Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Effects of light and water availability on the growth of Aglaia duperreana seedlings.

Aglaia duperreana, a species with a long cultivation history, is of high ornamental value. To understand the growth and photosynthetic changes of A. duperreana seedlings under variable environmental conditions, we conducted an experiment with light intensities adjusted at 70%, 50% and 30%, crossed with three moisture treatments at 70%, 50% and 30% of field capacity, and a control group which maintained 90% light intensity and 90% field capacity. The results showed that both drought stress and shading propensity significantly inhibited the growth of A. duperreana seedlings, with stronger impacts from drought stress. The increments in stem height and ground diameter, net photosynthetic rate, transpiration rate, stomatal conductance, and chlorophyll content were decreased with the maximum declines by 71.4%, 81.2%, 93.2%, 71.5%, 70.6% and 30.4%, respectively. Under severe drought stress (30% of field capacity), partial shading (50% of translucency) appeared to lessen the detrimental effects of drought. The combination of 70% translucency and 70% field capacity was optimal, resulting in higher increments in stem height, leaf area, net photosynthetic rate, transpiration rate, and stomatal conductance. The maximum fluorescence, variable fluorescence, PSⅡ potential activity, and PSⅡ maximum light energy conversion efficiency increased and then decreased with decreasing moisture. These findings suggested that A. duperreana could adapt to drought and shading stress by modulating growth, enhancing chlorophyll content, and adjusting photosynthetic system. Maintaining translucency at 70% and field moisture capacity at 70% could promote photosynthesis, with positive consequences on growth of A. duperreana.

Engineered Saccharomyces cerevisiae as a Biosynthetic Platform of Nucleotide Sugars.

Glycosylation of biomolecules can greatly alter their physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Glycosylation reactions rely on the stepwise addition of sugars using nucleotide diphosphate (NDP)-sugars. Making these substrates readily available will greatly accelerate the characterization of new glycosylation reactions, elucidation of their underlying regulation mechanisms, and production of glycosylated molecules. In this work, we engineered Saccharomyces cerevisiae to heterologously express nucleotide sugar synthases to access a wide variety of uridine diphosphate (UDP)-sugars from simple starting materials (i.e., glucose and galactose). Specifically, activated glucose, uridine diphosphate d-glucose (UDP-d-Glc), can be converted to UDP-d-glucuronic acid (UDP-d-GlcA), UDP-d-xylose (UDP-d-Xyl), UDP-d-apiose (UDP-d-Api), UDP-d-fucose (UDP-d-Fuc), UDP-l-rhamnose (UDP-l-Rha), UDP-l-arabinopyranose (UDP-l-Arap), and UDP-l-arabinofuranose (UDP-l-Araf) using the corresponding nucleotide sugar synthases of plant and microbial origins. We also expressed genes encoding the salvage pathway to directly activate free sugars to achieve the biosynthesis of UDP-l-Arap and UDP-l-Araf. We observed strong inhibition of UDP-d-Glc 6-dehydrogenase (UGD) by the downstream product UDP-d-Xyl, which we circumvented using an induction system (Tet-On) to delay the production of UDP-d-Xyl to maintain the upstream UDP-sugar pool. Finally, we performed a time-course study using strains containing the biosynthetic pathways to produce five non-native UDP-sugars to elucidate their time-dependent interconversion and the role of UDP-d-Xyl in regulating UDP-sugar metabolism. These engineered yeast strains are a robust platform to (i) functionally characterize sugar synthases in vivo, (ii) biosynthesize a diverse selection of UDP-sugars, (iii) examine the regulation of intracellular UDP-sugar interconversions, and (iv) produce glycosylated secondary metabolites and proteins.

HtrAs are essential for the survival of the haloarchaeon Natrinema gari J7-2 in response to heat, high salinity, and toxic substances.

