Research Centre in Biological Chemistry of Córdoba

AUTORES: Daniel J Klionsky1745,1749*, Kotb Abdelmohsen840, Akihisa Abe1237, Md Joynal Abedin1762, Hagai Abeliovich425,
\nAbraham Acevedo Arozena789, Hiroaki Adachi1800, Christopher M Adams1669, Peter D Adams57, Khosrow Adeli1981,
\nPeter J Adhihetty1625, Sharon G Adler700, Galila Agam67, Rajesh Agarwal1587, Manish K Aghi1537, Maria Agnello1826,
\nPatrizia Agostinis664, Patricia V Aguilar1960, Julio Aguirre-Ghiso784,786, Edoardo M Airoldi89,422, Slimane Ait-Si-Ali1376,
\nTakahiko Akematsu2010, Emmanuel T Akporiaye1097, Mohamed Al-Rubeai1394, Guillermo M Albaiceta1294,
\nChris Albanese363, Diego Albani561, Matthew L Albert517, Jesus Aldudo128, Hana Alg€ul1164, Mehrdad Alirezaei1198,
\nIraide Alloza642,888, Alexandru Almasan206, Maylin Almonte-Beceril524, Emad S Alnemri1212, Covadonga Alonso544,
\nNihal Altan-Bonnet848, Dario C Altieri1205, Silvia Alvarez1497, Lydia Alvarez-Erviti1395, Sandro Alves107,
\nGiuseppina Amadoro860, Atsuo Amano930, Consuelo Amantini1554, Santiago Ambrosio1458, Ivano Amelio756,
\nAmal O Amer918, Mohamed Amessou2089, Angelika Amon726, Zhenyi An1538, Frank A Anania291, Stig U Andersen6,
\nUsha P Andley2079, Catherine K Andreadi1690, Nathalie Andrieu-Abadie502, Alberto Anel2027, David K Ann58,
\nShailendra Anoopkumar-Dukie388, Manuela Antonioli832,858, Hiroshi Aoki1791, Nadezda Apostolova2007,
\nSaveria Aquila1500, Katia Aquilano1876, Koichi Araki292, Eli Arama2098, Agustin Aranda456, Jun Araya591,
\nAlexandre Arcaro1472, Esperanza Arias26, Hirokazu Arimoto1225, Aileen R Ariosa1749, Jane L Armstrong1930,
\nThierry Arnould1773, Ivica Arsov2120, Katsuhiko Asanuma675, Valerie Askanas1924, Eric Asselin1867, Ryuichiro Atarashi794,
\nSally S Atherton369, Julie D Atkin713, Laura D Attardi1131, Patrick Auberger1787, Georg Auburger379, Laure Aurelian1727,
\nRiccardo Autelli1992, Laura Avagliano1029,1755, Maria Laura Avantaggiati364, Limor Avrahami1166, Suresh Awale1986,
\nNeelam Azad404, Tiziana Bachetti568, Jonathan M Backer28, Dong-Hun Bae1933, Jae-sung Bae677, Ok-Nam Bae409,
\nSoo Han Bae2117, Eric H Baehrecke1729, Seung-Hoon Baek17, Stephen Baghdiguian1368,
\nAgnieszka Bagniewska-Zadworna2, Hua Bai90, Jie Bai667, Xue-Yuan Bai1133, Yannick Bailly884,
\nKithiganahalli Narayanaswamy Balaji473, Walter Balduini2002, Andrea Ballabio316, Rena Balzan1711, Rajkumar Banerjee239,
\nG abor B anhegyi1052, Haijun Bao2109, Benoit Barbeau1363, Maria D Barrachina2007, Esther Barreiro467, Bonnie Bartel997,
\nAlberto Bartolom e222, Diane C Bassham550, Maria Teresa Bassi1046, Robert C Bast Jr1273, Alakananda Basu1798,
\nMaria Teresa Batista1578, Henri Batoko1336, Maurizio Battino970, Kyle Bauckman2085, Bradley L Baumgarner1909,
\nK Ulrich Bayer1594, Rupert Beale1553, Jean-Fran¸cois Beaulieu1360, George R. Beck Jr48,294, Christoph Becker336,
\nJ David Beckham1595, Pierre-Andr e B edard749, Patrick J Bednarski301, Thomas J Begley1135, Christian Behl1419,
\nChristian Behrends757, Georg MN Behrens406, Kevin E Behrns1627, Eloy Bejarano26, Amine Belaid490,
\nFrancesca Belleudi1041, Giovanni B enard497, Guy Berchem706, Daniele Bergamaschi983, Matteo Bergami1401,
\nBen Berkhout1441, Laura Berliocchi714, Am elie Bernard1749, Monique Bernard1354, Francesca Bernassola1880,
