The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6.

The cellular activation of the NLRP3 inflammasome is spatiotemporally orchestrated by various organelles, but whether lysosomes contribute to this process remains unclear. Here, we show the vital role of the lysosomal membrane-tethered Ragulator complex in NLRP3 inflammasome activation. Deficiency of Lamtor1, an essential component of the Ragulator complex, abrogated NLRP3 inflammasome activation in murine macrophages and human monocytic cells. Myeloid-specific Lamtor1-deficient mice showed marked attenuation of NLRP3-associated inflammatory disease severity, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate (MSU)-induced arthritis. Mechanistically, Lamtor1 interacted with both NLRP3 and histone deacetylase 6 (HDAC6). HDAC6 enhances the interaction between Lamtor1 and NLRP3, resulting in NLRP3 inflammasome activation. DL-all-rac-α-tocopherol, a synthetic form of vitamin E, inhibited the Lamtor1-HDAC6 interaction, resulting in diminished NLRP3 inflammasome activation. Further, DL-all-rac-α-tocopherol alleviated acute gouty arthritis and MSU-induced peritonitis. These results provide novel insights into the role of lysosomes in the activation of NLRP3 inflammasomes by the Ragulator complex.

Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome.

Mitochondrial DNA (mtDNA) leakage into the cytoplasm can occur when cells are exposed to noxious stimuli. Specific sensors recognize cytoplasmic mtDNA to promote cytokine production. Cytoplasmic mtDNA can also be secreted extracellularly, leading to sterile inflammation. However, the mode of secretion of mtDNA out of cells upon noxious stimuli and its relevance to human disease remain unclear. Here, we show that pyroptotic cells secrete mtDNA encapsulated within exosomes. Activation of caspase-1 leads to mtDNA leakage from the mitochondria into the cytoplasm via gasdermin-D. Caspase-1 also induces intraluminal membrane vesicle formation, allowing for cellular mtDNA to be taken up and secreted as exosomes. Encapsulation of mtDNA within exosomes promotes a strong inflammatory response that is ameliorated upon exosome biosynthesis inhibition in vivo. We further show that monocytes derived from patients with Behçet's syndrome (BS), a chronic systemic inflammatory disorder, show enhanced caspase-1 activation, leading to exosome-mediated mtDNA secretion and similar inflammation pathology as seen in BS patients. Collectively, our findings support that mtDNA-containing exosomes promote inflammation, providing new insights into the propagation and exacerbation of inflammation in human inflammatory diseases.

Yes-associated protein 1 mediates initial cell survival during lorlatinib treatment through AKT signaling in ROS1-rearranged lung cancer.

Tyrosine kinase inhibitors (TKIs) that target the ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) gene have shown dramatic therapeutic effects in patients with ROS1-rearranged non-small-cell lung cancer (NSCLC). Nevertheless, advanced ROS1-rearranged NSCLC is rarely cured as a portion of the tumor cells can survive the initial stages of ROS1-TKI treatment, even after maximum tumor shrinkage. Therefore, understanding the mechanisms underlying initial cell survival during ROS1-TKI treatment is necessary to prevent cell survival and achieve a cure for ROS1-rearranged NSCLC. In this study, we clarified the initial survival mechanisms during treatment with lorlatinib, a ROS1 TKI. First, we established a patient-derived ezrin gene-ROS1-rearranged NSCLC cell line (KTOR71). Then, following proteomic analysis, we focused on yes-associated protein 1 (YAP1), which is a major mediator of the Hippo pathway, as a candidate factor involved in cell survival during early lorlatinib treatment. Yes-associated protein 1 was activated by short-term lorlatinib treatment both in vitro and in vivo. Genetic inhibition of YAP1 using siRNA, or pharmacological inhibition of YAP1 function by the YAP1-inhibitor verteporfin, enhanced the sensitivity of KTOR71 cells to lorlatinib. In addition, the prosurvival effect of YAP1 was exerted through the reactivation of AKT. Finally, combined therapy with verteporfin and lorlatinib was found to achieve significantly sustained tumor remission compared with lorlatinib monotherapy in vivo. These results suggest that YAP1 could mediate initial cell resistance to lorlatinib in KTOR71 cells. Thus, combined therapy targeting both YAP1 and ROS1 could potentially improve the outcome of ROS1-rearranged NSCLC.

