l-tyrosine modulates biofilm formation of Bacillus cereus ATCC 14579.

Bacillus cereus is a food-borne pathogen capable of producing biofilms. Following analysis of biofilm formation by B. cereus ATCC 14579 transposon mutants in defined medium (DM), a deletion mutant of bc2939 (Δbc2939) was constructed that showed decreased crystal violet biofilm staining and biofilm cell counts. In addition, Δbc2939 also produced smaller colony biofilms with lower cell counts and loss of wrinkly morphology. The bc2939 gene encodes for Prephenate dehydrogenase, which converts Prephenate to 4-Hydroxy-phenylpyruvate (4-HPPA) in the l-tyrosine branch of the Shikimate pathway. While growth of the mutant and WT in DM was similar, addition of l-tyrosine was required to restore WT-like (colony) biofilm formation. Comparative proteomics showed reduced expression of Tyrosine-protein kinase/phosphatase regulators and extracellular polysaccharide cluster 1 (EPS1) proteins, aerobic electron transfer chain cytochrome aa3/d quinol oxidases, and iso-chorismate synthase involved in menaquinone synthesis in DM grown mutant biofilm cells, while multiple oxidative stress-related catalases and superoxide dismutases were upregulated. Performance in shaking cultures showed a 100-fold lower concentration of menaquinone-7 and reduction in cell counts of DM grown Δbc2939 indicating increased oxygen sensitivity. Combining all results, points to an important role of Tyrosine-modulated EPS1 production and menaquinone-dependent aerobic respiration in B. cereus ATCC 14579 (colony) biofilm formation.

Arabidopsis clathrin adaptor EPSIN1 but not MODIFIED TRANSPORT TO THE VACOULE1 contributes to effective plant immunity against pathogenic Pseudomonas bacteria.

In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses.

Extracellular Polysaccharide from Rhizopus nigricans Inhibits Hepatocellular Carcinoma via miR-494-3p/TRIM36 Axis and Cyclin E Ubiquitination.

Background and Aims: This study was designed to uncover the mechanism for extracellular polysaccharide (EPS1-1)-mediated effects on hepatocellular carcinoma (HCC) development. Methods: HCC cells were treated with EPS1-1, miR-494-3p mimic, sh-TRIM36, and pcDNA3.1-TRIM36. The levels of miR-494-3p and TRIM36 were measured in normal hepatocytes, THLE-2, and HepG2 and HuH7HCC cell lines, along with the protein expression of cyclin D/E and p21. The proliferation, cell cycle, and apoptosis of HCC cells were assayed. The interactions between miR-494-3p and TRIM36, and between TRIM36 and cyclin E were assessed. Finally, the expression and localization of TRIM36 and cyclin E were monitored, and tumor apoptosis was detected, in tumor xenograft model. Results: EPS1-1 suppressed HCC cell proliferation and cyclin D/E expression and promoted apoptosis and p21 expression. miR-494-3p was upregulated and TRIM36 was downregulated in HCC cells. Transfection with miR-494-3p mimic or sh-TRIM36 facilitated HCC cell proliferation and the expression of cyclin D/E protein but they inhibited apoptosis and p21 expression in the presence of EPS1-1. Overexpression of TRIM36 further consolidated EPS1-1-mediated inhibition of HCC proliferation, cyclin D/E, and the promotion of apoptosis and p21 expression. Those effects were reversed by miR-494-3p overexpression. TRIM36 was a target gene of miR-494-3p, and TRIM36 induced cyclin E ubiquitination. EPS1-1 suppressed cyclin E expression, promoted TRIM36 expression and tumor apoptosis, all of which were abrogated by increasing the expression of miR-494-3p in vivo. Conclusions: EPS1-1 protected against HCC by limiting its proliferation and survival through the miR-494-3p/TRIM36 axis and by inducing cyclin E ubiquitination.

Role and Mechanism of Rhizopus Nigrum Polysaccharide EPS1-1 as Pharmaceutical for Therapy of Hepatocellular Carcinoma.