Bacterial and eukaryotic HtrAs can act as an extracytoplasmic protein quality control (PQC) system to help cells survive in stress conditions, but the functions of archaeal HtrAs remain unknown. Particularly, haloarchaea route most secretory proteins to the Tat pathway, enabling them to fold properly in well-controlled cytoplasm with cytosolic PQC systems before secretion. It is unclear whether HtrAs are required for haloarchaeal survival and stress response. The haloarchaeon Natrinema gari J7-2 encodes three Tat signal peptide-bearing HtrAs (NgHtrA, NgHtrB, and NgHtrC), and the signal peptides of NgHtrA and NgHtrC contain a lipobox. Here, the in vitro analysis reveals that the three HtrAs show different profiles of temperature-, salinity-, and metal ion-dependent proteolytic activities and could exhibit chaperone-like activities to prevent the aggregation of reduced lysozyme when their proteolytic activities are inhibited at low temperatures or the active site is disrupted. The gene deletion and complementation assays reveal that NgHtrA and NgHtrC are essential for the survival of strain J7-2 at elevated temperature and/or high salinity and contribute to the resistance of this haloarchaeon to zinc and inhibitory substances generated from tryptone. Mutational analysis shows that the lipobox mediates membrane anchoring of NgHtrA or NgHtrC, and both the membrane-anchored and free extracellular forms of the two enzymes are involved in the stress resistance of strain J7-2, depending on the stress conditions. Deletion of the gene encoding NgHtrB in strain J7-2 causes no obvious growth defect, but NgHtrB can functionally substitute for NgHtrA or NgHtrC under some conditions.IMPORTANCEHtrA-mediated protein quality control plays an important role in the removal of aberrant proteins in the extracytoplasmic space of living cells, and the action mechanisms of HtrAs have been extensively studied in bacteria and eukaryotes; however, information about the function of archaeal HtrAs is scarce. Our results demonstrate that three HtrAs of the haloarchaeon Natrinema gari J7-2 possess both proteolytic and chaperone-like activities, confirming that the bifunctional nature of HtrAs is conserved across all three domains of life. Moreover, we found that NgHtrA and NgHtrC are essential for the survival of strain J7-2 under stress conditions, while NgHtrB can serve as a substitute for the other two HtrAs under certain circumstances. This study provides the first biochemical and genetic evidence of the importance of HtrAs for the survival of haloarchaea in response to stresses.

Correction: Polycyclic polyprenylated acylphloroglucinols from Garcinia xanthochymus fruits exhibit antitumor effects through inhibition of the STAT3 signaling pathway.

Correction for 'Polycyclic polyprenylated acylphloroglucinols from Garcinia xanthochymus fruits exhibit antitumor effects through inhibition of the STAT3 signaling pathway' by Jing Xu et al., Food Funct., 2020, 11, 10568-10579, https://doi.org/10.1039/D0FO02535F.

Dissecting the Arginine and Lysine Biosynthetic Pathways and Their Relationship in Haloarchaeon Natrinema gari J7-2 via Endogenous CRISPR-Cas System-Based Genome Editing.

The evolutionary relationship between arginine and lysine biosynthetic pathways has been well established in bacteria and hyperthermophilic archaea but remains largely unknown in haloarchaea. Here, the endogenous CRISPR-Cas system was harnessed to edit arginine and lysine biosynthesis-related genes in the haloarchaeon Natrinema gari J7-2. The ΔargW, ΔargX, ΔargB, and ΔargD mutant strains display an arginine auxotrophic phenotype, while the ΔdapB mutant shows a lysine auxotrophic phenotype, suggesting that strain J7-2 utilizes the ArgW-mediated pathway and the diaminopimelate (DAP) pathway to synthesize arginine and lysine, respectively. Unlike the ArgD in Escherichia coli acting as a bifunctional aminotransferase in both the arginine biosynthesis pathway and the DAP pathway, the ArgD in strain J7-2 participates only in arginine biosynthesis. Meanwhile, in strain J7-2, the function of argB cannot be compensated for by its evolutionary counterpart ask in the DAP pathway. Moreover, strain J7-2 cannot utilize α-aminoadipate (AAA) to synthesize lysine via the ArgW-mediated pathway, in contrast to hyperthermophilic archaea that employ a bifunctional LysW-mediated pathway to synthesize arginine (or ornithine) and lysine from glutamate and AAA, respectively. Additionally, the replacement of a 5-amino-acid signature motif responsible for substrate specificity of strain J7-2 ArgX with that of its hyperthermophilic archaeal homologs cannot endow the ΔdapB mutant with the ability to biosynthesize lysine from AAA. The in vitro analysis shows that strain J7-2 ArgX acts on glutamate rather than AAA. These results suggest that the arginine and lysine biosynthetic pathways of strain J7-2 are highly specialized during evolution. IMPORTANCE Due to their roles in amino acid metabolism and close evolutionary relationship, arginine and lysine biosynthetic pathways represent interesting models for probing functional specialization of metabolic routes. The current knowledge with respect to arginine and lysine biosynthesis is limited for haloarchaea compared to that for bacteria and hyperthermophilic archaea. Our results demonstrate that the haloarchaeon Natrinema gari J7-2 employs the ArgW-mediated pathway and the DAP pathway for arginine and lysine biosynthesis, respectively, and the two pathways are functionally independent of each other; meanwhile, ArgX is a key determinant of substrate specificity of the ArgW-mediated pathway in strain J7-2. This study provides new clues about haloarchaeal amino acid metabolism and confirms the convenience and efficiency of endogenous CRISPR-Cas system-based genome editing in haloarchaea.