Dramatic dietary shift maintains sequestered toxins in chemically defended snakes.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins.

Enhancement of PET degradation by PET depolymerase with the microbe addition.

The degradation of polyethylene terephthalate (PET) by modified PET depolymerase has recently attracted much attention. We found that mixing a PET depolymerase with non-genetically modified Thermus sp. can enhance its PET-degrading activity by 7.7-fold. This approach is attractive for constructing a sustainable PET recycling system.

Improved ammonia production from soybean residues by cell surface-displayed l-amino acid oxidase on yeast.

Ammonia is critical for agricultural and chemical industries. The extracellular production of ammonia by yeast (Saccharomyces cerevisiae) using cell surface engineering can be efficient approach because yeast can avoid growth deficiencies caused by knockout of genes for ammonia assimilation. In this study, we produced ammonia outside the yeast cells by displaying an l-amino acid oxidase with a wide substrate specificity derived from Hebeloma cylindrosporum (HcLAAO) on yeast cell surfaces. The HcLAAO-displaying yeast successfully produced 12.6 m m ammonia from a mixture of 20 proteinogenic amino acids (the theoretical conversion efficiency was 63%). We also succeeded in producing ammonia from a food processing waste, soybean residues (okara) derived from tofu production. The conversion efficiency was 88.1%, a higher yield than reported in previous studies. Our study demonstrates that ammonia production outside of yeast cells is a promising strategy to utilize food processing wastes.

Accuracy of rapid antigen detection test for nasopharyngeal swab specimens and saliva samples in comparison with RT-PCR and viral culture for SARS-CoV-2 detection.

INTRODUCTION: Rapid antigen detection (RAD) tests are convenient tools for detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinics, and testing using saliva samples could decrease the risk of infection during sample collection. This study aimed to assess the accuracy of the SARS-CoV-2 RAD for testing of nasopharyngeal swab specimens and saliva samples in comparison with the RT-PCR tests and viral culture for detecting viable virus. METHODS: One hundred seventeen nasopharyngeal swab specimens and 73 saliva samples with positive results on RT-PCR were used. Residual samples were assayed using a commercially available RAD test immediately, and its positivity was determined at various time points during the clinical course. The concordance between 54 nasopharyngeal swab samples and saliva samples that were collected simultaneously was determined. Viral culture was performed on 117 samples and compared with the results of the RAD test. RESULTS: The positive rate of RAD test using saliva samples was low throughout the clinical course. Poor concordance was observed between nasopharyngeal swab specimens and saliva samples (75.9%, kappa coefficient 0.310). However, a substantially high concordance between the RAD test and viral culture was observed in both nasopharyngeal swab specimens (86.8%, kappa coefficient 0.680) and saliva samples (95.1%, kappa coefficient 0.643). CONCLUSIONS: The sensitivity of the SARS-CoV-2 RAD test was insufficient, particularly for saliva samples. However, a substantially high concordance with viral culture suggests its potential utility as an auxiliary test for estimating SARS-CoV-2 viability.

Pentenediol-Type Compounds Specifically Bind to Voltage-Dependent Anion Channel 1 in Saccharomyces cerevisiae Mitochondria.

Voltage-dependent anion channel 1 (VDAC1) situated in the outer mitochondrial membrane regulates the transfer of various metabolites and is a key player in mitochondria-mediated apoptosis. Although many small chemicals that modulate the functions of VDAC1 have been reported to date, most, if not all, of them cannot be regarded as specific reagents due to their interactions with other transporters or enzymes. By screening our chemical libraries using isolated Saccharomyces cerevisiae mitochondria, we found pentenediol (PTD)-type compounds (e.g., PTD-023) as new specific inhibitors of VDAC1. PTD-023 inhibited overall ADP-uptake/ATP-release reactions in isolated mitochondria at a single digit µM level. To identify the binding position of PTDs in VDAC1 by visualizing PTD-bound peptides, we conducted ligand-directed tosyl (LDT) chemistry using the synthetic LDT reagent t-PTD-023 derived from the parent PTD-023 in combination with mutagenesis experiments. t-PTD-023 made a covalent bond predominantly and subsidiarily with nucleophilic Cys210 and Cys130, respectively, indicating that PTDs bind to the region interactive with both residues. Site-directed mutations of hydrogen bond-acceptable Asp139 and Glu152 to Ala, which were selected as potential interactive partners of the critical pentenediol moiety based on the presumed binding model of PTDs in VDAC1, resulted in a decrease in susceptibility against PTD-023. This result strongly suggests that PTDs bind to VDAC1 through a specific hydrogen bond with the two residues. The present study is the first to demonstrate the binding position of specific inhibitors of VDAC1 at the amino acid level.

