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Iron is an indispensable micronutrient for all eukaryotic organisms due to its participation as a redox cofactor in many metabolic pathways. Iron imbalance leads to the most frequent human nutritional deficiency in the world. Adaptation to iron limitation requires a global reorganization of the cellular metabolism directed to prioritize iron utilization for essential processes. In response to iron scarcity, the conserved Saccharomyces cerevisiae mRNA-binding protein Cth2, which belongs to the tristetraprolin family of tandem zinc finger proteins, coordinates a global remodeling of the cellular metabolism by promoting the degradation of multiple mRNAs encoding highly iron-consuming proteins. In this work, we identify a critical mechanism for the degradation of Cth2 protein during the adaptation to iron deficiency. Phosphorylation of a patch of Cth2 serine residues within its amino-terminal region facilitates recognition by the SCFGrr1 ubiquitin ligase complex, accelerating Cth2 turnover by the proteasome. When Cth2 degradation is impaired by either mutagenesis of the Cth2 serine residues or deletion of GRR1, the levels of Cth2 rise and abrogate growth in iron-depleted conditions. Finally, we uncover that the casein kinase Hrr25 phosphorylates and promotes Cth2 destabilization. These results reveal a sophisticated posttranslational regulatory pathway necessary for the adaptation to iron depletion.IMPORTANCE Iron is a vital element for many metabolic pathways, including the synthesis of DNA and proteins, and the generation of energy via oxidative phosphorylation. Therefore, living organisms have developed tightly controlled mechanisms to properly distribute iron, since imbalances lead to nutritional deficiencies, multiple diseases, and vulnerability against pathogens. Saccharomyces cerevisiae Cth2 is a conserved mRNA-binding protein that coordinates a global reprogramming of iron metabolism in response to iron deficiency in order to optimize its utilization. Here we report that the phosphorylation of Cth2 at specific serine residues is essential to regulate the stability of the protein and adaptation to iron depletion. We identify the kinase and ubiquitination machinery implicated in this process to establish a posttranscriptional regulatory model. These results and recent findings for both mammals and plants reinforce the privileged position of E3 ubiquitin ligases and phosphorylation events in the regulation of eukaryotic iron homeostasis.

Heat shock factor 1 (HSF1) is a stress-responsive transcription factor that regulates expression of protein chaperones and cell survival factors. In cancer, HSF1 plays a unique role, hijacking the normal stress response to drive a cancer-specific transcriptional program. These observations suggest that HSF1 inhibitors could be promising therapeutics. However, HSF1 is activated through a complex mechanism, which involves release of a negative regulatory domain, leucine zipper 4 (LZ4), from a masked oligomerization domain (LZ1-3), and subsequent binding of the oligomer to heat shock elements (HSEs) in HSF1-responsive genes. Recent crystal structures have suggested that HSF1 oligomers are held together by extensive, buried contact surfaces, making it unclear whether there are any possible binding sites for inhibitors. Here, we have rationally designed a series of peptide-based molecules based on the LZ4 and LZ1-3 motifs. Using a plate-based, fluorescence polarization (FP) assay, we identified a minimal region of LZ4 that suppresses binding of HSF1 to the HSE. Using this information, we converted this peptide into a tracer and used it to understand how binding of LZ4 to LZ1-3 suppresses HSF1 activation. Together, these results suggest a previously unexplored avenue in the development of HSF1 inhibitors. Furthermore, the findings highlight how native interactions can inspire the design of inhibitors for even the most challenging protein-protein interactions (PPIs).

The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer.