Objective: This work is to study the effect of Rhizopus nigrum polysaccharide EPS1-1 on hepatocellular carcinoma (HCC) in vitro and in vivo. Methods: HepG2 and Huh-7 cells and nude mice models of liver cancers were used in this study. The cells and nude mice were treated with EPS1-1 at different concentrations. The CCK8 assays were used to measure the proliferation activities of cells, apoptosis was determined with flow cytometry, cell migration was measured by wound-healing assays, cell invasion was evaluated by Transwell assay, and the survival periods of different groups of tumor-bearing mice were compared. Real-time PCR and Western blot were used to measure the expression levels of mRNAs and proteins of the genes related to proliferation, apoptosis, migration, and invasion. Results: In vitro experiments revealed that when treated with EPS1-1, HepG2 and Huh-7 cell proliferation activities decreased, while there was an increase for the apoptosis rate, and the migration and invasion capabilities were significantly reduced. In vivo experiments showed that EPS1-1 could significantly reduce the tumor growth and lung metastasis of HCC, and prolong the survival periods of tumor-bearing nude mice. Furthermore, EPS1-1 has no apparent damage to the heart, liver, and kidney. Further studies showed that EPS1-1 could affect the expression of proliferation-related genes CCND1 and c-Myc, apoptosis-related genes BAX and Bcl-2, and migration and invasion related genes Vimentin and Slug, thereby affecting the biological process of HCC. Conclusion: EPS1-1 can inhibit the malignant process of HCC in vitro and in vivo, which indicates that EPS1-1 has the potential value of clinical application as chemotherapy or adjuvant in the treatment of liver cancer.

PBS3 and EPS1 Complete Salicylic Acid Biosynthesis from Isochorismate in Arabidopsis.

Salicylic acid (SA) is an important phytohormone mediating both local and systemic defense responses in plants. Despite over half a century of research, how plants biosynthesize SA remains unresolved. In Arabidopsis, a major part of SA is derived from isochorismate, a key intermediate produced by the isochorismate synthase, which is reminiscent of SA biosynthesis in bacteria. Whereas bacteria employ an isochorismate pyruvate lyase (IPL) that catalyzes the turnover of isochorismate to pyruvate and SA, plants do not contain an IPL ortholog and generate SA from isochorismate through an unknown mechanism. Combining genetic and biochemical approaches, we delineated the SA biosynthetic pathway downstream of isochorismate in Arabidopsis. We found that PBS3, a GH3 acyl adenylase-family enzyme important for SA accumulation, catalyzes ATP- and Mg2+-dependent conjugation of L-glutamate primarily to the 8-carboxyl of isochorismate and yields the key SA biosynthetic intermediate, isochorismoyl-glutamate A. Moreover, we discovered that EPS1, a BAHD acyltransferase-family protein with a previously implicated role in SA accumulation upon pathogen attack, harbors a noncanonical active site and an unprecedented isochorismoyl-glutamate A pyruvoyl-glutamate lyase activity that produces SA from the isochorismoyl-glutamate A substrate. Together, PBS3 and EPS1 form a two-step metabolic pathway to produce SA from isochorismate in Arabidopsis, which is distinct from how SA is biosynthesized in bacteria. This study closes a major knowledge gap in plant SA metabolism and would help develop new strategies for engineering disease resistance in crop plants.

Distinct structural changes in wild-type and amyloidogenic chicken cystatin caused by disruption of C95-C115 disulfide bond.

Human cystatin C (HCC) amyloid angiopathy (HCCAA) is characterized by tissue deposition of amyloid fibrils in blood vessels, which can lead to recurrent hemorrhagic stroke. Wild-type HCC forms part of the amyloid deposits in brain arteries of elderly people with amyloid angiopathy. A point mutation causing a glutamine to a leucine substitution at residue 68 in the HCC polypeptide chain greatly increases the amyloidogenic propensity of HCC and causes a more severe cerebral hemorrhage and premature death in young adults. In this study, we used molecular dynamics simulations to assess the importance of disulfide bridge formation upon the stability of chicken cystatin and how this may influence the propensity for amyloid formation. We found that disulfide bridge formation between Cys95 and Cys115 in human cystatin played a critical role in overall protein stability. Importantly, Cys95-Cys115 influenced cystatin structure in regions of the protein that play key roles in the protein-folding transitions that occur, which enable amyloid fibril formation. We hypothesized that correct disulfide bridge formation is a critical step in stabilizing cystatin toward its native conformation. Disrupting Cys95-Cys115 disulfide bridge formation within cystatin appears to significantly enhance the amyloidogenic properties of this protein. In addition, by combining in silico studies with our previous experimental results on Eps1, a molecular chaperone of the PDI family, we proposed that age-related HCCAA, may possess a different pathogenic mechanism compared with its amyloidogenic counterpart, the early onset amyloidogenic cystatin-related CAA.