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Reactive oxygen species (ROS) production activates unconventional secretion of antioxidant proteins

ABSTRACT: Signal-sequence-lacking superoxide dismutase 1 (SOD1) is secreted upon nutrient starvation, but the physiological relevance of this secretion is unclear. We now report that secreted SOD1 is functionally active. In addition, a secretome analysis has revealed number of other secreted cytoplasmic antioxidant enzymes, including both thioredoxins, (Trx1 and Trx2), and the peroxiredoxin Ahp1. Our data reveal that starvation triggers increased mitochondrial activity, leading to increased production of reactive oxygen species (ROS). Inhibiting mitochondrial activity or ROS, inhibited secretion of the antioxidants. We suggest elevated ROS that evade cytoplasmic antioxidants, can permeate across the plasma membrane to the extracellular space. Cells secrete antioxidants to prevent ROS-mediated damage to the extracellular space. These findings thus provide an understanding of why cells secrete signal sequence lacking proteins like SOD1 and the larger family of antioxidants.

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Eduard Sabidó

LAB HEAD: Amy Curwin

PROVIDER: PXD010849 | Pride | 2019-05-14

REPOSITORIES: Pride

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Project description:Environmental conditions are quite eﬀective during propagation and industrial application of Baker’s yeast (Saccharomyces cerevisiae). The change of temperature is one of the most important conditions which aﬀects the dough-leaving capacity of yeast cells. Accordingly, heat changes play an essential role in the commercial and economic impact of the yeast. Recent technologies in genomics have been used for analysis of global gene expression proﬁles such as microarray and RNA sequencing to solve the problems related with industrial application of yeast. Hereby, the aim of this study is to explore the eﬀects of heat stresses on global gene expression proﬁles and to identify the candidate genes for the heat stress response in commercial baker’s yeast by using microarray technology and comparative statistical data analyses. Data from all hybridizations and array normalization were analyzed using the GeneSpringGX 12.1 (Agilent) and the R 2.15.2 program language. In the analysis of this dataset, all required statistical methods are performed comparatively in each step and the best performed ones are used in further computations. Hence, as the ﬁrst step of the analyses, diﬀerent background normalization algorithms such as MAS5.0, RMA, MBEI and GC-RMA were applied and the algorithm which gives the most accurate ﬁndings was chosen for the normalization procedure. As a result, expression values, computed from CEL ﬁles, were processed by Robust Multiarray Analysis (RMA) which is the selected procedure for the normalization of the systematic diﬀerences between samples. In order to determine diﬀerentially expressed genes under heat stress treatments, two diﬀerent methods, namely, the fold-change and the hypothesis testing approaches are executed. In the fold-change method, various cut-oﬀ values are implemented while diﬀerent multiple testing procedures are performed for the hypothesis testing. Under the heat shock and temperature-shift stress conditions, up/down regulated diﬀerentially expressed probes were functionally categorized into the diﬀerent groups via the cluster analyses. Transcriptome changes under the heat shock and temperature-shift stress treatments show that the number of diﬀerentially up-regulated genes among the heat shock proteins (HSPs) and transcription factors (TFs) changed signiﬁcantly. Consequently, the identiﬁcation of thousands of genes related with heat stress treatments via microarray technology and comparative statistical analysis resulted in the creation of big picture which provides the understanding of the importance for the baker’s yeast industrial applications.

Project description:Pyruvate oxidase encoded by spxB is a major virulence factor in the human respiratory pathogen Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes large amounts of H2O2 and acetyl phosphate, which can serve as a phosphoryl group donor to response regulators and be converted to ATP. SpxB is the main source of the millimolar concentrations of H2O2 produced and tolerated by pneumococcus, despite its lack of a catalase. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen, similar to those used previously for phase variants, for spontaneous mutations that caused colonies of virulent serotype 2 strain D39 to change from a transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency of 3 x 10-5) were impaired for SpxB function. Modeling suggested that two mutations changed amino acids in SpxB required for FAD cofactor or subunit binding. One mutation deleted a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three independent mutations created the same frameshift near the start of a trans-acting regulatory gene designated as spxR. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in DNA, adenosine, and CoA compound binding, respectively, consistent with the idea that SpxR positively regulates spxB transcript amount in response to energy and metabolic state rather than oxidative state. Finally, microarray analyses of a null spxB or a spxR mutant revealed the presence of a new oxidative stress response in pneumococcus and unexpectedly demonstrated that SpxR strongly positively regulates the transcript amount of the strH exoglycosidase gene, which like spxB, has been implicated in host colonization. Keywords: genetic modification Bacterial strains were grown exponentially in rich (BHI) media at 37C and an atmosphere of 5% CO2, and were processed as described in the related Sample records. Samples were collected from three independent biological replicates and included one dye swap. Data were normalized using the Lowess (subgrid) method without background subtraction.

			Action	DRS
	150225_S_ACVM_01_01.msf	Msf
	150225_S_ACVM_01_01.raw	Raw
	150225_S_ACVM_02_01.msf	Msf
	150225_S_ACVM_02_01.raw	Raw
	150225_S_ACVM_03_01.msf	Msf

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Reactive oxygen species (ROS) production activates unconventional secretion of antioxidant proteins

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