Engineered geranyl diphosphate methyltransferase produces 2-methyl-dimethylallyl diphosphate as a noncanonical C6 unit for terpenoid biosynthesis.

Terpenoids constitute the largest class of natural products with complex structures, essential functions, and versatile applications. Creation of new building blocks beyond the conventional five-carbon (C5) units, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate, expands significantly the chemical space of terpenoids. Structure-guided engineering of an S-adenosylmethionine-dependent geranyl diphosphate (GPP) C2-methyltransferase from Streptomyces coelicolor yielded variants converting DMAPP to a new C6 unit, 2-methyl-DMAPP. Mutation of the Gly residue at the position 202 resulted in a smaller substrate-binding pocket to fit DMAPP instead of its native substrate GPP. Replacement of Phe residue at the position 222 with a Tyr residue contributed to DMAPP binding via hydrogen bond. Furthermore, using Escherichia coli as the chassis, we demonstrated that 2-methyl-DMAPP was accepted as a start unit to generate noncanonical trans- and cis-prenyl diphosphates (C5n+1) and terpenoids. This work provides insights into substrate recognition of prenyl diphosphate methyltransferases, and strategies to diversify terpenoids by expanding the building block portfolio.

Auto- and Hetero-Catalytic Processing of the N-Terminal Propeptide Promotes the C-Terminal Fibronectin Type III Domain-Mediated Dimerization of a Thermostable Vpr-like Protease.

Microbial Vpr-like proteases are extracellular multidomain subtilases with diverse functions and can form oligomers, but their maturation and oligomerization mechanisms remain to be elucidated. Here, we report a novel Vpr-like protease (BTV) from thermophilic bacterium Brevibacillus sp. WF146. The BTV precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain with an inserted protease-associated (PA) domain, two tandem fibronectin type III domains (Fn1 and Fn2), and a C-terminal propeptide. The BTV proform (pro-BTV) could be autoprocessed into the mature form (mBTV) via two intermediates lacking the N- or C-terminal propeptide, respectively, and the C-terminal propeptide delays the autocatalytic maturation of the enzyme. By comparison, pro-BTV is more efficiently processed into mBTV by protease TSS from strain WF146. Purified mBTV is a Ca2+-dependent thermostable protease, showing optimal activity at 60°C and retaining more than 60% of activity after incubation at 60°C for 8 h. The PA domain is important for enzyme stability and contributes to the substrate specificity of BTV by restricting the access of protein substrates to the active site. The proform and mature form of BTV exist as a monomer and a homodimer, respectively, and the dimerization is mediated by the Fn1 and Fn2 domains. The N-terminal propeptide of BTV not only acts as intramolecular chaperone and enzymatic inhibitor but also inhibits the homodimerization of the enzyme. The removal of the N-terminal propeptide leads to a structural adjustment of the enzyme and thus promotes enzyme dimerization. IMPORTANCE Vpr-like proteases are widely distributed in bacteria and fungi and are involved in processing lantibiotics, degrading collagen, keratin, and fibrin, and pathogenesis of microbes. The dissection of the roles of individual domains in enzyme maturation and oligomerization is crucial for understanding the action mechanisms of these multidomain proteases. Our results demonstrate that hetero-catalytic maturation of the extracellular Vpr-like protease BTV of Brevibacillus sp. WF146 is more efficient than autocatalytic maturation of the enzyme. Moreover, we found that the C-terminal tandem fibronectin type III domains rather than the PA domain mediate the dimerization of mature BTV, while the N-terminal propeptide inhibits the dimerization of the BTV proform. This study provides new insight into the activation and oligomerization mechanisms of Vpr-like proteases.

Effect of chronic ankle instability on lower extremity kinematics, dynamic postural stability, and muscle activity during unilateral jump-landing tasks: A systematic review and meta-analysis.

