Crystal structures of interleukin-2 tyrosine kinase and their implications for the design of selective inhibitors

Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fcepsilon. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.     

Pharmacologic inhibition of ALK5 causes selective induction of terminal differentiation in mouse keratinocytes expressing oncogenic HRAS

TGFβ has both tumor suppressive and oncogenic roles in cancer development. We previously showed that SB431542 (SB), a small molecule inhibitor of the TGFβ type I receptor (ALK5) kinase, suppressed benign epidermal tumor formation but enhanced malignant conversion. Here, we show that SB treatment of primary K5rTA/tetORASV12G bitransgenic keratinocytes did not alter HRASV12G-induced keratinocyte hyperproliferation. However, continuous SB treatment significantly enhanced HRASV12G-induced cornified envelope formation and cell death linked to increased expression of enzymes transglutaminase (TGM) 1 and TGM3 and constituents of the cornified envelope small proline-rich protein (SPR) 1A and SPR2H. In contrast, TGFβ1 suppressed cornified envelope formation in HRASV12G keratinocytes. Similar results were obtained in HRASV12G transgenic mice treated topically with SB or by coexpressing TGFβ1 and HRASV12G in the epidermis. Despite significant cell death, SB-resistant HRASV12G keratinocytes repopulated the primary culture that had overcome HRas-induced senescence. These cells expressed reduced levels of p16(ink4a) and were growth stimulated by SB but remained sensitive to a calcium-induced growth arrest. Together these results suggest that differential responsiveness to cornification may represent a mechanism by which pharmacologic blockade of TGFβ signaling can inhibit the outgrowth of preneoplastic lesions but may cause a more progressed phenotype in a separate keratinocyte population.     

VIPR: A probabilistic algorithm for analysis of microbial detection microarrays

Background  

All infectious disease oriented clinical diagnostic assays in use today focus on detecting the presence of a single, well defined target agent or a set of agents. In recent years, microarray-based diagnostics have been developed that greatly facilitate the highly parallel detection of multiple microbes that may be present in a given clinical specimen. While several algorithms have been described for interpretation of diagnostic microarrays, none of the existing approaches is capable of incorporating training data generated from positive control samples to improve performance.     

Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻⁵). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.     

The urban heat island and its impact on heat waves and human health in Shanghai

With global warming forecast to continue into the foreseeable future, heat waves are very likely to increase in both frequency and intensity. In urban regions, these future heat waves will be exacerbated by the urban heat island effect, and will have the potential to negatively influence the health and welfare of urban residents. In order to investigate the health effects of the urban heat island (UHI) in Shanghai, China, 30 years of meteorological records (1975–2004) were examined for 11 first- and second-order weather stations in and around Shanghai. Additionally, automatic weather observation data recorded in recent years as well as daily all-cause summer mortality counts in 11 urban, suburban, and exurban regions (1998–2004) in Shanghai have been used. The results show that different sites (city center or surroundings) have experienced different degrees of warming as a result of increasing urbanization. In turn, this has resulted in a more extensive urban heat island effect, causing additional hot days and heat waves in urban regions compared to rural locales. An examination of summer mortality rates in and around Shanghai yields heightened heat-related mortality in urban regions, and we conclude that the UHI is directly responsible, acting to worsen the adverse health effects from exposure to extreme thermal conditions.     

Aboveground resource allocation in response to root herbivory as affected by the arbuscular mycorrhizal symbiosis

Aims

Arbuscular mycorrhizal (AM) fungi associate with the majority of terrestrial plants, influencing their growth, nutrient uptake and defence chemistry. Consequently, AM fungi can significantly impact plant-herbivore interactions, yet surprisingly few studies have investigated how AM fungi affect plant responses to root herbivores. This study aimed to investigate how AM fungi affect plant tolerance mechanisms to belowground herbivory.

Methods

We examined how AM fungi affect plant (Saccharum spp. hybrid) growth, nutrient dynamics and secondary chemistry (phenolics) in response to attack from a root-feeding insect (Dermolepida albohirtum).

