Common Functions of Disordered Proteins across Evolutionary Distant Organisms.

Intrinsically disordered proteins and regions typically lack a well-defined structure and thus fall outside the scope of the classic sequence-structure-function relationship. Hence, classic sequence- or structure-based bioinformatic approaches are often not well suited to identify homology or predict the function of unknown intrinsically disordered proteins. Here, we give selected examples of intrinsic disorder in plant proteins and present how protein function is shared, altered or distinct in evolutionary distant organisms. Furthermore, we explore how examining the specific role of disorder across different phyla can provide a better understanding of the common features that protein disorder contributes to the respective biological mechanism

Improvement in self-reported exercise participation with the combination of tiotropium and rehabilitative exercise training in COPD patients

Steven Kesten1, Richard Casaburi2, David Kukafka3, Christopher B Cooper41Boehringer Ingelheim GmbH, Ingelheim, Germany; 2Harbor-UCLA Medical Center, Torrance, CA, USA; 3Northern Colorado Pulmonary Consultants PC, Fort Collins, CO, USA; 4UCLA School of Medicine, Los Angeles, CA, USABackground: Improvements in ventilatory mechanics with tiotropium increases exercise tolerance during pulmonary rehabilitation. We wondered whether tiotropium also increased physical activities outside of pulmonary rehabilitation.Methods: COPD patients participating in 8 weeks of pulmonary rehabilitation were studied in a randomized, double-blind, placebo-controlled trial of tiotropium 18 µg daily (tiotropium = 47, placebo = 44). Study drug was administered for 5 weeks prior to, 8 weeks during, and 12 weeks following pulmonary rehabilitation. Patients completed a questionnaire documenting participation in pre-defined activities outside of pulmonary rehabilitation during the 2 weeks prior to each visit. Patients who submitted an activity questionnaire at week 4 and on at least one subsequent visit were included in the analysis. For each patient, the number of sessions was multiplied with the duration of each activity and then summed to give overall activity duration.Results: Patients (n = 46) had mean age of 67 years, mean baseline FEV1 of 0.84 L (33% predicted). Mean (SE) increase in duration of activities (minutes during 2 weeks prior to each visit) from week 4 (prior to PR) to week 13 (end of PR) was 145 (84) minutes with tiotropium and 66 (96) minutes with placebo. The increase from week 4 to week 25 (end of follow-up) was 262 (96) and 60 (93) minutes for the respective groups. Increases in activity duration from week 4 to weeks 17, 21, and 25 were statistically significant with tiotropium. No statistical differences over time were observed within the placebo-treated group and differences between groups were not significant.Conclusions: Tiotropium appears to amplify the effectiveness of pulmonary rehabilitation as seen by increases in patient self-reported participation in physical activities.Keywords: activity, chronic obstructive pulmonary disease, exercise, pulmonary rehabilitation, tiotropiu

Development and implementation of treadmill exercise testing protocols in COPD

Christopher B Cooper1, Marlon Abrazado1, Daniel Legg2, Steven Kesten21David Geffen School of Medicine, University of California, Los Angeles, CA, USA; 2Boehringer Ingelheim Pharmaceuticals Inc., Ingelheim, GermanyBackground: Because treadmill exercise testing is more representative of daily activity than cycle testing, we developed treadmill protocols to be used in various clinical settings as part of a two-year, multicenter, chronic obstructive pulmonary disease (COPD) trial evaluating the effect of tiotropium on exercise.Methods: We enrolled 519 COPD patients aged 64.6 ± 8.3 years with a postbronchodilator forced expiratory volume in one second (FEV1) of 1.25 ± 0.42 L, 44.3% ± 11.9% predicted. The patients performed symptom-limited treadmill tests where work rate (W) was increased linearly using speed and grade adjustments every minute. On two subsequent visits, they performed constant W tests to exhaustion at 90% of maximum W from the incremental test.Results: Mean incremental test duration was 522 ± 172 seconds (range 20–890), maximum work rate 66 ± 34 watts. For the first and second constant W tests, both at 61 ± 33 watts, mean endurance times were 317 ± 61 seconds and 341 ± 184 seconds, respectively. The mean of two tests had an intraclass correlation coefficient of 0.85 (P < 0.001). During the second constant W test, 88.2% of subjects stopped exercise because of breathing discomfort; 87.1% for Global Initiative for Chronic Obstructive Lung Disease (GOLD) Stage II, 88.5% for GOLD Stage III, and 90.2% for GOLD Stage IV.Conclusion: The symptom-limited incremental and constant work treadmill protocol was well tolerated and appeared to be representative of the physiologic limitations of COPD.Keywords: chronic obstructive pulmonary disease, exercise testing, endurance, tiotropiu

