Cox17, a copper chaperone for cytochrome-c oxidase, is an essential and highly conserved protein in eukaryotic organisms. Yeast and mammalian Cox17 share six conserved cysteine residues, which are involved in complex redox reactions as well as in metal binding and transfer. Mammalian Cox17 exists in three oxidative states, each characterized by distinct metal-binding properties: fully reduced mammalian Cox17(0S-S) binds co-operatively to four Cu+; Cox17(2S-S), with two disulfide bridges, binds to one of either Cu+ or Zn2+; and Cox17(3S-S), with three disulfide bridges, does not bind to any metal ions. The E(m) (midpoint redox potential) values for two redox couples of Cox17, Cox17(3S-S)<-->Cox17(2S-S) (E(m1)) and Cox17(2S-S)<-->Cox17(0S-S) (E(m2)), were determined to be -197 mV and -340 mV respectively. The data indicate that an equilibrium exists in the cytosol between Cox17(0S-S) and Cox17(2S-S), which is slightly shifted towards Cox17(0S-S). In the IMS (mitochondrial intermembrane space), the equilibrium is shifted towards Cox17(2S-S), enabling retention of Cox17(2S-S) in the IMS and leading to the formation of a biologically competent form of the Cox17 protein, Cox17(2S-S), capable of copper transfer to the copper chaperone Sco1. XAS (X-ray absorption spectroscopy) determined that Cu4Cox17 contains a Cu4S6-type copper-thiolate cluster, which may provide safe storage of an excess of copper ions.

RNA silencing is a mechanism involved in gene regulation during development and anti-viral defense in plants and animals. Although many viral suppressors of this mechanism have been described up to now, this is not the case for endogenous suppressors. We have identified a novel endogenous suppressor in plants: RNase L inhibitor (RLI) of Arabidopsis thaliana. RLI is a very conserved protein among eukaryotes and archaea. It was first known as component of the interferon-induced mammalian 2'-5' oligoadenylate (2-5A) anti-viral pathway. This protein is in several organisms responsible for essential functions, which are not related to the 2-5A pathway, like ribosome biogenesis and translation initiation. Arabidopsis has two RLI paralogs. We have described in detail the expression pattern of one of these paralogs (AtRLI2), which is ubiquitously expressed in all plant organs during different developmental stages. Infiltrating Nicotiana benthamiana green fluorescent protein (GFP)-transgenic line with Agrobacterium strains harboring GFP and AtRLI2, we proved that AtRLI2 suppresses silencing at the local and at the systemic level, reducing drastically the amount of GFP small interfering RNAs.

Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metabolism. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd 2؉ and Co 2؉ ions. Both the 113 Cd NMR studies of 113 Cd 7 MT-4 and the spectroscopic characterization of Co 7 MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd 4 Cys 11 and Cd 3 Cys 9 clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd 7 MT-4 is cooperative and sequential, with the Cd 4 Cys 11 cluster being formed first, and that a distinct single-metal nucleation intermediate Cd 1 MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd 7 MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd 7 MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed. Metallothioneins (MTs)2 is a superfamily of low molecular mass cysteine-and metal-rich proteins or polypeptides conserved through evolution and present in all eukaryotes and certain prokaryotes (reviewed in Refs. 1 and 2). In mammals, the MT gene family consists of four subfamilies designated MT-1 through MT-4. Whereas in mouse only one member of each subfamily is present ( Fig. 1), in primates a significant genetic polymorphism exists with 17 genes and pseudogenes in humans. From these 17 genes, 10 are functional, including single MT-3 and MT-4 genes (3). Mammalian MTs are composed of a single polypeptide chain of 61-68 amino acids with a conserved array of 20 cysteines and no aromatic residues or histidine. In the structurally characterized MTs (MT-1-MT-3), all cysteines are present in reduced form and are involved in the binding of seven divalent (Zn 2ϩ , Cd 2ϩ ) and up to 12 monovalent metal ions (Cu ϩ ) forming two metal-thiolate clusters located in two independent protein domains (4 -6). Although still under debate, suggested functions for mammalian MTs include homeostasis and transport of physiologically essential metals (zinc, copper), detoxification of toxic metals (cadmium, mercury), protection against oxidative stress, regulation of cell proliferation and apoptosis, and the maintenance of intracellular redox balance (7-10). Differential expression of mammalian MT isoforms is tightly regulated during development and in pathological situations (9,11). The extensively studied mammalian MT-1 and MT-2 show ubiquitous expression regulated at the transcriptional level (12). Their biosynthesis is inducible by a variety of compounds and stress conditions, such as metals, glucocorticoids, cytokines, and reactive oxygen species (9). MT-3 and MT-4 are relatively unresponsiv...

Transcription factor IID (TFIID) activity can be regulated by cellular signals to specifically alter transcription of particular subsets of genes. Alternative splicing of TFIID subunits is often the result of external stimulation of upstream signaling pathways. We studied tissue distribution and cellular expression of different splice variants of TFIID subunit TAF4 mRNA and biochemical properties of its isoforms in human mesenchymal stem cells (hMSCs) to reveal the role of different isoforms of TAF4 in the regulation of proliferation and differentiation. Expression of TAF4 transcripts with exons VI or VII deleted, which results in a structurally modified hTAF4-TAFH domain, increases during early differentiation of hMSCs into osteoblasts, adipocytes and chondrocytes. Functional analysis data reveals that TAF4 isoforms with the deleted hTAF4-TAFH domain repress proliferation of hMSCs and preferentially promote chondrogenic differentiation at the expense of other developmental pathways. This study also provides initial data showing possible cross-talks between TAF4 and TP53 activity and switching between canonical and non-canonical WNT signaling in the processes of proliferation and differentiation of hMSCs. We propose that TAF4 isoforms generated by the alternative splicing participate in the conversion of the cellular transcriptional programs from the maintenance of stem cell state to differentiation, particularly differentiation along the chondrogenic pathway.