\nAnne Bertolotti791, Amanda S Bess272, S ebastien Besteiro1351, Saverio Bettuzzi1828, Savita Bhalla913,
\nShalmoli Bhattacharyya973, Sujit K Bhutia838, Caroline Biagosch1159, Michele Wolfe Bianchi520,1378,1381,
\nMartine Biard-Piechaczyk210, Viktor Billes298, Claudia Bincoletto1314, Baris Bingol350, Sara W Bird1128, Marc Bitoun1112,
\nIvana Bjedov1258, Craig Blackstone843, Lionel Blanc1183, Guillermo A Blanco1496, Heidi Kiil Blomhoff1812,
\nEmilio Boada-Romero1297, Stefan B€ockler1464, Marianne Boes1423, Kathleen Boesze-Battaglia1835, Lawrence H Boise286,287,
\nAlessandra Bolino2063, Andrea Boman693, Paolo Bonaldo1823, Matteo Bordi897, J€urgen Bosch608, Luis M Botana1308,
\nJoelle Botti1375, German Bou1405, Marina Bouch e1038, Marion Bouchecareilh1331, Marie-Jos ee Boucher1901,
\nMichael E Boulton481, Sebastien G Bouret1926, Patricia Boya133, Micha€el Boyer-Guittaut1345, Peter V Bozhkov1141,
\nNathan Brady374, Vania MM Braga469, Claudio Brancolini1997, Gerhard H Braus353, Jos e M Bravo-San Pedro299,393,508,1374,
\nLisa A Brennan322, Emery H Bresnick2022, Patrick Brest490, Dave Bridges1939, Marie-Agn es Bringer124, Marisa Brini1822,
\nGlauber C Brito1311, Bertha Brodin631, Paul S Brookes1872, Eric J Brown352, Karen Brown1690, Hal E Broxmeyer480,
\nAlain Bruhat486,1339, Patricia Chakur Brum1893, John H Brumell446, Nicola Brunetti-Pierri315,1171,
\nRobert J Bryson-Richardson781, Shilpa Buch1777, Alastair M Buchan1819, Hikmet Budak1022, Dmitry V Bulavin118,505,1789,
\nScott J Bultman1792, Geert Bultynck665, Vladimir Bumbasirevic1470, Yan Burelle1356, Robert E Burke216,217,
\nMargit Burmeister1750, Peter B€utikofer1473, Laura Caberlotto1987, Ken Cadwell896, Monika Cahova112, Dongsheng Cai24,
\nJingjing Cai2099, Qian Cai1018, Sara Calatayud2007, Nadine Camougrand1343, Michelangelo Campanella1700,
\nGrant R Campbell1525, Matthew Campbell1249, Silvia Campello556,1876, Robin Candau1769, Isabella Caniggia1983,
\nLavinia Cantoni560, Lizhi Cao116, Allan B Caplan1656, Michele Caraglia1051, Claudio Cardinali1043, Sandra Morais Cardoso1579, Jennifer S Carew208, Laura A Carleton874, Cathleen R Carlin101, Silvia Carloni2002,
\nSven R Carlsson1267, Didac Carmona-Gutierrez1643, Leticia AM Carneiro312, Oliana Carnevali971, Serena Carra1318,
\nAlice Carrier120, Bernadette Carroll900, Caty Casas1324, Josefina Casas1116, Giuliana Cassinelli324, Perrine Castets1462,
\nSusana Castro-Obregon214, Gabriella Cavallini1841, Isabella Ceccherini568, Francesco Cecconi253,555,1884,
\nArthur I Cederbaum459, Valent ın Ce~na199,1281, Simone Cenci1323,2064, Claudia Cerella444, Davide Cervia1996,
\nSilvia Cetrullo1478, Hassan Chaachouay2028, Han-Jung Chae187, Andrei S Chagin634, Chee-Yin Chai626,628,
\nGopal Chakrabarti1502, Georgios Chamilos1601, Edmond YW Chan1142, Matthew TV Chan181, Dhyan Chandra1003,
\nPallavi Chandra548, Chih-Peng Chang818, Raymond Chuen-Chung Chang1653, Ta Yuan Chang345, John C Chatham1434,
\nSaurabh Chatterjee1910, Santosh Chauhan527, Yongsheng Che62, Michael E Cheetham1263, Rajkumar Cheluvappa1783,
\nChun-Jung Chen1153, Gang Chen598,1676, Guang-Chao Chen9, Guoqiang Chen1078, Hongzhuan Chen1077, Jeff W Chen1514,