Unraveling the mechanism of TTL genes in cellulose biosynthesis

As sessile organisms, plants require mechanisms to sense and respond to the challenging environment, that encompass both biotic and abiotic factors that results in differential development. In these conditions is essential to balance growth and stress responses. As cell walls shape plant growth, this differential growth response cause alterations to the plant cell wall where cellulose is the major component. Therefore, understanding the mechanisms that regulate cellulose biosynthesis is essential to develop strategies to improve plant production. In Arabidopsis, the TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) gene family is composed by four members (TTL1 to TTL4) and mutations in TTL1, TTL3, and TTL4 genes cause reduced growth under salt and osmotic stress due to defects in plant cell wall integrity. We observe association of TTL3 with most core components in traducing BR signalling, such as LRR-RLK BRI1 or GSK3 BIN2 that modulate cellulose biosynthesis through phosphorylating cellulose synthases. Here, we show that ttl mutants present defects in the plant cell wall, particularly in Isoxaben, salt or sucrose stress. Spinning disk microscopy in etiolated hypocotyls reveals that, TTL proteins are responsible for the cellulose synthase complex (CSC) stability in plasma membrane (PM) upon sucrose stress. Moreover, TTL3 associates with LRR-RLKs that have been shown to be important for cellulose biosynthesis such as FEI1 in the FEI1/FEI2/SOS5 pathway. We aim to investigate the mechanisms by which TTL proteins regulate CesA stability in PM under stress, using a combination of genetics, biochemical, and molecular and cell biology approaches.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work was supported by grants from: (1) Ministerio de Ciencia e Innovación BIO2014-55380-R, BIO2014-56153-REDT; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256

Tiotropium reduces risk of exacerbations irrespective of previous use of inhaled anticholinergics in placebo-controlled clinical trials

Data have highlighted the potential bias introduced by withdrawal of inhaled corticosteroids at randomization in chronic obstructive pulmonary disease trials examining inhaled corticosteroids. Analyses were conducted to determine whether this was true of inhaled anticholinergic withdrawal in tiotropium trials.status: publishe

Soil-borne fungi alter the apoplastic purinergic signaling in plants by deregulating the homeostasis of extracellular ATP and its metabolite adenosine

Purinergic signaling activated by extracellular nucleotides and their derivative nucleosides trigger sophisticated signaling networks. The outcome of these pathways determine the capacity of the organism to survive under challenging conditions. Both extracellular ATP (eATP) and Adenosine (eAdo) act as primary messengers in mammals, essential for immunosuppressive responses. Despite the clear role of eATP as a plant damage-associated molecular pattern, the function of its nucleoside, eAdo, and of the eAdo/eATP balance in plant stress response remain to be fully elucidated. This is particularly relevant in the context of plant-microbe interaction, where the intruder manipulates the extracellular matrix. Here, we identify Ado as a main molecule secreted by the vascular fungus Fusarium oxysporum. We show that eAdo modulates the plant's susceptibility to fungal colonization by altering the eATP-mediated apoplastic pH homeostasis, an essential physiological player during the infection of this pathogen. Our work indicates that plant pathogens actively imbalance the apoplastic eAdo/eATP levels as a virulence mechanism

