A TrmBL2-like transcription factor mediates the growth phase-dependent expression of halolysin SptA in a concentration-dependent manner in Natrinema gari J7-2.

To cope with a high-salinity environment, haloarchaea generally employ the twin-arginine translocation (Tat) pathway to transport secretory proteins across the cytoplasm membrane in a folded state, including Tat-dependent extracellular subtilases (halolysins) capable of autocatalytic activation. Some halolysins, such as SptA of Natrinema gari J7-2, are produced at late-log phase to prevent premature enzyme activation and proteolytic damage of cellular proteins in haloarchaea; however, the regulation mechanism for growth phase-dependent expression of halolysins remains largely unknown. In this study, a DNA-protein pull-down assay was performed to identify the proteins binding to the 5'-flanking sequence of sptA encoding halolysin SptA in strain J7-2, revealing a TrmBL2-like transcription factor (NgTrmBL2). The ΔtrmBL2 mutant of strain J7-2 showed a sharp decrease in the production of SptA, suggesting that NgTrmBL2 positively regulates sptA expression. The purified recombinant NgTrmBL2 mainly existed as a dimer although monomeric and higher-order oligomeric forms were detected by native-PAGE analysis. The results of electrophoretic mobility shift assays (EMSAs) showed that NgTrmBL2 binds to the 5'-flanking sequence of sptA in a non-specific and concentration-dependent manner and exhibits an increased DNA-binding affinity with the increase in KCl concentration. Moreover, we found that a distal cis-regulatory element embedded in the neighboring upstream gene negatively regulates trmBL2 expression and thus participates in the growth phase-dependent biosynthesis of halolysin SptA. IMPORTANCE: Extracellular proteases play important roles in nutrient metabolism, processing of functional proteins, and antagonism of haloarchaea, but no transcription factor involved in regulating the expression of haloaechaeal extracellular protease has been reported yet. Here we report that a TrmBL2-like transcription factor (NgTrmBL2) mediates the growth phase-dependent expression of an extracellular protease, halolysin SptA, of haloarchaeon Natrinema gari J7-2. In contrast to its hyperthermophilic archaeal homologs, which are generally considered to be global transcription repressors, NgTrmBL2 functions as a positive regulator for sptA expression. This study provides new clues about the transcriptional regulation mechanism of extracellular protease in haloarchaea and the functional diversity of archaeal TrmBL2.

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.

Repositioning of Montelukast to inhibit proliferation of mutated KRAS pancreatic cancer through a novel mechanism that interfere the binding between KRAS and GTP/GDP.

Pancreatic cancer is one of the most lethal cancer types with 5-year survival rate of ∼10.8%. Various KRAS mutations exist in ∼85% pancreatic cancer cell lines. Mutated KRAS is a major cause that leads cancer cell proliferation. Chemotherapy is still the major treatment for pancreatic cancer. Alternatively, repositioning old drug to inhibit mutated KRAS may be a cost-effective way for pancreatic cancer treatment. In this study, we choose mutated KRAS (G12D) as a target. Based on mutated KRAS GTP binding domain (hydrolyze GTP to GDP), we perform virtual screening on FDA-approved drugs. Montelukast shows strong binding affinity to mutated KRAS as well as interfering both GTP and GDP binding to mutated KRAS. Furthermore, Montelukast shows very strong anti-proliferation effect on mutated KRAS pancreatic cancer cells both in vitro and in vivo. Our results support repositioning of Montelukast as single agent for pancreatic cancer treatment.

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.

Repositioning Lomitapide to block ZDHHC5-dependant palmitoylation on SSTR5 leads to anti-proliferation effect in preclinical pancreatic cancer models.

Palmitoylation of proteins plays important roles in various physiological processes, such as cell proliferation, inflammation, cell differentiation etc. However, inhibition of protein palmitoylation has led to few new drugs to date. ZDHHC5 serves as a key enzyme to catalyze palmitoylation on SSTR5 (a proven anti-proliferation receptor in pancreatic cells). Herein, we compare single-cell transcriptome data between pancreatic cancer tissues and normal pancreas tissues and identify that ZDHHC5 is a potential target to inhibit proliferation of pancreatic cancer cells. In addition, we report the repositioning of an orphan drug (Lomitapide) as an inhibitor of ZDHHC5, and we speculate that this inhibitor may be able to block palmitylation on SSTR5. Pharmacological blockade of ZDHHC5 with Lomitapide results in attenuated cancer cell growth and proliferation which collectively contributes to antitumor responses in vitro and in vivo. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of ZDHHC5 in pancreatic cancer, representing a new class of palmitoylation targeted therapy and laying a framework for paradigm-shifting therapies targeting cancer cell palmitoylation.

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.