Comparative Proteomic Analysis of Lithospermum erythrorhizon Reveals Regulation of a Variety of Metabolic Enzymes Leading to Comprehensive Understanding of the Shikonin Biosynthetic Pathway.

Plants produce a large variety of specialized (secondary) metabolites having a wide range of hydrophobicity. Shikonin, a red naphthoquinone pigment, is a highly hydrophobic metabolite produced in the roots of Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae. The shikonin molecule is formed by the coupling of p-hydroxybenzoic acid and geranyl diphosphate, catalyzed by a membrane-bound geranyltransferase LePGT at the endoplasmic reticulum, followed by cyclization of the geranyl chain and oxidations; the latter half of this biosynthetic pathway, however, has not yet been clarified. To shed light on these steps, a proteome analysis was conducted. Shikonin production in vitro was specifically regulated by illumination and by the difference in media used to culture cells and hairy roots. In intact plants, however, shikonin is produced exclusively in the root bark of L. erythrorhizon. These features were utilized for comparative transcriptome and proteome analyses. As the genome sequence is not known for this medicinal plant, sequences from de novo RNA-seq data with 95,861 contigs were used as reference for proteome analysis. Because shikonin biosynthesis requires copper ions and is sensitive to blue light, this methodology identified strong candidates for enzymes involved in shikonin biosynthesis, such as polyphenol oxidase, cannabidiolic acid synthase-like and neomenthol dehydrogenase-like proteins. Because acetylshikonin is the main end product of shikonin derivatives, an O-acetyltransferase was also identified. This enzyme may be responsible for end product formation in these plant species. Taken together, these findings suggest a putative pathway for shikonin biosynthesis.

Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides.

BACKGROUND: Clostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation. RESULTS: We cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8-19 cellulosomal proteins and 9-16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant. CONCLUSION: In this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides.

Epoxycyclohexenedione-Type Compounds Make Up a New Class of Inhibitors of the Bovine Mitochondrial ADP/ATP Carrier.

Through the extensive screening of our chemical library, we found epoxycyclohexenedione (ECHD)-type compounds (AMM-59 and -120) as unique inhibitors of the bovine heart mitochondrial ADP/ATP carrier (AAC). This study investigated the mechanism of inhibition of AAC by ECHDs using submitochondrial particles (SMPs). Proteomic analyses of ECHD-bound AAC as well as biochemical characterization using different SH reagents showed that ECHDs inhibit the function of AAC by covalently binding primarily to Cys57 and secondarily to Cys160. Interestingly, AAC remarkably aggregated in SMPs upon being incubated with high concentrations of ECHDs for a long period of time. This aggregation was observed under both oxidative and reductive conditions of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of SMP proteins, indicating that aggregation is not caused by intermolecular S-S linkages. ECHDs are the first chemicals, to the best of our knowledge, to induce prominent structural alteration in AAC without forming intermolecular S-S linkages. When all solvent-accessible cysteines (Cys57, Cys160, and Cys257) were previously modified by N-ethylmaleimide, the aggregation of AAC was completely suppressed. In contrast, when Cys57 or Cys160 is selectively modified by a SH reagent, the covalent binding of ECHDs to a residual free residue of the two cysteines is sufficient to induce aggregation. The aggregation-inducing ability of another ECHD analogue (AMM-124), which has an alkyl chain that is shorter than those of AMM-59 and -120, was significantly less efficient than that of the two compounds. On the basis of these results, the mechanism underlying the aggregation of AAC induced by ECHDs is discussed.

Comparative multi-omics analysis reveals diverse latex-based defense strategies against pests among latex-producing organs of the fig tree (Ficus carica).

