TAT fusion protein transduction into isolated mitochondria is accelerated by sodium channel inhibitors.

Stringent control of ion and protein transport across the mitochondrial membranes is required to maintain mitochondrial function and biogenesis. In particular, the inner mitochondrial membrane is generally impermeable to proteins entering the matrix except via tightly regulated protein import mechanisms. Recently, cell penetrant peptides have been shown to move across the inner mitochondrial membrane in a manner suggesting an independent mechanism. HIV-1 transactivator of transcription (TAT) is an arginine-rich cell penetrant peptide, 47YGRKKRRQRRR57, which can transduce full-length proteins not only across the cell membrane but also into intracellular organelles. In this study, we investigated the ability of a TAT-containing protein to move into the mitochondrial matrix. Using a novel FACS assay for isolated, purified mitochondria, we show that TAT can deliver a modified fluorescent protein, mMDH-GFP, to the matrix of mitochondria and it is subsequently processed by the matrix peptidases. In addition, transduction of TAT-mMDH-GFP into mitochondria is independent of canonical protein import pathways as well as mitochondrial membrane potential. In direct contrast to published reports regarding the cell membrane where the sodium channel inhibitor, amiloride, blocks endocytosis and inhibits TAT transduction, TAT transduction into mitochondria is markedly increased by this same sodium channel inhibitor. These results confirm that the cell penetrant peptide, TAT, can readily transduce a protein cargo into the mitochondrial matrix. These results also demonstrate a novel role for mitochondrial sodium channels in mediating TAT transduction into mitochondria that is independent of endocytotic mechanisms. The mechanism of TAT transduction into mitochondria therefore is distinctly different from transduction across the cell membrane.

Eyes absent represents a class of protein tyrosine phosphatases.

The Eyes absent proteins are members of a conserved regulatory network implicated in the development of the eye, muscle, kidney and ear. Mutations in the Eyes absent genes have been associated with several congenital disorders including the multi-organ disease bronchio-oto-renal syndrome, congenital cataracts and late-onset deafness. On the basis of previous analyses it has been shown that Eyes absent is a nuclear transcription factor, acting through interaction with homeodomain-containing Sine oculis (also known as Six) proteins. Here we show that Eyes absent is also a protein tyrosine phosphatase. It does not resemble the classical tyrosine phosphatases that use cysteine as a nucleophile and proceed by means of a thiol-phosphate intermediate. Rather, Eyes absent is the prototype for a class of protein tyrosine phosphatases that use a nucleophilic aspartic acid in a metal-dependent reaction. Furthermore, the phosphatase activity of Eyes absent contributes to its ability to induce eye formation in Drosophila.

Branchio-oto-renal syndrome associated mutations in Eyes Absent 1 result in loss of phosphatase activity.

The Eyes Absent (Eya) proteins are tyrosine phosphatases and transcriptional activators involved in cell-fate determination and organ development. Mutations in the gene encoding Eya homologue 1 have been implicated in the multi-organ developmental disorder branchio-oto-renal syndrome (BOR) and in ocular defects. Here we report that BOR-associated mutations lead to a loss of phosphatase activity in Eya1 proteins, while mutations associated with ocular defects yield Eya1 proteins with near normal levels of phosphatase activity. Furthermore we demonstrate that the N-terminal domain attenuates the catalytic activity of Eya suggesting a mechanism of regulation.

Formation of carcinogenic chromosomal rearrangements in human thyroid cells after induction of double-strand DNA breaks by restriction endonucleases.

Ionizing radiation (IR) exposure increases the risk of thyroid cancer and other cancer types. Chromosomal rearrangements, such as RET/PTC, are characteristic features of radiation-associated thyroid cancer and can be induced by radiation in vitro. IR causes double-strand breaks (DSBs), suggesting that such damage leads to RET/PTC, but the rearrangement mechanism has not been established. To study the mechanism, we explored the possibility of inducing RET/PTC by electroporation of restriction endonucleases (REs) into HTori-3 human thyroid cells. We used five REs, which induced DSB in a dose-dependent manner similar to that seen with IR. Although all but one RE caused DSB in one or more of the three genes involved in RET/PTC, rearrangement was detected only in cells electroporated with either PvuII (25 and 100  U) or StuI (100 and 250  U). The predominant rearrangement type was RET/PTC3, which is characteristic of human thyroid cancer arising early after Chernobyl-related radioactive iodine exposure. Both enzymes that produced RET/PTC had restriction sites only in one of the two fusion partner genes. Moreover, the two enzymes that produced RET/PTC had restriction sites present in clusters, which was not the case for RE that failed to induce RET/PTC. In summary, we establish a model of DSB induction by RE and report for the first time the formation of carcinogenic chromosomal rearrangements, predominantly RET/PTC3, as a result of DSB produced by RE. Our data also raise a possibility that RET/PTC rearrangement can be initiated by a complex DSB that is induced in one of the fusion partner genes.

Characterization of a plant, tyrosine-specific phosphatase of the aspartyl class.

The Arabidopsis thaliana homologue of the Eyes Absent genes (AtEYA) encodes a protein corresponding to the C-terminal conserved domain of the animal Eyes Absent proteins. We show here that AtEYA is a tyrosine-specific phosphatase that hydrolyzes its substrates in a metal-dependent reaction analogous to the phosphoserine phosphatases of the haloacid dehalogenase (HAD) family. The animal Eyes Absent proteins are a novel family of dual-function enzymes: they are transcription factors as well as phosphatases. They also represent a new mechanistic class of tyrosine phosphatases (PTPs) that do not have the Cys-containing signature motif. In contrast, AtEYA is only a tyrosine phosphatase and has no transactivation domain. Using the reaction mechanism of other HAD family enzymes as a model, we have conducted mutational analyses on AtEYA to query the roles of conserved residues. This analysis confirms the importance of the putative nucleophile, the general acid, and the metal-binding residues. Additionally, an inhibitory profile that is diagnostic of this new class of protein tyrosine phosphatases is described. The results of these studies on AtEYA reveal that while the animal and plant Eyes Absent proteins catalyze the same dephosphorylation reaction, the details of their specificity and active site environment, as well as their biological roles, are distinct.