions, including cancer, cardiovascular disease, and rheumatic disease. Existing evidence has shown that abnormal telomere shortening or telomerase activity abnormalities are present in the pathophysiological processes of most rheumatic diseases, including different disease stages and cell types. The impact of telomere shortening on rheumatic diseases is multifaceted. This review summarizes the current understanding of the link between telomere length and rheumatic diseases in clinical patients and examines probable telomere shortening in peripheral blood mononuclear cells and histiocytes. Therefore, understanding the intricate interaction between telomere shortening and various rheumatic diseases will help in designing personalized treatment and control measures for rheumatic disease.

Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB’s low specificity for Tv20S compared to the human proteasome and demonstrate CP-17’s higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.

Structural Analysis and Substrate Specificity of D-Carbamoylase from Pseudomonas.

The synthesis of enantiomeric forms of D-amino acids can be achieved by a two-step "hydantoinase process" based on the sequential catalysis of substrates by specific enzymes, D-carbamoylase and D-hydantoinase. Here, we describe the structural features of D-carbamoylase from Pseudomonas, the encoded gene of which was chemically synthesized and cloned into Escherichia coli. A significant fraction of the overexpressed recombinant protein forms insoluble inclusion bodies, which are partially converted to a soluble state upon treatment with N-lauroylsarcosine or upon incubation of cells at 28 °C. Purified His-tagged protein exhibits the highest activity towards N-carbamoyl-D-alanine and N-carbamoyl-D-tryptophan. Comprehensive virtual analysis of the interactions of bulky carbamylated amino acids with D-carbamoylase provided valuable information. Molecular docking analysis revealed the location of the substrate binding site in the three-dimensional structure of D-carbamoylase. Molecular dynamics simulations showed that the binding pocket of the enzyme in complex with N-carbamoyl-D-tryptophan was stabilized within 100 nanoseconds. The free energy data showed that Arg176 and Asn173 formed hydrogen bonds between the enzyme and substrates. The studies of D-carbamoylases and the properties of our previously obtained D-hydantoinase suggest the possibility of developing a harmonized biotechnological process for the production of new drugs and peptide hormones.

LRRK2 regulates production of reactive oxygen species in cell and animal models of Parkinson's disease.

Oxidative stress has long been implicated in Parkinson's disease (PD) pathogenesis, although the sources and regulation of reactive oxygen species (ROS) production are poorly defined. Pathogenic mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are associated with increased kinase activity and a greater risk of PD. The substrates and downstream consequences of elevated LRRK2 kinase activity are still being elucidated, but overexpression of mutant LRRK2 has been associated with oxidative stress, and antioxidants reportedly mitigate LRRK2 toxicity. Here, using CRISPR-Cas9 gene-edited HEK293 cells, RAW264.7 macrophages, rat primary ventral midbrain cultures, and PD patient-derived lymphoblastoid cells, we found that elevated LRRK2 kinase activity was associated with increased ROS production and lipid peroxidation and that this was blocked by inhibitors of either LRRK2 kinase or NADPH oxidase 2 (NOX2). Oxidative stress induced by the pesticide rotenone was ameliorated by LRRK2 kinase inhibition and was absent in cells devoid of LRRK2. In a rat model of PD induced by rotenone, a LRRK2 kinase inhibitor prevented the lipid peroxidation and NOX2 activation normally seen in nigral dopaminergic neurons in this model. Mechanistically, LRRK2 kinase activity was shown to regulate phosphorylation of serine-345 in the p47phox subunit of NOX2. This, in turn, led to translocation of p47phox from the cytosol to the membrane-associated gp91phox (NOX2) subunit, activation of the NOX2 enzyme complex, and production of ROS. Thus, LRRK2 kinase activity may drive cellular ROS production in PD through the regulation of NOX2 activity.

Investigating the interactions between an industrial lipase and anionic (bio)surfactants

In laundry formulations, synergies between amphiphiles and other additives such as enzymes increase sustainability through a large decrease in energy consumption. However, traditional surfactants are derived from petroleum, requiring chemical modifications (sulfonation, ethoxylation, or esterification) and generating environmental pollution through toxicity and low degradability. Use of biosurfactants removes these issues. To provide a firmer basis for the use of biosurfactants, we report on the interactions between the industrial lipase LIPEX® and three common biosurfactants, rhamnolipids, sophorolipids, and surfactin. The model surfactant sodium dodecyl sulfate (SDS) is included in the study for comparison. A thorough characterization by Small-angle X-ray scattering (SAXS) provides valuable information on the enzyme’s oligomerization and the surfactant micelles’ ellipsoidal morphology. Additionally, the enzymatic activity and complex formation in different surfactant mixtures are studied using isothermal titration calorimetry, activity assays, and SAXS. SDS activates the enzyme while promoting a controlled association of monomers while the biosurfactants inhibit the enzyme, independent of their effects on its quaternary structure. Rhamnolipids and surfactin promote lipase dimerization while sophorolipids have no significant effect on lipase quaternary structure. Based on these data, we propose a partial replacement that allows the enzyme to retain enzymatic activity while improving the environmental footprint of the formulation.

Decreased Mrpl42 expression exacerbates myocardial ischemia and reperfusion injury by inhibiting mitochondrial translation

Mammalian mitochondrial DNA (mtDNA) encodes a total of 13 proteins, all of which are subunits of enzyme complexes of the oxidative phosphorylation. The mtDNA-encoded protein synthesis depends on the mitochondrial ribosomal proteins (MRPs), which assemble to form a specialized form of ribosome. Some mtDNA-encoded proteins have been reported to be reduced after myocardial ischemic injury. However, the molecular mechanisms responsible for this decrease and whether this decrease is involved in myocardial ischemia/reperfusion (I/R) injury remains unknown. Here, we found that the mtDNA-encoded protein levels were significantly decreased after I/R injury, while the mRNA levels of these genes were either increased or had no significant change. Subsequently, by querying and analyzing public database resources, we found that the expression of many mitochondrial translation-related proteins tended to decrease after myocardial infarction injury, and the reduction in the expression of these proteins was most obvious for Mrpl42. Furthermore, we found that cardiac Mrpl42 knockdown aggravated I/R-induced cardiac contractile dysfunction and cardiomyocyte death, while restoring Mrpl42 expression in the heart reduced I/R injury. Mrpl42 knockdown impaired the translation of mtDNA-encoded genes, ultimately led to aberrations in mitochondrial morphology and respiratory function. In addition, we found that the decrease in the expression of Mrpl42 after I/R injury was caused by the downregulation of Nrf2, which directly regulates Mrpl42 transcription. Our study revealed that ischemic downregulation of Mrpl42 expression and subsequent inhibition of mitochondrial translation contribute to cardiac I/R injury. Targeting Mrpl42 may be a novel therapeutic intervention for cardiac I/R injury and myocardial infarction.