MAIN CONCLUSION: Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ. To determine the diversity of latex-based defense strategies in fig tree (Ficus carica) organs, we conducted comparative proteomic, transcriptomic and metabolomic analyses on latexes isolated from immature fruit, young petioles and lignified trunks of F. carica after constructing a unigene sequence library using RNA-seq data. Trypsin inhibitors were the most abundant proteins in petiole latex, while cysteine proteases ("ficins") were the most abundant in immature fruit and trunk latexes. Galloylglycerol, a possible defense-related metabolite, appeared to be highly accumulated in all three latexes. The expression levels of pathogenesis-related proteins were highest in the latex of trunk, suggesting that this latex had adapted a defensive role against microbe attacks. Although young petioles and immature fruit are both unlignified soft organs, and potential food for herbivorous insects, unigenes for the sesquiterpenoid pathway, which likely produces defense-associated volatiles, and the phenylpropanoid pathway, which produces toxic furanocoumarins, were expressed less in immature fruit latex. This difference may indicate that while petioles and fruit protect the plant from attack by herbivores, the fruit must also attract insect pollinators at younger stages and animals after ripening. We also suggest possible candidate transcription factors and signal transduction proteins that are involved in the differential expression of the unigenes.

FRI-4 carbapenemase-producing Enterobacter cloacae complex isolated in Tokyo, Japan.

Objectives: A carbapenem-resistant Enterobacter cloacae complex isolated in Tokyo, Japan, produced a carbapenemase that was detected by a Carba NP test and a modified carbapenem inactivation method, but none of the 'Big Five' carbapenemase genes was detected by PCR. This study aimed to identify the carbapenemase. Methods: Carbapenemase genes were screened by WGS. Next, we generated a recombinant plasmid in which the carbapenemase gene was inserted. We also extracted the carbapenemase gene-carrying plasmid from the E. cloacae complex. The effects of both plasmids on the antibiotic susceptibility of Escherichia coli were then tested. The carbapenemase gene-carrying plasmid in the E. cloacae complex was completely sequenced. Results: A novel carbapenemase gene, blaFRI-4, encoded an amino acid sequence that was 93.2% identical to French imipenemase (FRI-1). E. coli transformed with blaFRI-4 showed reduced carbapenem susceptibility. A complete sequence of the blaFRI-4-carrying 98 508 bp IncFII/IncR plasmid (pTMTA61661) showed that blaFRI-4 and the surrounding region (18.7 kb) were duplicated. Conclusions: The FRI-4-producing E. cloacae complex was isolated in Japan, whereas all other FRI variants have been found in Europe, suggesting that the spread of FRI carbapenemases is global.

Apoptosis-derived membrane vesicles drive the cGAS-STING pathway and enhance type I IFN production in systemic lupus erythematosus.

OBJECTIVE: Despite the importance of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) pathogenesis, the mechanisms of IFN-I production have not been fully elucidated. Recognition of nucleic acids by DNA sensors induces IFN-I and interferon-stimulated genes (ISGs), but the involvement of cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) and stimulator of interferon genes (STING) in SLE remains unclear. We studied the role of the cGAS-STING pathway in the IFN-I-producing cascade driven by SLE serum. METHODS: We collected sera from patients with SLE (n=64), patients with other autoimmune diseases (n=31) and healthy controls (n=35), and assayed them using a cell-based reporter system that enables highly sensitive detection of IFN-I and ISG-inducing activity. We used Toll-like receptor-specific reporter cells and reporter cells harbouring knockouts of cGAS, STING and IFNAR2 to evaluate signalling pathway-dependent ISG induction. RESULTS: IFN-I bioactivity and ISG-inducing activities of serum were higher in patients with SLE than in patients with other autoimmune diseases or healthy controls. ISG-inducing activity of SLE sera was significantly reduced in STING-knockout reporter cells, and STING-dependent ISG-inducing activity correlated with disease activity. Double-stranded DNA levels were elevated in SLE. Apoptosis-derived membrane vesicles (AdMVs) from SLE sera had high ISG-inducing activity, which was diminished in cGAS-knockout or STING-knockout reporter cells. CONCLUSIONS: AdMVs in SLE serum induce IFN-I production through activation of the cGAS-STING pathway. Thus, blockade of the cGAS-STING axis represents a promising therapeutic target for SLE. Moreover, our cell-based reporter system may be useful for stratifying patients with SLE with high ISG-inducing activity.

