An enrichment method for heme-less mutants of Saccharomyces cerevisiae based on photodynamic propeties of Zn-protoporphyrin

Summary A new and widely applicable technique has been elaborated for enrichment of mutants of Saccharomyces cerevisiae deficient in porphyrin and heme synthesis. The method is based on selective photooxidative killing of the wild-type cells, sensitized by Zn-protoporphyrin synthesized and accumulated in wild-type cells under defined conditions.Using a single cycle of selection, heme-deficient mutants have been obtained with a frequency of 2–2.3%.     

A molecular switch on an arrestin-like protein relays glucose signaling to transporter endocytosis

Endocytosis regulates the plasma membrane protein landscape in response to environmental cues. In yeast, the endocytosis of transporters depends on their ubiquitylation by the Nedd4-like ubiquitin ligase Rsp5, but how extracellular signals trigger this ubiquitylation is unknown. Various carbon source transporters are known to be ubiquitylated and endocytosed when glucose-starved cells are exposed to glucose. We show that this required the conserved arrestin-related protein Rod1/Art4, which was activated in response to glucose addition. Indeed, Rod1 was a direct target of the glucose signaling pathway composed of the AMPK homologue Snf1 and the PP1 phosphatase Glc7/Reg1. Glucose promoted Rod1 dephosphorylation and its subsequent release from a phospho-dependent interaction with 14-3-3 proteins. Consequently, this allowed Rod1 ubiquitylation by Rsp5, which was a prerequisite for transporter endocytosis. This paper therefore demonstrates that the arrestin-related protein Rod1 relays glucose signaling to transporter endocytosis and provides the first molecular insights into the nutrient-induced activation of an arrestin-related protein through a switch in post-translational modifications.     

Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphoenolpyruvate β-glucoside phosphotransferase system of Escherichia coli

β-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme II^bgl appeared as the target of N-ethylmaleimide inactivation. The same feature was found in the case of methyl-α-d-glucoside uptake via enzyme II^glc.It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-α-d-glucoside only sensitizes enzyme II^bglc and $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ only sensitizes enzyme II^bgl towards N-ethylmaleimide inactivation.The inactivation of enzyme II^bgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ phosphorylation. Both results suggest that the inactivator resistent form of enzyme II^bgl is an energized form of the enzyme.     

Sulforaphane promotes chlamydial infection by suppressing mitochondrial protein oxidation and activation of complement C3

Sulforaphane (SFN), a phytochemical found in broccoli and other cruciferous vegetables, is a potent antioxidant and anti‐inflammatory agent with reported effects in cancer chemoprevention and suppression of infection with intracellular pathogens. Here we report on the impact of SFN on infection with Chlamydia trachomatis (Ct), a common sexually transmitted pathogen responsible for 131 million new cases annually worldwide. Astoundingly, we find that SFN as well as broccoli sprouts extract (BSE) promote Ct infection of human host cells. Both the number and size of Ct inclusions were increased when host cells were pretreated with SFN or BSE. The initial investigations presented here point to both the antioxidant and thiol alkylating properties of SFN as regulators of Ct infection. SFN decreased mitochondrial protein sulfenylation and promoted Ct development, which were both reversed by treatment with mitochondria‐targeted paraquat (MitoPQ). Inhibition of the complement component 3 (complement C3) by SFN was also identified as a mechanism by which SFN promotes Ct infections. Mass spectrometry analysis found alkylation of cysteine 1010 (Cys1010) in complement C3 by SFN. The studies reported here raise awareness of the Ct infection promoting activity of SFN, and also identify potential mechanisms underlying this activity.     

A Phase I Double Blind,Placebo-Controlled,Randomized Study of the Safety and Immunogenicity of Electroporated HIV DNA with or without Interleukin 12 in Prime-Boost Combinations with an Ad35 HIV Vaccine in Healthy HIV-Seronegative African Adults

Background

Strategies to enhance the immunogenicity of DNA vaccines in humans include i) co-administration of molecular adjuvants, ii) intramuscular administration followed by in vivo electroporation (IM/EP) and/or iii) boosting with a different vaccine. Combining these strategies provided protection of macaques challenged with SIV; this clinical trial was designed to mimic the vaccine regimen in the SIV study.

Methods

Seventy five healthy, HIV-seronegative adults were enrolled into a phase 1, randomized, double-blind, placebo-controlled trial. Multi-antigenic HIV (HIVMAG) plasmid DNA (pDNA) vaccine alone or co-administered with pDNA encoding human Interleukin 12 (IL-12) (GENEVAX IL-12) given by IM/EP using the TriGrid Delivery System was tested in different prime-boost regimens with recombinant Ad35 HIV vaccine given IM.

Results

All local reactions but one were mild or moderate. Systemic reactions and unsolicited adverse events including laboratory abnormalities did not differ between vaccine and placebo recipients. No serious adverse events (SAEs) were reported. T cell and antibody response rates after HIVMAG (x3) prime—Ad35 (x1) boost were independent of IL-12, while the magnitude of interferon gamma (IFN-γ) ELISPOT responses was highest after HIVMAG (x3) without IL-12. The quality and phenotype of T cell responses shown by intracellular cytokine staining (ICS) were similar between groups. Inhibition of HIV replication by autologous T cells was demonstrated after HIVMAG (x3) prime and was boosted after Ad35. HIV specific antibodies were detected only after Ad35 boost, although there was a priming effect with 3 doses of HIVMAG with or without IL-12. No anti-IL-12 antibodies were detected.

Conclusion

The vaccines were safe, well tolerated and moderately immunogenic. Repeated administration IM/EP was well accepted. An adjuvant effect of co-administered plasmid IL-12 was not detected.

Trial Registration

ClinicalTrials.gov NCT01496989