\nJian-Kang Chen370,371, Min Chen249, Mingzhou Chen2104, Peiwen Chen1823, Qi Chen1674, Quan Chen172,
\nShang-Der Chen138, Si Chen325, Steve S-L Chen10, Wei Chen2125, Wei-Jung Chen829, Wen Qiang Chen979, Wenli Chen1113,
\nXiangmei Chen1133, Yau-Hung Chen1157, Ye-Guang Chen1250, Yin Chen1447, Yingyu Chen953,955, Yongshun Chen2135,
\nYu-Jen Chen712, Yue-Qin Chen1145, Yujie Chen1208, Zhen Chen339, Zhong Chen2123, Alan Cheng1702,
\nChristopher HK Cheng184, Hua Cheng1728, Heesun Cheong814, Sara Cherry1836, Jason Chesney1703,
\nChun Hei Antonio Cheung817, Eric Chevet1359, Hsiang Cheng Chi140, Sung-Gil Chi656, Fulvio Chiacchiera308,
\nHui-Ling Chiang958, Roberto Chiarelli1826, Mario Chiariello235,567,577, Marcello Chieppa835, Lih-Shen Chin290,
\nMario Chiong1285, Gigi NC Chiu878, Dong-Hyung Cho676, Ssang-Goo Cho650, William C Cho982, Yong-Yeon Cho105,
\nYoung-Seok Cho1064, Augustine MK Choi2095, Eui-Ju Choi656, Eun-Kyoung Choi387,400,685, Jayoung Choi1563,
\nMary E Choi2093, Seung-Il Choi2116, Tsui-Fen Chou412, Salem Chouaib395, Divaker Choubey1574, Vinay Choubey1936,
\nKuan-Chih Chow822, Kamal Chowdhury730, Charleen T Chu1856, Tsung-Hsien Chuang827, Taehoon Chun657,
\nHyewon Chung652, Taijoon Chung978, Yuen-Li Chung1194, Yong-Joon Chwae18, Valentina Cianfanelli254,
\nRoberto Ciarcia1775, Iwona A Ciechomska886, Maria Rosa Ciriolo1876, Mara Cirone1042, Sofie Claerhout1694,
\nMichael J Clague1698, Joan Cl aria1457, Peter GH Clarke1687, Robert Clarke361, Emilio Clementi1045,1398, C edric Cleyrat1781,
\nMiriam Cnop1366, Eliana M Coccia574, Tiziana Cocco1459, Patrice Codogno1375, J€orn Coers271, Ezra EW Cohen1533,
\nDavid Colecchia235,567,577, Luisa Coletto25, N uria S Coll123, Emma Colucci-Guyon516, Sergio Comincini1829,
\nMaria Condello578, Katherine L Cook2073, Graham H Coombs1929, Cynthia D Cooper2076, J Mark Cooper1395,
\nIsabelle Coppens601, Maria Tiziana Corasaniti1387, Marco Corazzari485,1884, Ramon Corbalan1566,
\nElisabeth Corcelle-Termeau251, Mario D Cordero1899, Cristina Corral-Ramos1289, Olga Corti507,1109, Andrea Cossarizza1767,
\nPaola Costelli1993, Safia Costes1518, Susan L Cotman721, Ana Coto-Montes946, Sandra Cottet566,1688, Eduardo Couve1301,
\nLori R Covey1015, L Ashley Cowart762, Jeffery S Cox1536, Fraser P Coxon1427, Carolyn B Coyne1846, Mark S Cragg1919,
\nRolf J Craven1679, Tiziana Crepaldi1995, Jose L Crespo1300, Alfredo Criollo1285, Valeria Crippa558, Maria Teresa Cruz1576,
\nAna Maria Cuervo26, Jose M Cuezva1277, Taixing Cui1907, Pedro R Cutillas987, Mark J Czaja27, Maria F Czyzyk-Krzeska1572,
\nRuben K Dagda2068, Uta Dahmen1404, Chunsun Dai800, Wenjie Dai1187, Yun Dai2059, Kevin N Dalby1940,
\nLuisa Dalla Valle1822, Guillaume Dalmasso1340, Marcello D’Amelio557, Markus Damme188, Arlette Darfeuille-Michaud1340,
\nCatherine Dargemont950, Victor M Darley-Usmar1433, Srinivasan Dasarathy205, Biplab Dasgupta202, Srikanta Dash1254,
\nCrispin R Dass242, Hazel Marie Davey8, Lester M Davids1560, David D avila227, Roger J Davis1731, Ted M Dawson604,
\nValina L Dawson606, Paula Daza1898, Jackie de Belleroche470, Paul de Figueiredo1180,1182,
\nRegina Celia Bressan Queiroz de Figueiredo135, Jos e de la Fuente1023, Luisa De Martino1775,
\nAntonella De Matteis1171, Guido RY De Meyer1443, Angelo De Milito631, Mauro De Santi2002,