OBJECTIVE: To determine if individuals with chronic ankle instability (CAI) demonstrate altered landing kinematics, muscle activity, and impaired dynamic postural stability during a unilateral jump-landing task. METHODS: 21 studies were included from PubMed, MEDLINE, Embase and CINAHL searched on September 26, 2021. Mean differences in joint angles and muscle activity between CAI and controls were analysed as continuous variables and pooled using a random-effects model to obtain standardised mean differences and 95% confidence intervals. Dynamic postural stability measured using time to stabilisation (TTS) was assessed qualitatively. RESULTS: We found greater plantarflexion (pooled SMD = 0.33, 95%CI [0.02,0.65]), reduced knee flexion (pooled SMD = -0.67, 95%CI [-0.97, -0.37]), and reduced hip flexion (pooled SMD = -0.52, 95%CI [-0.96, -0.07]) in CAI after landing. Regarding muscle activity, we observed reduced peroneus longus muscle activation (pooled SMD = -0.77, 95% CI [-1.17, -0.36]) in CAI prior to landing. CONCLUSION: Our study provides preliminary evidence of altered landing kinematics in the sagittal plane and reduced peroneus muscle activity in CAI during a dynamic jump-landing task. These results may have clinical implications in the development of more effective and targeted rehabilitation programmes for patients with CAI.

Sec-Dependent Secretion of Subtilase SptE in Haloarchaea Facilitates Its Proper Folding and Heterocatalytic Processing by Halolysin SptA Extracellularly.

In response to high-salt conditions, haloarchaea export most secretory proteins through the Tat pathway in folded states; however, it is unclear why some haloarchaeal proteins are still routed to the Sec pathway. SptE is an extracellular subtilase of Natrinema sp. strain J7-2. Here, we found that SptE precursor comprises a Sec signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE) containing seven domains (C1 to C7). SptE is produced extracellularly as a mature form (M180) in strain J7-2 and a proform (ΔS) in the ΔsptA mutant strain, indicating that halolysin SptA mediates the conversion of the secreted proform into M180. The proper folding of ΔS is more efficient in the presence of NaCl than KCl. ΔS requires SptA for cleavage of the N-terminal propeptide and C-terminal C6 and C7 domains to generate M180, accompanied by the appearance of autoprocessing product M120 lacking C5. At lower salinities or elevated temperatures, M180 and M120 could be autoprocessed into M90, which comprises the catalytic and C1 domains and has a higher activity than M180. When produced in Haloferax volcanii, SptE could be secreted as a properly folded proform, but its variant (TSptE) with a Tat signal peptide does not fold properly and suffers from severe proteolysis extracellularly; meanwhile, TSptE is more inclined to aggregate intracellularly than SptE. Systematic domain deletion analysis reveals that the long CTE is an important determinant for secretion of SptE via the Sec rather than Tat pathway to prevent enzyme aggregation before secretion. IMPORTANCE While Tat-dependent haloarchaeal subtilases (halolysins) have been extensively studied, the information about Sec-dependent subtilases of haloarchaea is limited. Our results demonstrate that proper maturation of Sec-dependent subtilase SptE of Natrinema sp. strain J7-2 depends on the action of halolysin SptA from the same strain, yielding multiple hetero- and autocatalytic mature forms. Moreover, we found that the different extra- and intracellular salt types (NaCl versus KCl) of haloarchaea and the long CTE are extrinsic and intrinsic factors crucial for routing SptE to the Sec rather than Tat pathway. This study provides new clues about the secretion and adaptation mechanisms of Sec substrates in haloarchaea.

Discovery of Novel Polycyclic Polyprenylated Acylphloroglucinols from the Fruits of Garcinia xanthochymus as Antitumor Agents by Suppressing the STAT3 Signaling.

Pharmacologic studies have revealed that polycyclic polyprenylated acylphloroglucinols (PPAPs) collectively exhibit a broad range of biological activities, including antineoplastic potential. Here, six new PPAPs, named garcixanthochymones F-K (3, 5, 7, 8, 11, and 15), together with nine known analogues were isolated from the fruits of Garcinia xanthochymus. Their structures were elucidated based on the spectroscopic data, including UV, HRESIMS, and NMR, and quantum chemical calculations. All the isolated PPAPs were tested for anti-proliferative activity against four human tumor cell lines, including SGC7901, A549, HepG2, and MCF-7. Most of the PPAPs possessed high anti-proliferative activity with IC50 values in the range of 0.89 to 36.98 µM, and significant apoptosis was observed in MCF-7 cells exposed to compounds 2 and 5. Besides, docking results showed that compounds 2 and 5 could strongly combine with the Src homology 2 (SH2) domain of STAT3 via hydrogen bond and hydrophobic interaction, which is one of the key oncogenes and crucial therapeutic targets. Furthermore, compounds 2 and 5 efficiently downregulated the expression of p-STAT3Tyr705 and pivotal effector proteins involved in oncogenic signaling pathways of MCF-7 cells.