Pinpoint Chemical Modification of the Quinone-Access Channel of Mitochondrial Complex I via a Two-Step Conjugation Reaction.

We previously showed that a bulky ring-strained cycloalkyne possessing a rhodamine fluorophore directly reacts (via strain-promoted click chemistry) with the azido group incorporated (via ligand-directed tosyl chemistry) into Asp160 in the 49 kDa subunit of complex I in bovine heart submitochondrial particles [Masuya, T., et al. (2014) Biochemistry 53, 7816-7823]. This two-step conjugation may be a promising technique for specific chemical modifications of the quinone-access channel in complex I by various molecular probes, which would lead to new methodologies for studying the enzyme. However, because the reactivities of ring-strained cycloalkynes are generally high, they also react with other nucleophilic amino acids in mitochondrial proteins, resulting in significant undesired side reactions. To minimize side reactions and achieve precise pinpoint chemical modification of 49 kDa Asp160, we investigated an optimal pair of chemical tags for the two-step conjugation reaction. We found that instead of strain-promoted click chemistry, Diels-Alder cycloaddition of a pair of cyclopropene incorporated into 49 kDa Asp160 (via ligand-directed tosyl chemistry) and externally added tetrazine is more efficient for the pinpoint modification. An excess of quinone-site inhibitors did not interfere with Diels-Alder cycloaddition between the cyclopropene and tetrazine. These results along with the previous findings (cited above) strongly suggest that in contrast to the predicted quinone-access channel modeled by X-ray crystallographic and single-particle cryo-electron microscopic studies, the channel is open or undergoes large structural rearrangements to allow bulky ligands into the proximity of 49 kDa Asp160.

Definitive screening design enables optimization of LC-ESI-MS/MS parameters in proteomics.

In proteomics, more than 100,000 peptides are generated from the digestion of human cell lysates. Proteome samples have a broad dynamic range in protein abundance; therefore, it is critical to optimize various parameters of LC-ESI-MS/MS to comprehensively identify these peptides. However, there are many parameters for LC-ESI-MS/MS analysis. In this study, we applied definitive screening design to simultaneously optimize 14 parameters in the operation of monolithic capillary LC-ESI-MS/MS to increase the number of identified proteins and/or the average peak area of MS1. The simultaneous optimization enabled the determination of two-factor interactions between LC and MS. Finally, we found two parameter sets of monolithic capillary LC-ESI-MS/MS that increased the number of identified proteins by 8.1% or the average peak area of MS1 by 67%. The definitive screening design would be highly useful for high-throughput analysis of the best parameter set in LC-ESI-MS/MS systems.

Reduction of Synthetic Ubiquinone QT Catalyzed by Bovine Mitochondrial Complex I Is Decoupled from Proton Translocation.

We previously succeeded in site-specific chemical modifications of the inner part of the quinone binding pocket of bovine mitochondrial complex I through ligand-directed tosylate (LDT) chemistry using specific inhibitors as high-affinity ligands for the enzyme [Masuya, T., et al. (2014) Biochemistry 53, 2304-2317, 7816-7823]. To investigate whether a short-chain ubiquinone, in place of these specific inhibitors, serves as a ligand for LDT chemistry, we herein synthesized a LDT reagent QT possessing ubiquinone scaffold and performed LDT chemistry with bovine heart submitochondrial particles (SMP). Detailed proteomic analyses revealed that QT properly guides the tosylate group into the quinone binding pocket and transfers a terminal alkyne to nucleophilic amino acids His150 and Asp160 in the 49 kDa subunit. This result clearly indicates that QT occupies the inner part of the quinone binding pocket. Nevertheless, we noted that QT is a unique electron acceptor from complex I distinct from typical short-chain ubiquinones such as ubiquinone-1 (Q1) for several reasons; for example, QT reduction in NADH-QT oxidoreduction was almost completely insensitive to quinone-site inhibitors (such as bullatacin and piericidin A), and this reaction did not produce a membrane potential. On the basis of detailed comparisons of the electron transfer features between QT and typical short-chain quinones, we conclude that QT may accept electrons from an N2 cluster at a position different from that of typical short-chain quinones because of its unique side-chain structure; accordingly, QT reduction is unable to induce putative structural changes inside the quinone binding pocket, which are critical for driving proton translocation. Thus, QT is the first ubiquinone analogue, to the best of our knowledge, the catalytic reduction of which is decoupled from proton translocation through the membrane domain. Implications for mechanistic studies on QT are also discussed.