Overcoming stress: new insights in the regulation of cell wall biosynthesis

In addition to being crucial in plant development and defence, cellulose is the most abundant organic compound of all biomass on Earth1. Therefore, it is essential to elucidate the regulation of its biosynthesis to improve crop's tolerance to biotic and abiotic stresses. We have found the Tetratricopeptide Thioredoxin-Like (TTL)2 proteins as new players in the regulation of the cellulose synthase complex (CSC), identifying its dynamic association with the CSC under cellulose-deficient conditions3. We have found that TTLs are essential to maintain cellulose synthesis under salt stress, mediated by a stress-resilient cortical microtubule array and the stabilization of the CSCs at the plasma membrane. TTLs carry this out by interacting with Cellulose Synthase 1 and promoting the polymerization of microtubules. This dynamic behaviour of TTLs is not specific to salinity stress, and other modifications that cause reduced cellulose content also lead to the re-localization from the cytosol to the CSC. We conclude that TTLs act as intermediates between stress perception and regulation of cellulose biosynthesis to overcome adverse environmental conditions. All TTL proteins contain an Intrinsic Disordered Region at the end terminus, and we are now investigating how changes in phosphorylation regulate the activity and dynamic localization of these proteins.This work was funded by the Spanish Ministry for Science and Innovation (MCIN/AEI/ 10.13039/501100011033) (PGC2018-098789-B-I00) and (PID2019-107657RB-C22) to MAB, NRL and AC respectively. The Andalusian Research Plan co-financed by the European Union (PAIDI 2020-PY20_00084) to MAB and Junta de Andalucía UMA-FEDER project (grant UMA18-FEDERJA-154) to NRL, and the Swiss National foundation to CSR (SNF 31003A_163065/1 to AM). CK was supported by a Peter und Traudl Engelhorn-Stiftung fellowship, an ETH Career Seed Grant (SEED-05 19-2) of the ETH Foundation, an Emerging Investigator grant (NNF20OC0060564) of the Novo Nordisk Foundation, and an Experiment grant (R346-2020-1546) of the Lundbeck foundation. AGM and FP were supported by BES-2015-071256 and FPU19/02219 fellowships respectively, and meeting attendance was supported by Plan Propio de Investigación, Transfe-rencia y Divulgación Científica de la Universidad de Málaga (UMA) Campus de Excelencia Internacional Andalucía Tech. VAS was supported by an Emerging Investigator research project (UMA20-FEDERJA-007) and co-financed by the “Programa Operativo FEDER 2014-2020” and by the “Consejería de Economía y Conocimiento de la Junta de Andalucía”. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Calling for reinforcements: the role of TTL proteins in the regulación of cell wall biosynthesis

Cellulose is the most abundant organic compound of all biomass on Earth1, with highly relevant roles in plant development and defence. Hence, it is essential to understand the regulation of its biosynthesis to improve the crop's tolerance to biotic and abiotic stresses. Tetratricopeptide Thioredoxin-Like (TTL)2 proteins have been identified as new players in regulating the cellulose synthase complex (CSC), uncovering their dynamic association with the CSC under cellulose-deficient conditions3. We show that TTLs are essential to maintain cellulose synthesis under salt stress, mediated by a reinforced cortical microtubule array and the stabilization of the CSCs at the plasma membrane. To perform this, TTLs interact with Cellulose Synthase 1 and promote microtubules polymerization. This dynamic behaviour of TTLs is not specific to salinity stress, and other factors that cause defects in cellulose also cause the re-localization from the cytosol to the CSC. We conclude that TTLs act as intermediates between stress sensing and the regulation of cellulose biosynthesis to overcome adverse environmental conditions. We are now investigating how changes in phosphorylation of the Intrinsic Disordered Region at the end terminus of TTLs regulate their activity and dynamic localization.This work was funded by the Spanish Ministry for Science and Innovation (MCIN/AEI/ 10.13039/501100011033) (PGC2018-098789-B-I00) and (PID2019-107657RB-C22) to MAB, NRL and AC respectively. The Andalusian Research Plan co-financed by the European Union (PAIDI 2020-PY20_00084) to MAB and Junta de Andalucía UMA-FEDER project (grant UMA18-FEDERJA-154, UMA20-FEDERJA-023) to NRL and MAB respectively, and the Swiss National foundation to CSR (SNF 31003A_163065/1 to AM). CK was supported by a Peter und Traudl Engelhorn-Stiftung fellowship, an ETH Career Seed Grant (SEED-05 19-2) of the ETH Foundation, an Emerging Investigator grant (NNF20OC0060564) of the Novo Nordisk Foundation, and an Experiment grant (R346-2020-1546) of the Lundbeck foundation. AGM and FP were supported by BES-2015-071256 and FPU19/02219 fellowships respectively, and meeting attendance was supported by Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. VAS was supported by an Emerging Investigator research project (UMA20-FEDERJA-007) and co-financed by the “Programa Operativo FEDER 2014-2020” and by the “Consejería de Economía y Conocimiento de la Junta de Andalucía”