Genome-wide identification of cis DNA methylation quantitative trait loci in three Southeast Asian Populations.

DNA methylation (DNAm) is an epigenetic modification that acts to regulate gene transcription, is essential for cellular processes and plays an important role in complex traits and disease. Variation in DNAm levels is influenced by both genetic and environmental factors. Several studies have examined the extent to which common genetic variation influences DNAm (i.e. mQTLs), however, an improved understanding of mQTLs across diverse human populations is needed to increase their utility in integrative genomic studies in order to further our understanding of complex trait and disease biology. Here, we systematically examine cis-mQTLs in three Southeast Asian populations in the Singapore Integrative Omics (iOmics) Study, comprised of Chinese (n = 93), Indians (n = 83) and Malays (n = 78). A total of 24 851 cis-mQTL probes were associated with at least one SNP in meta- and ethnicity-specific analyses at a stringent significance level. These cis-mQTL probes show significant differences in local SNP heritability between the ethnicities, enrichment in functionally relevant regions using data from the Roadmap Epigenomics Mapping Consortium and are associated with nearby genes and complex traits due to pleiotropy. Importantly, DNAm prediction performance and the replication of cis-mQTLs both within iOmics and between two independent mQTL studies in European and Bangladeshi individuals is best when the genetic distance between the ethnicities is small, with differences in cis-mQTLs likely due to differences in allele frequency and linkage disequilibrium. This study highlights the importance of, and opportunities from, extending investigation of the genetic control of DNAm to Southeast Asian populations.

Polycyclic polyprenylated acylphloroglucinols from Garcinia xanthochymus fruits exhibit antitumor effects through inhibition of the STAT3 signaling pathway.

The fruits of Garcinia xanthochymus can be eaten raw or processed into jams, preserves and vinegar. They provide not only vitamin and protein nutrients, but also pharmacologically active compounds, among which polycyclic polyprenylated acylphloroglucinols (PPAPs) are a major class. According to the literature, PPAPs exhibited good anti-cancer effects. This study investigated the antitumor effects and the underlying mechanism of S1 (the regioisomeric mixture of xanthochymol and guttiferone E) and S2 (the regioisomeric mixture of isoxanthochymol and cycloxanthochymol) isolated from the fruits of G. xanthochymus. In an H22 allograft mouse model, S1 and S2 could suppress the liver tumor growth and phosphorylation of STAT3. Computational modeling showed that S1 and S2 could form hydrogen bonds with the SH2 domain of STAT3. In HepG2 and MCF-7 cell lines, S1 and S2 downregulated the expression of p-STAT3Tyr705. Moreover, S1 and S2 inhibited the phosphorylation of JAK2 and Src, which are the upstream kinases of STAT3, and the expression of various STAT3-regulated genes, including anti-apoptotic (Bcl-XL, Mcl-1 and survivin), proliferative (cyclin D1) and angiogenic (VEGF) genes. As a result, S1 and S2 arrested the cell cycle and induced cell apoptosis, which were proved by the activation of cleaved caspase-3 and caspase-8. These results demonstrated that S1 and S2 from G. xanthochymus exhibited antitumor effects through the inactivation of STAT3, and could be promising candidates for cancer treatment.

Investigation of Indigoidine Synthetase Reveals a Conserved Active-Site Base Residue of Nonribosomal Peptide Synthetase Oxidases.

Nonribosomal peptide synthetase (NRPS) oxidase (Ox) domains oxidize protein-bound intermediates to install crucial structural motifs in bioactive natural products. The mechanism of this domain remains elusive. Here, by studying indigoidine synthetase, a single-module NRPS involved in the biosynthesis of indigoidine and several other bacterial secondary metabolites, we demonstrate that its Ox domain utilizes an active-site base residue, tyrosine 665, to deprotonate a protein-bound l-glutaminyl residue. We further validate the generality of this active-site residue among NRPS Ox domains. These findings not only resolve the biosynthetic pathway mediated by indigoidine synthetase but enable mechanistic insight into NRPS Ox domains.

Corrigendum: RfpA, RfpB, and RfpC are the Master Control Elements of Far-Red Light Photoacclimation (FaRLiP).