Specific Methylation of Asp160 (49 kDa subunit) Located inside the Quinone Binding Cavity of Bovine Mitochondrial Complex I.

Asp160 in the 49 kDa subunit of bovine mitochondrial complex I, which is located in the inner part of the quinone binding cavity, is considered to be an essential residue for energy conversion of the enzyme. To elucidate the catalytic function of this residue, we attempted to specifically methylate 49 kDa Asp160 [Asp(COO)-CH3] through a ligand-directed tosyl (LDT) chemistry technique with an acetogenin derivative (ALM) as a high-affinity ligand. We confirmed the specific methylation of 49 kDa Asp160 through liquid chromatography-tandem mass spectrometry analysis of the tryptic digests of the 49 kDa subunit. The binding affinity of a quinazoline-type inhibitor ([(125)I]AzQ) occupying the quinone binding cavity was not affected by methylation, indicating that this chemical modification does not induce significant structural changes inside the quinone binding cavity. The methylation of 49 kDa Asp160 did not lead to the complete loss of catalytic activity; the modified enzyme retained partial electron transfer and proton translocation activities. These results along with the fact that 49 kDa Asp160 elicits a very strong nucleophilicity against various LDT reagents in the local protein environment strongly suggest that this residue is free from strict interactions (such as electrostatic interaction) arising from nearby residue(s) and is functionally important but not essential for the energy conversion of complex I.

Inactivation of the antifungal and immunomodulatory properties of human cathelicidin LL-37 by aspartic proteases produced by the pathogenic yeast Candida albicans.

Constant cross talk between Candida albicans yeast cells and their human host determines the outcome of fungal colonization and, eventually, the progress of infectious disease (candidiasis). An effective weapon used by C. albicans to cope with the host defense system is the release of 10 distinct secreted aspartic proteases (SAPs). Here, we validate a hypothesis that neutrophils and epithelial cells use the antimicrobial peptide LL-37 to inactivate C. albicans at sites of candidal infection and that C. albicans uses SAPs to effectively degrade LL-37. LL-37 is cleaved into multiple products by SAP1 to -4, SAP8, and SAP9, and this proteolytic processing is correlated with the gradual decrease in the antifungal activity of LL-37. Moreover, a major intermediate of LL-37 cleavage-the LL-25 peptide-is antifungal but devoid of the immunomodulatory properties of LL-37. In contrast to LL-37, LL-25 did not affect the generation of reactive oxygen species by neutrophils upon treatment with phorbol esters. Stimulating neutrophils with LL-25 (rather than LL-37) significantly decreased calcium flux and interleukin-8 production, resulting in lower chemotactic activity of the peptide against neutrophils, which may decrease the recruitment of neutrophils to infection foci. LL-25 also lost the function of LL-37 as an inhibitor of neutrophil apoptosis, thereby reducing the life span of these defense cells. This study indicates that C. albicans can effectively use aspartic proteases to destroy the antimicrobial and immunomodulatory properties of LL-37, thus enabling the pathogen to survive and propagate.

Inactivation of human kininogen-derived antimicrobial peptides by secreted aspartic proteases produced by the pathogenic yeast Candida albicans.

Ten secreted aspartic proteases (Saps) of Candida albicans cleave numerous peptides and proteins in the host organism and deregulate its homeostasis. Human kininogens contain two internal antimicrobial peptide sequences, designated NAT26 and HKH20. In our current study, we characterized a Sap-catalyzed cleavage of kininogen-derived antimicrobial peptides that results in the loss of the anticandidal activity of these peptides. The NAT26 peptide was effectively inactivated by all Saps, except Sap10, whereas HKH20 was completely degraded only by Sap9. Proteolytic deactivation of the antifungal potential of human kininogens can help the pathogens to modulate or evade the innate immunity of the host.