Gangliosides are sialic acid-containing glycosphingolipids that are present on all mammalian plasma membranes where they participate in recognition and signaling activities. We have established mutant mice that lack GM3 synthase (CMP-NeuAc:lactosylceramide alpha2,3-sialyltransferase; EC 2.4.99.-). These mutant mice were unable to synthesize GM3 ganglioside, a simple and widely distributed glycosphingolipid. The mutant mice were viable and appeared without major abnormalities but showed a heightened sensitivity to insulin. A basis for the increased insulin sensitivity in the mutant mice was found to be enhanced insulin receptor phosphorylation in skeletal muscle. Importantly, the mutant mice were protected from high-fat diet-induced insulin resistance. Our results show that GM3 ganglioside is a negative regulator of insulin signaling, making it a potential therapeutic target in type 2 diabetes.

The microtubule-associated protein (MAP) tau is abnormally hyperphosphorylated in Alzheimer disease and accumulates in neurons undergoing neurofibrillary degeneration. In the present study, the associations of the Alzheimer-hyperphosphorylated tau (AD P-tau) with the high molecular weight MAPs (HMW-MAPs) MAP1 and MAP2 were investigated. The AD P-tau was found to aggregate with MAP1 and MAP2 in solution. The association of AD P-tau to the MAPs resulted in inhibition of MAP-promoted microtubule assembly. However, unlike the coaggregation of AD P-tau and normal tau, the association between AD P-tau and the HMW-MAPs did not result in the formation of filaments/tangles. The affinity of the tau-AD P-tau association was higher than that of HMW-MAPs-AD P-tau because normal tau inhibited the latter binding. The association between AD P-tau and the HMW-MAPs also appeared to occur in situ because these proteins cosedimented from the Alzheimer brain extracts, and, in the sediment, the levels of the HMW-MAPs correlated with the levels of AD P-tau. These studies suggested that the abnormally phosphorylated tau can sequester both normal tau and HMW-MAPs and disassemble microtubules but, under physiological conditions, can form tangles of filaments only from tau.

Abstract The ability of CD8+ T lymphocytes to eliminate tumors is limited by their ability to engender an immunosuppressive microenvironment. Here we describe a subset of tumor-infiltrating CD8+ T cells marked by high expression of the immunosuppressive ATP ecto-nucleotidase CD39. The frequency of CD39highCD8+ T cells increased with tumor growth but was absent in lymphoid organs. Tumor-infiltrating CD8+ T cells with high CD39 expression exhibited features of exhaustion, such as reduced production of TNF and IL2 and expression of coinhibitory receptors. Exhausted CD39+CD8+ T cells from mice hydrolyzed extracellular ATP, confirming that CD39 is enzymatically active. Furthermore, exhausted CD39+CD8+ T cells inhibited IFNγ production by responder CD8+ T cells. In specimens from breast cancer and melanoma patients, CD39+CD8+ T cells were present within tumors and invaded or metastatic lymph nodes, but were barely detectable within noninvaded lymph nodes and absent in peripheral blood. These cells exhibited an exhausted phenotype with impaired production of IFNγ, TNF, IL2, and high expression of coinhibitory receptors. Although T-cell receptor engagement was sufficient to induce CD39 on human CD8+ T cells, exposure to IL6 and IL27 promoted CD39 expression on stimulated CD8+ T cells from human or murine sources. Our findings show how the tumor microenvironment drives the acquisition of CD39 as an immune regulatory molecule on CD8+ T cells, with implications for defining a biomarker of T-cell dysfunction and a target for immunotherapeutic intervention. Significance: The tumor microenvironment elicits a subset of functionally exhausted CD8+ T cells by creating conditions that induce cell surface expression of CD39, an immunosuppressive molecule that can be therapeutically targeted to restore effector T-cell function. Cancer Res; 78(1); 115–28. ©2017 AACR.

Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controls that are influenced by redox regulation. Here we show that the Arabidopsis (Arabidopsis thaliana) low vitamin C mutants, vtc1 and vtc2, which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR) proteins, and have enhanced basal resistance against infections caused by Pseudomonas syringae. The mutants have a delayed senescence phenotype with smaller leaf cells than the wild type at maturity. The vtc leaves have more glutathione than the wild type, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused to the nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants. Fluorescence was observed in the nuclei of 6- to 8-week-old GFP-NPR1 vtc1 plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the time when constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in vtc plants does not result from activation of early senescence. Moreover, H2O2-sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold for activation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program.

Accumulation of free L-proline (Pro) is a typical stress response incited by osmotic injuries in plants and microorganisms. Although the protective role of Pro in osmotic stress is not well understood, it is thought to function as compatible osmolyte or as a scavenger of reactive oxygen species (ROS). Here we show that, in Arabidopsis thaliana, Pro biosynthesis can be activated by incompatible plant-pathogen interactions triggering a hypersensitive response (HR). Pro accumulates in leaf tissues treated with Pseudomonas syringae pv. tomato avirulent strains (avrRpt2 and avrRpm1) but remains unchanged in leaves infected with isogenic virulent bacteria. Incompatible interactions lead to transcriptional activation of AtP5CS2, but not AtP5CS1, encoding the rate limiting enzyme in Pro biosynthesis pyrroline-5-carboxylate synthase (P5CS). AtP5CS2:GUS and AtP5CS2:LUC transgenes were induced inside and around the HR lesions produced by avirulent Pseudomonas spp. in transgenic plants. Pro accumulation was faster and stronger when stimulated by avrRpm1 than by avrRpt2, and was compromised in the low-salicylic acid plants NahG and eds5 when signaled through the RPS2-dependent pathway. In addition, Pro content and AtP5CS2 expression were enhanced by ROS in wild-type plants, suggesting that ROS may function as an intermediate signal in AtP5CS2-mediated Pro accumulation.

The interaction between ligands and proteins usually induces changes in protein thermal stability with modifications in the midpoint denaturation temperature, enthalpy of unfolding, and heat capacity. These modifications are due to the coupling of unfolding with binding equilibrium. Furthermore, they can be attained by changes in protein structure and conformational flexibility induced by ligand interaction. To study these effects we have used bovine serum albumin (BSA) interacting with three different anilinonaphthalene sulfonate derivatives (ANS). These ligands have different effects on protein stability, conformation, and dynamics. Protein stability was studied by differential scanning calorimetry and fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism and infrared spectroscopy including kinetics of hydrogen/deuterium exchange. The order of calorimetric midpoint of denaturation was: 1,8-ANS-BSA > 2,6-ANS-BSA > free BSA >> (nondetected) bis-ANS-BSA. Both 1,8-ANS and 2,6-ANS did not substantially modify the secondary structure of BSA, whereas bis-ANS induced a distorted alpha-helix conformation with an increase of disordered structure. Protein flexibility followed the order: 1,8-ANS-BSA < 2,6-ANS-BSA < free BSA << bis-ANS-BSA, indicating a clear correlation between stability and conformational flexibility. The structure induced by an excess of bis-ANS to BSA is compatible with a molten globule-like state. Within the context of the binding landscape model, we have distinguished five conformers (identified by subscript): BSA(1,8-ANS), BSA(2,6-ANS), BSA(free), BSA(bis-ANS), and BSA(unfolded) among the large number of possible states of the conformational dynamic ensemble. The relative population of each distinguishable conformer depends on the type and concentration of ligand and the temperature of the system.

