Structural and functional analysis of Hikeshi, a new nuclear transport receptor of Hsp70s.

Hikeshi is a nuclear transport receptor required for cell survival after stress. It mediates heat-shock-induced nuclear import of 70 kDa heat-shock proteins (Hsp70s) through interactions with FG-nucleoporins (FG-Nups), which are proteins in nuclear pore complexes (NPCs). Here, the crystal structure of human Hikeshi is presented at 1.8 Šresolution. Hikeshi forms an asymmetric homodimer that is responsible for the interaction with Hsp70s. The asymmetry of Hikeshi arises from the distinct conformation of the C-terminal domain (CTD) and the flexibility of the linker regions of each monomer. Structure-guided mutational analyses showed that both the flexible linker region and the CTD are important for nuclear import of Hsp70. Pull-down assays revealed that only full-length Hsp70s can interact with Hikeshi. The N-terminal domain (NTD) consists of a jelly-roll/ß-sandwich fold structure which contains hydrophobic pockets involved in FG-Nup recognition. A unique extended loop (E-loop) in the NTD is likely to regulate the interactions of Hikeshi with FG-Nups. The crystal structure of Hikeshi explains how Hikeshi participates in the regulation of nuclear import through the recognition of FG-Nups and which part of Hikeshi affects its binding to Hsp70. This study is the first to yield structural insight into this highly unique import receptor.

Structural basis for the selective nuclear import of the C2H2 zinc-finger protein Snail by importin ß.

Snail contributes to the epithelial-mesenchymal transition by suppressing E-cadherin in transcription processes. The Snail C2H2-type zinc-finger (ZF) domain functions both as a nuclear localization signal which binds to importin ß directly and as a DNA-binding domain. Here, a 2.5 Šresolution structure of four ZF domains of Snail1 complexed with importin ß is presented. The X-ray structure reveals that the four ZFs of Snail1 are required for tight binding to importin ß in the nuclear import of Snail1. The shape of the ZFs in the X-ray structure is reminiscent of a round snail, where ZF1 represents the head, ZF2-ZF4 the shell, showing a novel interaction mode, and the five C-terminal residues the tail. Although there are many kinds of C2H2-type ZFs which have the same fold as Snail, nuclear import by direct recognition of importin ß is observed in a limited number of C2H2-type ZF proteins such as Snail, Wt1, KLF1 and KLF8, which have the common feature of terminating in ZF domains with a short tail of amino acids.

Crystallization and preliminary X-ray diffraction analysis of human importin ß-Snail zinc finger domain complex.

Snail is a C2H2-type zinc finger transcriptional repressor that induces epithelial-mesenchymal transition by repression of E-cadherin expression levels during embryonic development and tumour progression. Snail is imported into the nucleus by importin ß through direct binding with its four zinc finger domain. The complex between importin ß and Snail four zinc finger domain was crystallized in order to understand the nuclear transport mechanism of Snail. The constituents of the complex were separately expressed and were then co-purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 228.2, b = 77.5, c = 72.0 Å, ß = 100.9° and diffracted to 2.5 Šresolution.

Crystallization and preliminary X-ray crystallographic study of the human MST2 SARAH domain.

The SARAH domain at the C-terminus of human MST2 (residues 436-484) was overproduced and purified using an Escherichia coli expression system. The purified domain was crystallized using the hanging-drop vapour-diffusion technique. Two crystal forms were obtained. The crystals belonged to space group P2, with unit-cell parameters a = 62.0, b = 119.2, c = 62.0 Å, α = 90.0, ß = 90.5, γ = 90.0°, or to space group P6(1)22, with unit-cell parameters a = 54.5, b = 54.5, c = 303.1 Å. These crystals diffracted to 2.7 and 3.0 Å resolution, respectively.

Identification of the Thioredoxin-Like 2 Autoantibody as a Specific Biomarker for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has a higher risk of death within 5 years of being diagnosed than the other forms of breast cancer. It is the second leading cause of death due to cancer among women. Currently, however, no diagnostic blood-based biomarker exists to identify the early stages of TNBC. To address this point, we utilized a human protein microarray system to identify serum autoantibodies that showed different expression patterns between TNBC and normal serum samples, and identified five autoantibodies showing TNBC-specific expression. Among them, we selected the thioredoxin-like 2 (TXNL2) autoantibody and evaluated its diagnostic relevance by dot blot analysis with the recombinant TXNL2 protein. We demonstrated that the TXNL2 autoantibody showed 2- to 6-fold higher expression in TNBC samples than in normal samples suggesting that serum TXNL2 autoantibodies are potential biomarkers for TNBC.

