Gout based on hyperuricemia is a common disease with a genetic predisposition, which causes acute arthritis. The ABCG2/BCRP gene, located in a gout-susceptibility locus on chromosome 4q, has been identified by recent genome-wide association studies of serum uric acid concentrations and gout. Urate transport assays demonstrated that ABCG2 is a high-capacity urate secretion transporter. Sequencing of the ABCG2 gene in 90 hyperuricemia patients revealed several nonfunctional ABCG2 mutations, including Q126X. Quantitative trait locus analysis of 739 individuals showed that a common dysfunctional variant of ABCG2, Q141K, increases serum uric acid. Q126X is assigned to the different disease haplotype from Q141K and increases gout risk, conferring an odds ratio of 5.97. Furthermore, 10% of gout patients (16 out of 159 cases) had genotype combinations resulting in more than 75% reduction of ABCG2 function (odds ratio, 25.8). Our findings indicate that nonfunctional variants of ABCG2 essentially block gut and renal urate excretion and cause gout.

The oomycete pathogen Phytophthora infestans causes potato late blight, one of the most economically damaging plant diseases worldwide. P. infestans produces AVR3a, an essential modular virulence effector with an N-terminal RXLR domain that is required for host-cell entry. In host cells, AVR3a stabilizes and inhibits the function of the E3 ubiquitin ligase CMPG1, a key factor in host immune responses including cell death triggered by the pathogen-derived elicitor protein INF1 elicitin. To elucidate the molecular basis of AVR3a effector function, we determined the structure of Phytophthora capsici AVR3a4, a close homolog of P. infestans AVR3a. Our structural and functional analyses reveal that the effector domain of AVR3a contains a conserved, positively charged patch and that this region, rather than the RXLR domain, is required for binding to phosphatidylinositol monophosphates (PIPs) in vitro. Mutations affecting PIP binding do not abolish AVR3a recognition by the resistance protein R3a but reduce its ability to suppress INF1-triggered cell death in planta. Similarly, stabilization of CMPG1 in planta is diminished by these mutations. The steadystate levels of non-PIP-binding mutant proteins in planta are reduced greatly, although these proteins are stable in vitro. Furthermore, overexpression of a phosphatidylinositol phosphate 5-kinase results in reduction of AVR3a levels in planta. Our results suggest that the PIP-binding ability of the AVR3a effector domain is essential for its accumulation inside host cells to suppress CMPG1-dependent immunity.R protein | NMR | programmed cell death

The zinc finger CW (zf-CW) domain is a motif of about 60 residues that is frequently found in proteins involved in epigenetic regulation. Here, we determined the NMR solution structure of the zf-CW domain of the human zf-CW and PWWP domain containing protein 1 (ZCWPW1). The zf-CW domain adopts a new fold in which a zinc ion is coordinated tetrahedrally by four conserved Cys ligand residues. The tertiary structure of the zf-CW domain partially resembles that adopted by the plant homeo domain (PHD) finger bound to the histone tail, suggesting that the zf-CW domain and the PHD finger have similar functions. The solution structure of the complex of the zf-CW domain with the histone H3 tail peptide (1-10) with trimethylated K4 clarified its binding mode. Our structural and biochemical studies have identified the zf-CW domain as a member of the histone modification reader modules for epigenetic regulation.

The WRKY family transcription factors regulate plant-specific reactions that are mostly related to biotic and abiotic stresses. They share the WRKY domain, which recognizes a DNA element (TTGAC(C/T)) termed the W-box, in target genes. Here, we determined the solution structure of the C-terminal WRKY domain of Arabidopsis WRKY4 in complex with the W-box DNA by NMR. A four-stranded β-sheet enters the major groove of DNA in an atypical mode termed the β-wedge, where the sheet is nearly perpendicular to the DNA helical axis. Residues in the conserved WRKYGQK motif contact DNA bases mainly through extensive apolar contacts with thymine methyl groups. The importance of these contacts was verified by substituting the relevant T bases with U and by surface plasmon resonance analyses of DNA binding.

In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology.

Rhesus TRIM5␣ (TRIM5␣ rh ) is a cytosolic protein that potently restricts HIV-1 at an early postentry stage, prior to reverse transcription. The ability of TRIM5␣ rh to block HIV-1 infection has been correlated with a decrease of pelletable HIV-1 capsid during infection. To genetically dissect the ability of TRIM5␣ to block reverse transcription, we studied a set of TRIM5␣ rh RING domain mutants that potently restrict HIV-1 but allow the occurrence of reverse transcription. These TRIM5␣ rh RING variants blocked HIV-1 infection after reverse transcription but prior to integration, as suggested by the routing of nuclear viral DNA to circularization in the form of 2-long terminal repeat (2-LTR) circles. The folding of RING domain variants was similar to that of the wild type, as evaluated by nuclear magnetic resonance. RING domain changes that allowed the occurrence of reverse transcription were impaired in their ability to decrease the amount of pelletable capsid compared with wild-type TRIM5␣. Similar effects of this particular group of mutations were observed with human TRIM5␣ inhibition of N-tropic murine leukemia virus (N-MLV). Interestingly, TRIM5␣ rh RING domain variants also prevented the degradation of TRIM5␣ rh that occurs following cell entry of HIV-1. These data correlated the block of reverse transcription with the ability of TRIM5␣ to accelerate uncoating. Collectively, these results suggest that TRIM5␣ rh blocks HIV-1 reverse transcription by inducing premature viral uncoating in target cells. Several newly discovered proteins endogenously expressed in primates show the ability to dominantly block retroviral infection and cross-species transmission by interfering with the early phase of viral replication (27,46,51). Of particular interest are members of the tripartite motif (TRIM) family of proteins (43). The splicing variant alpha of TRIM5 from rhesus macaque (TRIM5␣ rh ) is an ϳ53-kDa cytosolic protein that potently restricts HIV-1 (24, 49). TRIM5␣ rh blocks HIV-1 and certain other retroviruses soon after viral entry but prior to reverse transcription (24, 51). The retroviral capsid protein (CA) is the viral determinant for susceptibility to restriction by TRIM5␣ (38). Studies on the fate of the HIV-1 capsid in the cytosol of infected cells have correlated restriction with a decreased amount of cytosolic particulate capsid (10, 13, 41, 52), suggesting that TRIM5␣ rh acts by inducing premature uncoating in target cells.TRIM5␣ rh is composed of four distinct domains: RING, B-box 2, coiled-coil, and B30.2(SPRY) (43). The RING domain of TRIM5␣ rh is an E3 ubiquitin ligase (12,23,26,28,31,32,57). The E3 ligase activity of TRIM5␣ rh correlates with the ability of TRIM5␣ rh to block HIV-1 (31). The B-box 2 domain of TRIM5␣ rh and other TRIM proteins, such as TRIM63, self-associates into dimeric complexes that are important for TRIM5␣ higher-order self-association (HOSA) and capsid binding avidity; these B-box 2 domain functions are essential for full and potent restriction of 14,20,22,34,39). The coiled-c...

Human Transformer2-β (hTra2-β) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-β specifically binds to two types of RNA sequences [the CAA and (GAA)2 sequences]. We determined the solution structure of the hTra2-β RRM (spanning residues Asn110–Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the β-sheet surface. We then solved the complex structure of the hTra2-β RRM with the (GAA)2 sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the β-sheet surface. Further NMR experiments revealed that the hTra2-β RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-β RRM recognizes two types of RNA sequences in different RNA binding modes.

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