Parkinson's disease is an age-related movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-amino-acid protein AS (α-synuclein). AS fibrillation follows a nucleation polymerization pathway involving diverse transient prefibrillar species varying in size and morphology. Similar to other neurodegenerative diseases, cytotoxicity is currently attributed to these prefibrillar species rather than to the insoluble aggregates. Nevertheless, the underlying molecular mechanisms responsible for cytotoxicity remain elusive and structural studies may contribute to the understanding of both the amyloid aggregation mechanism and oligomer-induced toxicity. It is already recognized that soluble oligomeric AS species adopt β-sheet structures that differ from those characterizing the fibrillar structure. In the present study we used ATR (attenuated total reflection)-FTIR (Fourier-transform infrared) spectroscopy, a technique especially sensitive to β-sheet structure, to get a deeper insight into the β-sheet organization within oligomers and fibrils. Careful spectral analysis revealed that AS oligomers adopt an antiparallel β-sheet structure, whereas fibrils adopt a parallel arrangement. The results are discussed in terms of regions of the protein involved in the early β-sheet interactions and the implications of such conformational arrangement for the pathogenicity associated with AS oligomers.

AIMS: This work was conducted to identify the antifungal compounds produced by two previously isolated Bacillus sp. strains: ARP(2) 3 and MEP(2) 18. Both strains were subjected to further analysis to determine their taxonomic position and to identify the compounds responsible for their antifungal activity as well as to evaluate the efficiency of these strains to control sclerotinia stem rot in soybean. METHODS AND RESULTS: The antifungal compounds were isolated by acid precipitation of cell-free supernatants, purified by RP-HPLC and then tested for antagonistic activity against Sclerotinia sclerotiorum. Mass spectra from RP-HPLC eluted fractions showed the presence of surfactin C(15) , fengycins A (C(16) -C(17)) and B (C(16)) isoforms in supernatants from strain ARP(2) 3 cultures, whereas the major lipopeptide produced by strain MEP(2) 18 was iturin A C(15) . Alterations in mycelial morphology and sclerotial germination were observed in the presence of lipopeptides-containing supernatants from Bacillus strains cultures. Foliar application of Bacillus amyloliquefaciens strains on soybean plants prior to S. sclerotiorum infection resulted in significant protection against sclerotinia stem rot compared with noninoculated plants or plants inoculated with a nonlipopeptide-producing B. subtilis strain. CONCLUSIONS: Both strains, renamed as B. amyloliquefaciens ARP(2) 3 and MEP(2) 18, were able to produce antifungal compounds belonging to the cyclic lipopeptide family. Our data suggest that the foliar application of lipopeptide-producing B. amyloliquefaciens strains could be a promising strategy for the management of sclerotinia stem rot in soybean. SIGNIFICANCE AND IMPACT OF THE STUDY: Sclerotinia stem rot was ranked as one of the most severe soybean disease in Argentina and worldwide. The results of this study showed the potential of B. amyloliquefaciens strains ARP(2) 3 and MEP(2) 18 to control plant diseases caused by S. sclerotiorum.

The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature Tm, of thermally induced denaturation of STV from 75 degrees C in unliganded protein to 112 degrees C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a Ka approximately 10(15) reproduce the behavior observed, but the estimated values of Tm are 15-20 degrees C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions. The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability.

L-proline (Pro) catabolism is activated in plants recovering from abiotic stresses associated with water deprivation. In this catabolic pathway, Pro is converted to glutamate by two reactions catalyzed by proline dehydrogenase (ProDH) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH), with Δ(1)-pyrroline-5-carboxylate (P5C) as the intermediate. Alternatively, under certain conditions, the P5C derived from Pro is converted back to Pro by P5C reductase, thus stimulating the Pro-P5C cycle, which may generate reactive oxygen species (ROS) as a consequence of the ProDH activity. We previously observed that Pro biosynthesis is altered in Arabidopsis (Arabidopsis thaliana) tissues that induce the hypersensitive response (HR) in response to Pseudomonas syringae. In this work, we characterized the Pro catabolic pathway and ProDH activity in this model. Induction of ProDH expression was found to be dependent on salicylic acid, and an increase in ProDH activity was detected in cells destined to die. To evaluate the role of ProDH in the HR, ProDH-silenced plants were generated. These plants displayed reduced ROS and cell death levels as well as enhanced susceptibility in response to avirulent pathogens. Interestingly, the early activation of ProDH was accompanied by an increase in P5C reductase but not in P5CDH transcripts, with few changes occurring in the Pro and P5C levels. Therefore, our results suggest that in wild-type plants, ProDH is a defense component contributing to HR and disease resistance, which apparently potentiates the accumulation of ROS. The participation of the Pro-P5C cycle in the latter response is discussed.

