Crystal structures of native and xylosaccharide-bound alkali thermostable xylanase from an alkalophilic Bacillus sp. NG-27: structural insights into alkalophilicity and implications for adaptation to polyextreme conditions

Crystal structures are known for several glycosyl hydrolase family 10 (GH10) xylanases. However, none of them is from an alkalophilic organism that can grow in alkaline conditions. We have determined the crystal structures at 2.2 Angstroms of a GH10 extracellular endoxylanase (BSX) from an alkalophilic Bacillus sp. NG-27, for the native and the complex enzyme with xylosaccharides. The industrially important enzyme is optimally active and stable at 343 K and at a pH of 8.4. Comparison of the structure of BSX with those of other thermostable GH10 xylanases optimally active at acidic or close to neutral pH showed that the solvent-exposed acidic amino acids, Asp and Glu, are markedly enhanced in BSX, while solvent-exposed Asn was noticeably depleted. The BSX crystal structure when compared with putative three-dimensional homology models of other extracellular alkalophilic GH10 xylanases from alkalophilic organisms suggests that a protein surface rich in acidic residues may be an important feature common to these alkali thermostable enzymes. A comparison of the surface features of BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in the polyextremophilicity of proteins, where understanding the structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. The work brings to the fore the role of the nature and composition of solvent-exposed residues in the adaptation of enzymes to polyextreme conditions, as in BSX.     

Design and characterization of modular scaffolds for tubulin assembly

In cells, microtubule dynamics is regulated by stabilizing and destabilizing factors. Whereas proteins in both categories have been identified, their mechanism of action is rarely understood at the molecular level. This is due in part to the difficulties faced in structural approaches to obtain atomic models when tubulin is involved. Here, we design and characterize new stathmin-like domain (SLD) proteins that sequester tubulins in numbers different from two, the number of tubulins bound by stathmin or by the SLD of RB3, two stathmin family members that have been extensively studied. We established rules for the design of tight tubulin-SLD assemblies and applied them to complexes containing one to four tubulin heterodimers. Biochemical and structural experiments showed that the engineered SLDs behaved as expected. The new SLDs will be tools for structural studies of microtubule regulation. The larger complexes will be useful for cryo-electron microscopy, whereas crystallography or nuclear magnetic resonance will benefit from the 1:1 tubulin-SLD assembly. Finally, our results provide new insight into SLD function, suggesting that a major effect of these phosphorylatable proteins is the programmed release of sequestered tubulin for microtubule assembly at the specific cellular locations of members of the stathmin family.     

A new species of Neurocalyx (Rubiaceae) from Peninsular India

A new species of Rubiaceae from the Kalakkad-Mundanthurai Tiger Reserve of Tamil Nadu, India, Neumcalyx bremeri, is described and illustrated. Conservation status assigned as Critically Endangered by applying IUCN Red List Category criteria is provided.     

Elevational gradients of reptile richness in the southern Western Ghats of India: Evaluating spatial and bioclimatic drivers

Exploring elevational patterns in species richness and their underlying mechanisms is a major goal in biogeography and community ecology. Reptiles can be powerful model organisms to examine biogeographical patterns. In this study, we examine the elevational patterns of reptile species richness and test a series of hypotheses that may explain them. We sampled reptile communities along a tropical elevational gradient (100–1,500 m a.s.l.) in the Western Ghats of India using time‐constrained visual encounter surveys at each 100‐m elevation zone for 3 years. First, we investigated species richness patterns across elevation and the support of mid‐domain effect and Rapoport's rule. Second, we tested whether a series of bioclimatic (temperature and tree density) and spatial (mid‐domain effect and area) hypotheses explained species richness. We used linear regression and AICc to compare competing models for all reptiles, and each of the subgroups: snakes, lizards, and Western Ghats’ endemics. Overall reptile richness and lizard richness both displayed linear declines with elevation, which was best explained by temperature. Snake richness and endemic species richness did not systematically vary across elevation, and none of the potential hypotheses explained variation in them. This is the first standardized sampling of reptiles along an elevational gradient in the Western Ghats, and our results agree with the global view that temperature is the primary driver of ectotherm species richness. By establishing strong reptile diversity–temperature associations across elevation, our study also has implications for the impact of future climate change on range‐restricted species in the Western Ghats.     

Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage

How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.     

Mechanism of rapid elimination of lysophosphatidic acid and related lipids from the circulation of mice

Lysophosphatidic acid (LPA) is a bioactive lipid mediator. Concentrations of the major LPA species in mouse plasma decreased uniformly following administration of a potent selective inhibitor of the LPA-generating lysophospholipase D autotaxin, identifying an active mechanism for removal of LPA from the circulation. LPA, akylglycerol phosphate (AGP), sphingosine 1-phosphate (S1P), and a variety of structural mimetics of these lipids, including phosphatase-resistant phosphonate analogs of LPA, were rapidly eliminated (t1/2 < 30 s) from the circulation of mice following intravenous administration of a single bolus dose without significant metabolism in situ in the blood. These lipids accumulated in the liver. Elimination of intravenously administered LPA was blunted by ligation of the hepatic circulation, and ∼90% of LPA administered through the portal vein was accumulated by the isolated perfused mouse liver at first pass. At early times following intravenous administration, more LPA was associated with a nonparenchymal liver cell fraction than with hepatocytes. Primary cultures of nonparenchymal liver cells rapidly assimilated exogenously provided LPA. Our results identify hepatic uptake as an important determinant of the bioavailability of LPA and bioactive lysophospholipid mimetics and suggest a mechanism to explain changes in circulating LPA levels that have been associated with liver dysfunction in humans.     

UV-absorbing bacteria in coral mucus and their response to simulated temperature elevations

Reef-building corals encompass various strategies to defend against harmful ultraviolet (UV) radiation. Coral mucus contains UV-absorbing compounds and has rich prokaryotic diversity associated with it. In this study, we isolated and characterized the UV-absorbing bacteria from the mucus of the corals Porites lutea and Acropora hyacinthus during the pre-summer and summer seasons. A total of 17 UV-absorbing bacteria were isolated and sequenced. The UV-absorbing bacteria showed UV absorption at wavelengths ranging from λ _max = 333 nm to λ _min = 208 nm. Analysis of the DNA sequences revealed that the majority of the UV-absorbing bacteria belonged to the family Firmicutes and the remaining belonged to the family Proteobacteria (class Gammaproteobacteria). Comparison of the sequences with the curated database yielded four distinct bacterial groups belonging to the genus Bacillus, Staphylococcus, Salinicoccus and Vibrio. The absorption peaks for the UV-absorbing bacteria shifted to the UV-A range (320–400 nm) when they were incubated at higher temperatures. Deciphering the complex relationship between corals and their associated bacteria will help us to understand their adaptive strategies to various stresses.     

Glycine decarboxylase activity drives non-small cell lung cancer tumor-initiating cells and tumorigenesis

Identification of the factors critical to the tumor-initiating cell (TIC) state may open new avenues in cancer therapy. Here we show that the metabolic enzyme glycine decarboxylase (GLDC) is critical for TICs in non-small cell lung cancer (NSCLC). TICs from primary NSCLC tumors express high levels of the oncogenic stem cell factor LIN28B and GLDC, which are both required for TIC growth and tumorigenesis. Overexpression of GLDC and other glycine/serine enzymes, but not catalytically inactive GLDC, promotes cellular transformation and tumorigenesis. We found that GLDC induces dramatic changes in glycolysis and glycine/serine metabolism, leading to changes in pyrimidine metabolism to regulate cancer cell proliferation. In the clinic, aberrant activation of GLDC correlates with poorer survival in lung cancer patients, and aberrant GLDC expression is observed in multiple cancer types. This link between glycine metabolism and tumorigenesis may provide novel targets for advancing anticancer therapy.