[This corrects the article DOI: 10.3389/fmicb.2015.01303.].

Adamantyl and homoadamantyl derivatives from Garcinia multiflora fruits.

Nine undescribed caged polycyclic polyprenylated acylphloroglucinols (PPAPs), including adamantane type PPAPs (1-2), and homoadamantane type PPAPs (3-9), were isolated from the fruits of Garcinia multiflora, along with three known analogues. A new epimeric pair of isohypersampsonone B (5) and epi-isohypersampsonone B (6), featuring an unusual hexahydrofuro[2,3-b]furan-diepoxy ring system fused in a homoadamantane skeleton, was not separated due to the rapid equilibration between the two isomeric forms. All new caged PPAPs (1-9), sharing a common isogeranyl group, were determined on the basis of comprehensive NMR and MS spectroscopic data. Their cytotoxicity against three human tumor cell lines (SGC-7901, HepG2, HCT-116) and the nitric oxide production inhibitory activity of lipopolysaccharides-stimulated RAW 264.7 cells were tested. Compounds 8 and 12 displayed mild cytotoxicity against three human cancer cell lines with IC50 values of 10-20 µM. Furthermore, compounds 8 and 12 also exhibited NO production inhibitory effect with an IC50 value of 18.24 and 12.50 µM respectively.

Activities of recombinant human bleomycin hydrolase on bleomycins and engineered analogues revealing new opportunities to overcome bleomycin-induced pulmonary toxicity.

The bleomycins (BLMs) are widely used in combination therapies for the treatment of various cancers. Dose-dependent and cumulative pulmonary toxicity is the major cause of BLM-associated morbidity, limiting the broad uses of BLMs as anticancer drugs. The organ specificity of BLM-induced toxicity has been correlated with the expression of the hBLMH gene, encoding the human bleomycin hydrolase (hBLMH), which is poorly expressed in the lung. hBLMH hydrolyzes BLMs into the biologically inactive deamido BLMs, thereby protecting organs from BLM-induced toxicity. Here we report (i) expression of hBLMH and production and isolation of recombinant human bleomycin hydrolase (rhBLMH) from E. coli, (ii) structural characterization of deamido BLM A2 and B2 isolated from rhBLMH-catalyzed hydrolysis of BLM A2 and B2, and (iii) kinetic characterization of the rhBLMH-catalyzed hydrolysis of BLM A2 and B2, in comparison with five BLM analogues. rhBLMH from E. coli catalyzes rapid and efficient hydrolysis of all BLMs tested, exhibiting a superior catalytic efficiency for BLM B2. These findings reveal new opportunities to overcome BLM-induced pulmonary toxicity in chemotherapies, potentially by exploring BLM B2 as the preferred congener, engineering designer BLMs with optimized activity for rhBLMH, or co-administrating rhBLMH directly into the lung as a potential protein therapeutic.

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells.

Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in nonphotosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium Microcoleus sp. PCC 7113. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibits a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for the design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells, tissues, and whole organisms with light across the visible spectrum and into the near IR.

Novel dual-targeting anti-proliferative dihydrotriazine-chalcone derivatives display suppression of cancer cell invasion and inflammation by inhibiting the NF-κB signaling pathway.

Chalcones present in edible plants possess anti-cancer and anti-inflammatory properties, with the Michael acceptor moiety reported to be responsible for their biological activities. In this study, two novel dihydrotriazine-chalcone compounds previously identified to exert anti-proliferative effects through dual-targeting of dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR), were evaluated for their anti-invasive and anti-inflammatory abilities. At non-lethal concentrations, the compounds suppressed in vitro migration of MDA-MB-231 breast carcinoma cells, which was correlated with a dose-dependent downregulation of phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) expression and secretion. At similar concentrations, these chalcone-based compounds suppressed expression of inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharides (LPS)-stimulated murine macrophage-like RAW 264.7 cells, as well as tumor necrosis factor alpha (TNF-α) in LPS-stimulated human monocytes isolated from healthy donors. Mechanistically, inhibition of cancer cell invasion and inflammation by the compounds were mediated through suppression of the nuclear factor-kappaB (NF-κB) signaling pathway, which corroborated with the reported mechanism of action of chalcones. Their abilities to target multiple biological mediators relevant to multi-step carcinogenesis and with bioactivities stronger than those of the parent chalcone scaffold have warranted dihydrotriazine-chalcone compounds as promising candidates for use in pharmacological intervention of aggressive cancers.