Membrane proteins exit the endoplasmic reticulum (ER) in COPII-transport vesicles. ER export is a selective process in which transport signals present in the cytoplasmic tail (CT) of cargo membrane proteins must be recognized by coatomer proteins for incorporation in COPII vesicles. Two classes of ER export signals have been described for type I membrane proteins, the diacidic and the dihydrophobic motifs. Both motifs participate in the Sar1-dependent binding of Sec23p-Sec24p complex to the CTs during early steps of cargo selection. However, information concerning the amino acids in the CTs that interact with Sar1 is lacking. Herein, we describe a third class of ER export motif, [RK](X)[RK], at the CT of Golgi resident glycosyltransferases that is required for these type II membrane proteins to exit the ER. The dibasic motif is located proximal to the transmembrane border, and experiments of cross-linking in microsomal membranes and of binding to immobilized peptides showed that it directly interacts with the COPII component Sar1. Sar1GTP-bound to immobilized peptides binds Sec23p. Collectively, the present data suggest that interaction of the dibasic motif with Sar1 participates in early steps of selection of Golgi resident glycosyltransferases for transport in COPII vesicles.

In cultured neurons, axon formation is preceded by the appearance in one of the multiple neurites of a large growth cone containing a labile actin network and abundant dynamic microtubules. The invasion-inducing T-lymphoma and metastasis 1 (Tiam1) protein that functions as a guanosine nucleotide exchange factor for Rac1 localizes to this neurite and its growth cone, where it associates with microtubules. Neurons overexpressing Tiam1 extend several axon-like neurites, whereas suppression of Tiam1 prevents axon formation, with most of the cells failing to undergo changes in growth cone size and in cytoskeletal organization typical of prospective axons. Cytochalasin D reverts this effect leading to multiple axon formation and penetration of microtubules within neuritic tips devoid of actin filaments. Taken together, these results suggest that by regulating growth cone actin organization and allowing microtubule invasion within selected growth cones, Tiam1 promotes axon formation and hence participates in neuronal polarization.

Plant tissues display major alterations upon the perception of microbial pathogens. Changes of cytoplasmic and apoplastic components that sense and transduce plant defenses have been extensively characterized. In contrast, less information is available about modifications affecting the plant nuclear genome under these circumstances. Here, we investigated whether the Arabidopsis thaliana DNA methylation status is altered in tissues responding to the attack of Pseudomonas syringae pv. tomato DC3000. We applied amplified fragment length polymorphism analysis to monitor cytosine methylation at anonymous 5'-CCGG-3' and 5'-GATC-3' sites in naive and infected samples. Plant genomic fragments reducing methylation upon infection, including peri/centromeric repeats such as the 180-bp unit, Athila retrotansposon, and a portion of the nuclear insertion of mitochondrial DNA, were isolated and characterized. P. syringae pv. tomato-induced hypomethylation was detected by high-performance liquid chromatography assays and at the molecular level it did not seem to equally affect all 5-methyl cytosine (5-mC) residues. Nuclei from challenged tissues displayed structural chromatin alterations, including loosening of chromocenters, which also were stimulated by avirulent P. syringae pv. tomato, but not by the P. syringae pv. tomato hrpL- mutant. Finally, P. syringae pv. tomato-induced hypomethylation was found to occur in the absence of DNA replication, suggesting that it involves an active demethylation mechanism. All these responses occurred at 1 day postinfection, largely preceding massive plant cell death generated by pathogen attack.

Salicylic acid (SA) is a stress-induced hormone involved in the activation of defense genes. Here we analyzed the early genetic responses to SA of wild type and npr1-1 mutant Arabidopsis seedlings, using Complete Arabidopsis Transcriptome MicroArray (CATMAv2) chip. We identified 217 genes rapidly induced by SA (early SAIGs); 193 by a NPR1-dependent and 24 by a NPR1-independent pathway. These two groups of genes also differed in their functional classification, expression profiles and over-representation of cis-elements, supporting differential pathways for their activation. Examination of the expression patterns for selected early SAIGs from both groups indicated that their activation by SA required TGA2/5/6 subclass of transcription factors. These genes were also activated by Pseudomonas syringae pv. tomato AvrRpm1, suggesting that they might play a role in defense against bacteria. This study gives a global idea of the early response to SA in Arabidopsis seedlings, expanding our knowledge about SA function in plant defense.