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus

Peripheral membrane TTL proteins safeguard cellulose synthesis under stress.

Land plants provide around eighty percent of biomass on Earth and roughly one-third corresponds to cellulose (Bar-On et al 2018). Despite its biological and societal importance, many aspects of cellulose biosynthesis and regulation remain elusive. Controlled primary cell wall remodeling allows plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is not well understood (Colin et al 2023). In this work, we identify that Tetratricopeptide Thioredoxin-Like (TTL) proteins, which we previous describe as a scaffold of brassinosteroids signalling components, are also new members of the cellulose synthase complex (CSC) and we describe their unique and hitherto unknown dynamic association with the CSC under cellulose-deficient conditions (Amorim-Silva et al 2019 and Kesten, García-Moreno, Amorim-Silva et al 2022). We found out that TTLs are essential for maintaining cellulose synthesis under high salinity conditions, establishing a stress-resilient cortical microtubule array, and stabilizing CSCs at the plasma membrane. To fulfill these functions, TTLs interact with Cellulose Synthase1 (CESA1) and engage with cortical microtubules to promote their polymerization. We propose that TTLs function as bridges connecting stress perception with dynamic regulation of cellulose biosynthesis at the plasma membrane. In addition, we are currently working to identify and characterize new components involved in TTLs function and dynamics during cellulose biosynthesis under saline stress conditions. References: Amorim-Silva et al. 2019 The Plant Cell Bar-On et al. 2018 Proc. Natl. Acad. Sci. Colin et al. 2023 The Plant Cell Kesten, García-Moreno, Amorim-Silva et al. 2022 Sci. Adv.Meeting attendance was supported by Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work was funded by the Spanish Ministry for Science and Innovation (MCIN/AEI/ 10.13039/501100011033) (PGC2018-098789-B-I00) and (PID2019-107657RB-C22) to MAB, NRL and AC respectively. The Andalusian Research Plan co-financed by the European Union (PAIDI 2020-PY20_00084) to MAB andde Andalucía UMA-FEDER project (grant UMA18-FEDERJA-154) to NRL, and the Swiss National foundation to CSR (SNF 31003A_163065/1 to AM). CK was supported by a Peter und Traudl Engelhorn-Stiftung fellowship, an ETH Career Seed Grant (SEED-05 19-2) of the ETH Foundation, an Emerging Investigator grant (NNF20OC0060564) of the Novo Nordisk Foundation, and an Experiment grant (R346-2020-1546) of the Lundbeck foundation. AGM and FP were supported by BES-2015-071256 and FPU19/02219 fellowships respectively. VAS was supported by an Emerging Investigator research project (UMA20-FEDERJA-007) and co-financed by the “Programa Operativo FEDER 2014-2020” and by the “Consejería de Economía y Conocimiento de la Junta de Andalucía”.