Tumorigenesis driven by the oncogene BRAF.sup.V600E is shown both to depend on the BRAF substrates MEK1/2 associating with copper, and to be sensitive to copper-chelating drugs, suggesting merit in testing such drugs for the treatment of BRAF mutation-positive cancers. A role for copper in BRAF cancers A large proportion of melanomas and some other cancers harbour mutations in the BRAF gene, most of them at codon 600, causing constitutive activation of the MAPK (mitogen-activated protein kinase) pathway. Following the discovery that copper transport promotes MAPK signalling in Drosophila by binding to and activating the kinase MEK, Chris Counter and colleagues now show that oncogenic signalling by mutant BRAF requires copper binding to MEK, promoting activation of ERK1/2, the next kinases in the cascade. Interfering with copper availability by genetic means or with copper-chelating agents reduces BRAF-driven tumour growth in vivo in mouse models, and also of cancer cells that have become resistant to BRAF inhibitors. Thus copper chelators, already in the clinic for other indications, may prove useful for the treatment of BRAF-mutant tumours in combination with BRAF inhibitors, and potentially to prevent resistance. The BRAF kinase is mutated, typically Val 600[right arrow]Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers.sup.1,2. BRAF.sup.V600Ephosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer.sup.3. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma.sup.4, an effect that is increased when administered together with a BRAF.sup.V600Einhibitor.sup.5. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction.sup.6. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF.sup.V600E-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease.sup.7decreased tumour growth of human or murine cells transformed by BRAF.sup.V600E or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF.sup.V600Emutation., Author(s): Donita C. Brady [sup.1] , Matthew S. Crowe [sup.1] , Michelle L. Turski [sup.1] , G. Aaron Hobbs [sup.2] , Xiaojie Yao [sup.3] , Apirat Chaikuad [sup.4] , Stefan [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2006.09.003 Byline: Yasuhiro Nose, Erin M. Rees, Dennis J. Thiele Abstract: Copper is essential for biological processes such as free radical detoxification, mitochondrial respiration and iron metabolism. A central player in copper homeostasis is the high-affinity integral plasma membrane copper transporter Ctr1. However, the precise mechanisms by which Ctr1 functions are not known. Here, we highlight an important breakthrough in our understanding of how Ctr1 facilitates Cu(I) movement across membranes: the publication of structural details for human Ctr1 obtained from 2D crystallography and electron microscopy. Author Affiliation: Department of Pharmacology and Cancer Biology, Sarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center, Research Drive-LSRC C351, Durham, NC 27710-3813, USA

Yeast prions are non-Mendelian genetic elements that are conferred by altered and self-propagating protein conformations. Such a protein conformation-based transmission is similar to that of [PrP.sub.Sc], the infectious protein responsible for prion diseases. Despite recent progress in understanding the molecular nature and epigenetic transmission of prions, the underlying mechanisms governing prion conformational switch and determining prion 'strains' are not understood. We report here that the evolutionarily conserved heat-shock transcription factor (HSF) strongly influences yeast prion formation and strain determination. An hall mutant lacking the amino-terminal activation domain inhibits the yeast prion [[PSI.sup.+]] formation whereas a mutant lacking the carboxyl-terminal activation domain promotes [[PSI.sup.+]] formation. Moreover, specific [[PSI.sup.+]] strains are preferentially formed in these mutants, demonstrating the importance of genetic makeup in determining de novo appearance of prion strains. Although these hsf1 mutants preferentially support the formation of certain [[PSI.sup.+]] strains, they are capable of receiving and faithfully propagating nonpreferable strains, suggesting that prion initiation and propagation are distinct processes requiring different cellular components. Our findings establish the importance of HSF in prion initiation and strain determination and imply a similar regulatory role of mammalian HSFs in the complex etiology of prion disease.

A novel and paradoxical role is described for metal-responsive transcription factor (MTF-1) in Drosophila wherein it also mediates transcriptional activation of Ctr1B, a copper importer, upon copper depletion. Findings show that a single transcription factor, MTF-1, played a direct role in copper detoxification and acquisition by inducing the expression of metallothioneins and of copper importer, respectively.

The loop domain of heat shock transcription factor (HSF) 1 has been found to determine DNA-binding specificity and heat stress responses. The mechanistic basis for differences between HSF1 and HSF2 in DNA binding to the hsp70 heat shock elements (HSEs) and its effect on transcription has been studied. HSPs are heat shock proteins.

In vivo footprinting and chromatin-mapping experiments have shown that the single metal response element (MRE) plays a critical role in transcriptional autoactivation by the Amt1 copper metaloregulatory transcription factor in Candida glabrata. Transcription occurs when MRE binds to the Amt1 gene, resulting in protection from toxic levels of copper. The AMT1 promoter region within a nucleosome offers a mechanism for rapid transcription factor access to evoke response to copper.