Parkinson's disease (PD) is an age-related movement disorder characterized by a progressive degeneration of dopaminergic neurons in the midbrain. Although the presence of amyloid deposits of α-synuclein (α-syn) is the main pathological feature, PD brains also present a severe permanent inflammation, which largely contributes to neuropathology. Although α-syn has recently been implicated in this process, the molecular mechanisms underlying neuroinflammation remain unknown. In the present study, we investigated the ability of different α-syn aggregates to trigger inflammatory responses. We showed that α-syn induced inflammation through activation of Toll-like receptor 2 (TLR2) and the nucleotide oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome only when folded as amyloid fibrils. Oligomeric species, thought to be the primary species responsible for the disease, were surprisingly unable to trigger the same cascades. As neuroinflammation is a key player in PD pathology, these results put fibrils back to the fore and rekindles discussions about the primary toxic species contributing to the disease. Our data also suggest that the inflammatory properties of α-syn fibrils are linked to their intrinsic structure, most probably to their cross-β structure. Since fibrils of other amyloids induce similar immunological responses, we propose that the canonical fibril-specific cross-β structure represents a new generic motif recognized by the innate immune system.

The product of the incorporation of [14C]tyrosine as single unit into a protein of the soluble fraction of rat brain homogenate was purified by following a procedure used to purify tubulin. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified material showed a single protein band containing all the radioactivity. Purification data indicate that this protein accounts for 10.2% of the total protein of the supernatant fraction. This is in good agreement with the amount found for tubulin by the [3H]colchicine-binding method (10.5% of the total protein). The incorporated [14C]-tyrosine was found in the alpha-subunit of tubulin. Protein labelled with [3H]colchicine and [14C]tyrosine was precipatated with vinblastine sulphate and the radioactivity of 3H and that of 14C were quantitatively recovered in the precipitate (98%). Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the vinblastine precipitate showed that the 14C radioactivity moved with the tubulin band. Results obtained in experiments with phenylalanine and 3,4-dihydroxyphenylalanine were identical to those obtained for tyrosine. Bineing of colchicine did not interfere with the incorporation of tyrosine. About 30% of tubulin from rat brain supernatant fraction can incorporate tyrosine as single unit.

In eukaryotic genomes, gene expression and DNA recombination are affected by structural chromatin traits. Chromatin structure is shaped by the activity of enzymes that either introduce covalent modifications in DNA and histone proteins or use energy from ATP to disrupt histone-DNA interactions. The genomic 'marks' that are generated by covalent modifications of histones and DNA, or by the deposition of histone variants, are susceptible to being altered in response to stress. Recent evidence has suggested that proteins generating these epigenetic marks play crucial roles in the defence against pathogens. Histone deacetylases are involved in the activation of jasmonic acid- and ethylene-sensitive defence mechanisms. ATP-dependent chromatin remodellers mediate the constitutive repression of the salicylic acid-dependent pathway, whereas histone methylation at the WRKY70 gene promoter affects the activation of this pathway. Interestingly, bacterial-infected tissues show a net reduction in DNA methylation, which may affect the disease resistance genes responsible for the surveillance against pathogens. As some epigenetic marks can be erased or maintained and transmitted to offspring, epigenetic mechanisms may provide plasticity for the dynamic control of emerging pathogens without the generation of genomic lesions.

CREB3 family of transcription factors are ER localized proteins that belong to the bZIP family. They are transported from the ER to the Golgi, cleaved by S1P and S2P proteases and the released N-terminal domains act as transcription factors. CREB3 family members regulate the expression of a large variety of genes and according to their tissue-specific expression profiles they play, among others, roles in acute phase response, lipid metabolism, development, survival, differentiation, organelle autoregulation, and protein secretion. They have been implicated in the ER and Golgi stress responses as regulators of the cell secretory capacity and cell specific cargos. In this review we provide an overview of the diverse functions of each member of the family (CREB3, CREB3L1, CREB3L2, CREB3L3, CREB3L4) with special focus on their role in the central nervous system.

We engineered a machine learning approach, MSHub, to enable auto-deconvolution of gas chromatography-mass spectrometry (GC-MS) data. We then designed workflows to enable the community to store, process, share, annotate, compare and perform molecular networking of GC-MS data within the Global Natural Product Social (GNPS) Molecular Networking analysis platform. MSHub/GNPS performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization and quantifies the reproducibility of fragmentation patterns across samples.