Low pH-driven folding of WW45-SARAH domain leads to stabilization of the WW45-Mst2 complex.

The scaffolding protein Salvador (Sav) plays a key role in the Hippo (Hpo) signalling pathway, which controls tissue growth by inhibiting cell proliferation and promoting apoptosis. Dysregulation of the Hippo pathway contributes to cancer development. Since the identification of the first Sav gene in 2002, very little is known regarding the molecular basis of Sav-SARAH mediating interactions due to its insolubility. In this study, refolding of the first Sav (known as WW45)-SARAH provided insight into the biochemical and biophysical properties, indicating that WW45-SARAH exhibits properties of a disordered protein, when the domain was refolded at a neutral pH. Interestingly, WW45-SARAH shows folded and rigid conformations relative to the decrease in pH. Further, diffracting crystals were obtained from protein refolded under acidic pH, suggesting that the refolded WW45 protein at low pH has a homogeneous and stable conformation. A comparative analysis of molecular properties found that the acidic-stable fold of WW45-SARAH enhances a heterotypic interaction with Mst2-SARAH. In addition, using an Mst2 mutation that disrupts homotypic dimerization, we showed that the monomeric Mst2-SARAH domain could form a stable complex of 1:1 stoichiometric ratio with WW45 refolded under acidic pH.

Structure of human PRL-3, the phosphatase associated with cancer metastasis.

PRL-3, a novel class protein of prenylated tyrosine phosphatase, is important in cancer metastasis. Due to its high levels of expression in metastatic tumors, PRL-3 may constitute a useful marker for metastasis and might be a new therapeutic target. Here, we present the solution structure of the phosphatase domain of a human PRL-3 (residues 1-162) in phosphate-free state. The nuclear magnetic resonance (NMR) structure of PRL-3 is similar to that of other known phosphatases with minor differences in the secondary structure. But the conformation and flexibility of the loops comprising the active site differ significantly. When phosphate ions or sodium orthovanadate, which is a known inhibitor, are added to the apo PRL-3, the NMR signals from the residues in the active site appeared and could be assigned, indicating that the conformation of the residues has been stabilized.

Crystallization and preliminary X-ray crystallographic study of human Hikeshi, a new nuclear transport receptor for Hsp70.

Hikeshi is a new nuclear transport receptor that plays an important role in the nuclear import of Hsp70 heat-shock proteins under thermal stress. Wild-type human Hikeshi and its Phe97Ala mutant were overproduced and purified using an Escherichia coli expression system. The purified proteins were crystallized using the hanging-drop vapour-diffusion technique. Wild-type crystals grew in space group C2221, with unit-cell parameters a = 61.1, b = 137.8, c = 97.9 Å, α = 90.0, ß = 90.0, γ = 90.0°. Phe97Ala mutant crystals were obtained in space group P32, with unit-cell parameters a = 85.7, b = 85.7, c = 69.1 Å, α = 90.0, ß = 90.0, γ = 120.0°. These crystals diffracted to 1.8 and 2.5 Šresolution, respectively. This study is the first to yield structural insight into this highly unusual fourth import receptor after importins, NTF2 and TAP.

Structure and interaction of ubiquitin-associated domain of human Fas-associated factor 1.

Fas-associated factor (FAF)-1 is a multidomain protein that was first identified as a member of the Fas death-inducing signaling complex, but later found to be involved in various biological processes. Although the exact mechanisms are not clear, FAF1 seems to play an important role in cancer, asbestos-induced mesotheliomas, and Parkinson's disease. It interacts with polyubiquitinated proteins, Hsp70, and p97/VCP (valosin-containing protein), in addition to the proteins of the Fas-signaling pathway. We have determined the crystal structure of the ubiquitin-associated domain of human FAF1 (hFAF1-UBA) and examined its interaction with ubiquitin and ubiquitin-like proteins using nuclear magnetic resonance. hFAF1-UBA revealed a canonical three-helical bundle that selectively binds to mono- and di-ubiquitin (Lys48-linked), but not to SUMO-1 (small ubiquitin-related modifier 1) or NEDD8 (neural precursor cell expressed, developmentally down-regulated 8). The interaction between hFAF1-UBA and di-ubiquitin involves hydrophobic interaction accompanied by a transition in the di-ubiquitin conformation. These results provide structural insight into the mechanism of polyubiquitin recognition by hFAF1-UBA.