The role of post-transcriptional modulators of metalloproteins in response to metal deficiencies.

Copper and iron proteins have a wide range of functions in living organisms. Metal assembly into metalloproteins is a complex process, where mismetalation is detrimental and energy consuming to cells. Under metal deficiency, metal distribution is expected to reach a metalation ranking, prioritizing essential versus dispensable metalloproteins, while avoiding interference with other metals and protecting metal-sensitive processes. In this review, we propose that post-transcriptional modulators of metalloprotein mRNA (ModMeR) are good candidates in metal prioritization under metal-limited conditions. ModMeR target high quota or redundant metalloproteins and, by adjusting their synthesis, ModMeR act as internal metal distribution valves. Inappropriate metalation of ModMeR targets could compete with metal delivery to essential metalloproteins and interfere with metal-sensitive processes, such as chloroplastic photosynthesis and mitochondrial respiration. Regulation of ModMeR targets could increase or decrease the metal flow through interconnected pathways in cellular metal distribution, helping to achieve adequate differential metal requirements. Here, we describe and compare ModMeR that function in response to copper and iron deficiencies. Specifically, we describe copper-miRNAs from Arabidopsis thaliana and diverse iron ModMeR from yeast, mammals, and bacteria under copper and iron deficiencies, as well as the influence of oxidative stress. Putative functions derived from their role as ModMeR are also discussed.

Dissecting mRNA decay and translation inhibition during iron deficiency.

Iron participates as a vital cofactor in multiple metabolic pathways. Despite its abundance, iron bioavailability is highly restricted in aerobic and alkaline environments. Therefore, living organisms have evolved multiple adaptive mechanisms to respond to iron scarcity. These strategies include a global remodeling of iron metabolism directed to optimize iron utilization. In the baker's yeast Saccharomyces cerevisiae, this metabolic reorganization is accomplished to a large extent by an mRNA-binding protein called Cth2. Yeast Cth2 belongs to a conserved family of tandem zinc finger containing proteins that specifically bind to transcripts with AU-rich elements and promote their turnover. A recent study has revealed that Cth2 also inhibits the translation of its target mRNAs (Ramos-Alonso et al., PLoS Genet 14:e1007476, https://doi.org/10.1371/journal.pgen.1007476 , 2018). Interestingly, the mammalian Cth2 ortholog known as tristetraprolin (aka TTP/TIS11/ZFP36), which is also implicated in controlling iron metabolism, promotes the decay and prevents the translation of its regulated transcripts. These observations open the possibility to study the relative contribution of altering mRNA stability and translation to the physiological adaptation to iron deficiency, the function played by the different domains within the mRNA-binding protein, and the potential factors implicated in coordinating both post-transcriptional events.

Structural model of a2-subunit N-terminus and its binding interface for Arf-GEF CTH2: Implication for regulation of V-ATPase, CTH2 function and rational drug design.

We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N1-402) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Grüber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N1-402 and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber Icyt-subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N1-352 fragment. Next, we combined it with the determined NMR structures of peptides a2N368-395 and a2N386-402 of the C-terminal section of a2N1-402. The complete molecular model of a2N1-402 revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N1-402. Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N1-402 interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N1-17 and a2N35-49 is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function.

Central roles of iron in the regulation of oxidative stress in the yeast Saccharomyces cerevisiae.

Oxygen is essential for aerobic organisms but causes cytotoxicity probably through the generation of reactive oxygen species (ROS). In this study, we screened for the genes that regulate oxidative stress in the yeast Saccharomyces cerevisiae, and found that expression of CTH2/TIS11 caused an increased resistance to ROS. CTH2 is up-regulated upon iron starvation and functions to remodel metabolism to adapt to iron starvation. We showed here that increased resistance to ROS by CTH2 would likely be caused by the decreased ROS production due to the decreased activity of mitochondrial respiration, which observation is consistent with the fact that CTH2 down-regulates the mitochondrial respiratory proteins. We also found that expression of CTH1, a paralog of CTH2, also caused an increased resistance to ROS. This finding supported the above view, because mitochondrial respiratory proteins are the common targets of CTH1 and CTH2. We further showed that supplementation of iron in medium augmented the growth of S. cerevisiae under oxidative stress, and expression of CTH2 and supplementation of iron collectively enhanced its growth under oxidative stress. Since CTH2 is regulated by iron, these findings suggested that iron played crucial roles in the regulation of oxidative stress in S. cerevisiae.

The yeast mRNA-binding protein Cth2 post-transcriptionally modulates ergosterol biosynthesis in response to iron deficiency.

Sterol synthesis is an iron-dependent metabolic pathway in eukaryotes. Consequently, fungal ergosterol biosynthesis (ERG) is down-regulated in response to iron deficiency. In this report, we show that, upon iron limitation or overexpression of the iron-regulated mRNA-binding protein Cth2, the yeast Saccharomyces cerevisiae down-regulates the three initial enzymatic steps of ergosterol synthesis (ERG1, ERG7 and ERG11). Mechanistically, we show that Cth2 protein limits the translation and promotes the decrease in the mRNA levels of these specific ERG genes, which contain consensus Cth2-binding sites defined as AU-rich elements (AREs). Thus, expression of CTH2 leads to the accumulation of initial sterol intermediates, such as squalene, and to the drop of ergosterol levels. Changes in CTH2 expression levels disturb the response of yeast cells to stresses related to membrane integrity such as high ethanol and sorbitol concentrations. Therefore, CTH2 should be considered as a critical regulatory factor of ergosterol biosynthesis during iron deficiency.