Big data and artificial intelligence discover novel drugs targeting proteins without 3D structure and overcome the undruggable targets.

The discovery of targeted drugs heavily relies on three-dimensional (3D) structures of target proteins. When the 3D structure of a protein target is unknown, it is very difficult to design its corresponding targeted drugs. Although the 3D structures of some proteins (the so-called undruggable targets) are known, their targeted drugs are still absent. As increasing crystal/cryogenic electron microscopy structures are deposited in Protein Data Bank, it is much more possible to discover the targeted drugs. Moreover, it is also highly probable to turn previous undruggable targets into druggable ones when we identify their hidden allosteric sites. In this review, we focus on the currently available advanced methods for the discovery of novel compounds targeting proteins without 3D structure and how to turn undruggable targets into druggable ones.

Artificial intelligence and big data facilitated targeted drug discovery.

Different kinds of biological databases publicly available nowadays provide us a goldmine of multidiscipline big data. The Cancer Genome Atlas is a cancer database including detailed information of many patients with cancer. DrugBank is a database including detailed information of approved, investigational and withdrawn drugs, as well as other nutraceutical and metabolite structures. PubChem is a chemical compound database including all commercially available compounds as well as other synthesisable compounds. Protein Data Bank is a crystal structure database including X-ray, cryo-EM and nuclear magnetic resonance protein three-dimensional structures as well as their ligands. On the other hand, artificial intelligence (AI) is playing an important role in the drug discovery progress. The integration of such big data and AI is making a great difference in the discovery of novel targeted drug. In this review, we focus on the currently available advanced methods for the discovery of highly effective lead compounds with great absorption, distribution, metabolism, excretion and toxicity properties.

The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant.

Environmental abiotic stresses are limiting factors for less tolerant organisms, including soil plants. Abiotic stress tolerance-associated genes from prokaryotic organisms are supposed to have a bright prospect for transgenic application. The drought-adapted cyanobacterium Nostoc flagelliforme is arising as a valuable prokaryotic biotic resource for gene excavation. In this study, we evaluated the salt-tolerant function and application potential of a candidate gene drnf1 from N. flagelliforme, which contains a P-loop NTPase (nucleoside-triphosphatase) domain, through heterologous expression in two model organisms Synechocystis sp. PCC 6803 and Arabidopsis thaliana. It was found that DRNF1 could confer significant salt tolerance in both transgenic organisms. In salt-stressed transgenic Synechocystis, DRNF1 could enhance the respiration rate; slow-down the accumulation of exopolysaccharides; up-regulate the expression of salt tolerance-related genes at a higher level, such as those related to glucosylglycerol synthesis, Na⁺/H⁺ antiport, and sugar metabolism; and maintain a better K⁺/Na⁺ homeostasis, as compared to the wild-type strain. These results imply that DRNF1 could facilitate salt tolerance by affecting the respiration metabolism and indirectly regulating the expression of important salt-tolerant genes. Arabidopsis was employed to evaluate the salt tolerance-conferring potential of DRNF1 in plants. The results show that it could enhance the seed germination and shoot growth of transgenic plants under saline conditions. In general, a novel prokaryotic salt-tolerant gene from N. flagelliforme was identified and characterized in this study, enriching the candidate gene pool for genetic engineering in plants.

No enhancing effect of vitamin A administration on iron absorption or body total iron content in preschool children from Chengdu, China.

To explore the effect of vitamin A supplements on iron metabolic homeostasis for preschoolers. This was a randomized, placebo-controlled and blinded intervention trial with 3- to 6-y old preschoolers. A total of 445 subjects were randomly divided into four groups: a vitamin A supplementation group (group 1, a single oral dose of vitamin A as retinol 200,000 IU), an iron supplement group (group 2, daily oral supplement with the elemental iron 1-2 mg/kg/d for 5 d a week, lasting for 6 mo) a combined vitamin A and iron (group 3) and administration of no vitamin A or iron as a placebo-control (group 4). A total of 387 (95, 98, 90 and 104 from groups 1, 2, 3 and 4) children completed the intervention. After intervention, serum retinol levels of children in group 1 and group 3 was markedly higher than those of children in groups 2 and 4 (p<0.05). The serum ferritin level of children in group 1 significantly decreased after intervention (p<0.05), but increased in group 2 (p<0.05). The sTfR-SF index (TFR-F) and total body iron content (BTIC) showed the same change after intervention. In group 2 and group 3, the levels of TRF-F index and BTIC had statistically increased to the same degree after intervention (p<0.05). The impact of vitamin A intervention on iron metabolic homeostasis was mainly manifested in storage and mobilization; there was no direct effect on total body iron content or iron absorption in the intestine.