Results

Root herbivory reduced root mass by almost 27%. In response, plants augmented investment in aboveground biomass by 25%, as well as increasing carbon concentrations. The AM fungi increased aboveground biomass, phosphorus and carbon. Meanwhile, root herbivory increased foliar phenolics by 31% in mycorrhizal plants, and increased arbuscular colonisation of roots by 75% overall. AM fungi also decreased herbivore performance, potentially via increasing root silicon concentrations.

Conclusions

Our results suggest that AM fungi may be able to augment plant tolerance to root herbivory via resource allocation aboveground and, at the same time, enhance plant root resistance by increasing root silicon. The ability of AM fungi to facilitate resource allocation aboveground in this way may be a more widespread strategy for plants to cope with belowground herbivory.

     

Hydrophobic and Basic Domains Target Proteins to Lipid Droplets

In recent years, progress in the study of the lateral organization of the plasma membrane has led to the proposal that mammalian cells use two different organelles to store lipids: intracellular lipid droplets (LDs) and plasma membrane caveolae. Experimental evidence suggests that caveolin (CAV) may act as a sensitive lipid-organizing molecule that physically connects these two lipid-storing organelles. Here, we determine the sequences necessary for efficient sorting of CAV to LDs. We show that targeting is a process cooperatively mediated by two motifs. CAV's central hydrophobic domain (Hyd) anchors CAV to the endoplasmic reticulum (ER). Next, positively charged sequences (Pos-Seqs) mediate sorting of CAVs into LDs. Our findings were confirmed by identifying an equivalent, non-conserved but functionally interchangeable Pos-Seq in ALDI, a bona fide LD-resident protein. Using this information, we were able to retarget a cytosolic protein and convert it to an LD-resident protein. Further studies suggest three requirements for targeting via this mechanism: the positive charge of the Pos-Seq, physical proximity between Pos-Seq and Hyd and a precise spatial orientation between both motifs. The study uncovers remarkable similarities with the signals that target proteins to the membrane of mitochondria and peroxisomes     

FTY720-enhanced T cell homing is dependent on CCR2, CCR5, CCR7, and CXCR4: evidence for distinct chemokine compartments

FTY720 stimulates CCR7-driven T cell homing to peripheral lymph nodes (LN) by direct activation of sphingosine 1-phosphate receptors, along with the participation of multidrug transporters, 5-lipoxygenase, and G protein-coupled receptors for chemokines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell chemotaxis to CCR2-CCL2, but not to a variety of other chemokines, including CCR5-CCL3/4/5 and CXCR4-CXCL12. FTY720 influences CCR2-CCL2-driven migration through activation of the multidrug transporters, Abcb1 and Abcc1, and through 5-lipoxygenase activity. In vivo administration of FTY720 induces chemokine-dependent migration of T cells in the thymus, peripheral blood, LN, and spleen. The CCR7 and CCR2 chemokine ligands are required for both T cell sequestration in LN and thymic T cell egress following FTY720 administration. Furthermore, FTY720 administration uncovers a requirement for CXCR4 ligands for LN homing, but not for thymic egress, and CCR5 for thymic egress, but not LN homing. FTY720-driven splenic and peripheral blood T cell egress are both independent of CCR2, CCR5, CCR7, or CXCR4. These results indicate that FTY720- and sphingosine 1-phosphate receptor-stimulated T cell migration are dependent on the restricted usage of chemokine receptor-ligand pairs within discrete anatomic compartments.     

Importin alpha: a multipurpose nuclear-transport receptor

The importin alpha/beta heterodimer targets hundreds of proteins to the nuclear-pore complex (NPC) and facilitates their translocation across the nuclear envelope. Importin alpha binds to classical nuclear localization signal (cNLS)-containing proteins and links them to importin beta, the karyopherin that ferries the ternary complex through the NPC. A second karyopherin, the exportin CAS, recycles importin alpha back to the cytoplasm. In this article, we discuss control mechanisms that importin alpha exerts over the assembly and disassembly of the ternary complex and we describe how new groups of importin alpha genes arose during the evolution of metazoan animals to function in development and differentiation. We also describe activities of importin alpha that seem to be distinct from its housekeeping functions in nuclear transport.     

NAR Breakthrough Article: Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1

The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2′,5′-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2′,5′-phosphodiester recognition and explain why the enzyme lacks activity toward 3′,5′-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.