[Effect of vitamin A supplements on iron metabolic homeostasis for preschoolers].

OBJECTIVE: To explore the effect of vitamin A (VA) combined iron supplements on iron metabolic homeostasis for preschoolers. METHODS: About 445 preschoolers with aged 3-6 years old from eight kindergartens in Pixian county, Chengdu were recruited into this trial from March to September, 2011. All subjects met the inclusion criteria were randomly divided into four groups using random number table: sole VA supplementation group (VA group, a single oral dose of VA at about 200 000 units), sole iron supplementation group (FE group, daily oral supplementation with the element iron 1-2 mg·kg(-1)·d(-1) for five days a week, lasting for 6 months), the combined supplementation of VA and iron group (VF group) and control group (CO group, no VA and iron supplementation). The concentration of serum VA, serum ferritin (SF), serum transferring receptor (sTfR), C-reactive protein (CRP) and hemoglobin (Hb) were measured from 3 ml vein blood. The sTfR-SF index (TFR-F index) and total body iron content (TBIC) before and after intervention were calculated. The differences of these indexes between groups before and after intervention were analyzed. RESULTS: The level of sTfR before intervention in VA group ((1.78 ± 0.17) mg/L) was significantly higher than that of after intervention ((1.18 ± 0.11)mg/L) (t = 28.88, P < 0.01). The levels of TFR-F index and TBIC in FE and VF groups before intervention ( (1.59 ± 0.37), (1.63 ± 0.40) and (9.04 ± 2.71), (9.26 ± 2.33) mg/kg, respectively) were all lower than those of after intervention (TFR-F index:(1.84 ± 0.51), (1.87 ± 0.45) and TBIC:(12.42 ± 3.49), (13.01 ± 2.98)mg/kg) (t values were 3.93, 3.78, 7.57 and 9.41, respectively, all P values were <0.01). The incidence of iron deficiency in VA, FE, and VF groups before intervention were 26% (25/95), 31% (30/98) and 31% (28/90) and were 41% (39/95), 10% (10/98) and 18% (16/90) for after intervention, respectively. The difference of this index in VA, FE and VF groups were significant (χ(2) values were 4.59, 12.50 and 4.31, respectively, all the P values were <0.05). CONCLUSION: Combined VA and iron was as effective as VA alone or iron alone in decreasing the iron deficiency, the impact of VA intervention on iron metabolic homeostasis was mainly manifested in iron usage and mobilization, but showed no effect on total body iron content.

Quality evaluation of the edible blue-green alga Nostoc flagelliforme using a chlorophyll fluorescence parameter and several biochemical markers.

Nostoc flagelliforme is an edible blue-green alga with herbal and dietary values. Due to the diminishing supply of natural N. flagelliforme and the large investment on the development of its cultivation technology, it is anticipated that artificially cultured N. flagelliforme will soon sustain the market supply. Once this change occurs, the storage-associated quality problem will become the focus of attention for future trade. In this paper, we used a chlorophyll fluorescence parameter, maximum quantum efficiency of Photosystem II (Fv/Fm), and several biomarkers to evaluate the quality of several N. flagelliforme samples. It was found that longer storage times resulted in darker coloured solutions (released pigments) and decreased amounts of chlorophyll a (Chl a) and water-soluble sugars (WSS). Additionally, a higher Fv/Fm value suggests better physiological recovery and quality. In actual application, determination of Fv/Fm would be the first step for evaluating the quality of N. flagelliforme, and the biochemical indexes would serve as good secondary markers.

Effect of simultaneous supplementation of vitamin A and iron on diarrheal and respiratory tract infection in preschool children in Chengdu City, China.

OBJECTIVE: The goal of this study was to investigate whether vitamin A combined with iron supplementation for preschool children resulted in improved changes in children's infectious morbidity. METHOD: In this randomized placebo-controlled and blinded field intervention trial, totally 445 preschoolers, ages 3 to 6 y old, were randomly selected. All children were randomly divided into four groups: vitamin A supplement-only group (group I), iron supplement-only group (group II), vitamin A and iron supplement group (group III), and no vitamin A and ferrous sulfate as placebo-control (group IV) for 6 mo. The morbidity of diarrhea and respiratory infections, were collected during supplementation. RESULTS: There was evidence of the lowest incidence rate of respiratory-related illnesses and fewest symptoms of runny nose, cough, and fever for children in group III compared with children in groups I, II and IV (P < 0.05). Moreover, despite the undistinguished incidence rate of vomiting, nausea, and stomach pain, the rate of diarrhea-related illness was significantly lower for children in group III than for those in the other three groups. CONCLUSION: The beneficial affects on infectious morbidity over 6 mo, highlight the potential of vitamin A plus an iron supplement for preschool-aged children.