Elucidating the role of post-translational modifications of alpha-synuclein using semisynthesis:phosphorylation at Tyrosine 125 and monoubiquitination at Lysine 6

Alpha-synuclein (α-syn) is a natively unfolded protein that is closely linked to Parkinson’s disease (PD) by genetic, neuropathologic and biochemical evidence. Aggregated and fibrillar forms of α-syn are the main components of intracellular protein inclusions found in PD patients’ brains, termed Lewy Bodies (LB). Both in animal models and in vitro, α-syn forms fibrillar aggregates that resemble those observed in PD brain tissues. Although disease-associated mutations have been shown to promote the fibrillization of α-syn, the exact mechanisms responsible for triggering α-syn aggregation and toxicity in sporadic PD remain unknown. Addressing this gap of knowledge is crucial for understanding the molecular basis of the disease and developing effective therapies for the treatment of PD and other synucleinopathies. This project was initiated on the basis of the working hypothesis that post-translational modifications (PTM) may play important roles in modulating α-syn function and/or regulating its aggregation and toxicity. More specifically, α-syn is ubiquitously N-terminally acetylated, and phosphorylated (serines 87 and S129), ubiquitinated (lysines 12, 21 and 23) and truncated forms of α-syn have been observed in association with wild-type α-syn in LB and in brain tissues from PD patients and transgenic animals. Other modifications, such as phosphorylation at tyrosine 125 (Y125), were significantly reduced in diseased brains. Despite the discovery of candidate enzymes that mediate α-syn phosphorylation, ubiquitination and truncation, little is known about how each of these modifications alters α-syn structure, function, aggregation and toxicity in vivo. This is primarily due to the lack of tools that allow site-specific introduction of these modifications and the lack of natural mutations that can mimic the effect of these modifications, including phosphorylation. The primary focus of this thesis was to develop strategies to overcome these limitations so as to elucidate the effect of phosphorylation at Y125 and monoubiquitination at lysine 6 (K6) on α-syn structure, fibril formation, membrane binding and subcellular localization. Towards this goal, we developed two semisynthetic strategies that allow site-specific introduction of PTM in α-syn based on the ligation of synthetic peptides containing the desired modified amino acid with recombinantly expressed proteins using Expressed Protein Ligation. This approach enables the introduction of single or multiple PTM in the N- or C-terminal regions of α-syn, and the preparation of modified α-syn in milligram quantities. Using these approaches, we were able to show for the first time that ubiquitination stabilizes the monomeric form of the protein and inhibits, rather than promotes, α-syn aggregation, while phosphorylation at Y125 does not significantly change the structure and aggregation propensity of α-syn. With the semisynthetic pY125 α-syn in our hands, we were also able to investigate for the first time the sub-cellular localization of pY125 α-syn through its microinjection into primary neurons. This was not previously possible due to technical limitations related to the absence of appropriate antibodies against pY125 α-syn for immunocytochemical studies and difficulties in generating sitespecifically modified α-syn. Furthermore, we were able to investigate the effect of α-syn phosphorylation on its interactions with other proteins, by probing the effect of pS129 and pY125 on the binding to a nanobody that was specifically selected by phage-display to tightly bind to the C-terminal domain of α-syn. Accordingly, we demonstrated that phosphorylation at a single residue is capable of disrupting the binding of full-length α-syn to another protein. These results have wide-ranging implications for the potential role of phosphorylation and other PTM in regulating α-syn’s function(s). We also exploited our ability to combine semisynthetic and enzymatic approaches to investigate potential cross-talk between different PTM, namely phosphorylation at Y125 and S129 and monoubiquitination at K6. These advances would eventually allow investigating the effect of cross-talk between other N and C-terminal modifications on α-syn’s properties and to investigate PTM-dependent protein-protein and protein-ligand interactions in vitro and in cellular models of synucleinopathies. Together, our semisynthetic approaches provide novel means for the introduction of site-specific modifications in the N- and C-termini of α-syn. Current efforts in our laboratory are focused on extending these approaches to investigate the dynamics of PTM through the use of photocaged modified amino acids and to prepare novel fluorescently labeled α-syn variants to investigate its folding at the single-molecule level in living cells. The results presented in this thesis and preliminary studies from our group demonstrate that semisynthetic α-syn can provide unique opportunities to investigate structure-function of α-syn and the role of PTM in the biology of α-syn in health and disease

Chemical strategies for controlling protein folding and elucidating the molecular mechanisms of amyloid formation and toxicity

It has been more than a century since the first evidence linking the process of amyloid formation to the pathogenesis of Alzheimer's disease. During the last three decades in particular, increasing evidence from various sources (pathology, genetics, cell culture studies, biochemistry, and biophysics) continues to point to a central role for the pathogenesis of several incurable neurodegenerative and systemic diseases. This is in part driven by our improved understanding of the molecular mechanisms of protein misfolding and aggregation and the structural properties of the different aggregates in the amyloid pathway and the emergence of new tools and experimental approaches that permit better characterization of amyloid formation in vivo. Despite these advances, detailed mechanistic understanding of protein aggregation and amyloid formation in vitro and in vivo presents several challenges that remain to be addressed and several fundamental questions about the molecular and structural determinants of amyloid formation and toxicity and the mechanisms of amyloid-induced toxicity remain unanswered. To address this knowledge gap and technical challenges, there is a critical need for developing novel tools and experimental approaches that will not only permit the detection and monitoring of molecular events that underlie this process but also allow for the manipulation of these events in a spatial and temporal fashion both in and out of the cell. This review is primarily dedicated in highlighting recent results that illustrate how advances in chemistry and chemical biology have been and can be used to address some of the questions and technical challenges mentioned above. We believe that combining recent advances in the development of new fluorescent probes, imaging tools that enabled the visualization and tracking of molecular events with advances in organic synthesis, and novel approaches for protein synthesis and engineering provide unique opportunities to gain a molecular-level understanding of the process of amyloid formation. We hope that this review will stimulate further research in this area and catalyze increased collaboration at the interface of chemistry and biology to decipher the mechanisms and roles of protein folding, misfolding, and aggregation in health and disease

High-Temperature and Drought Stress Effects on Growth, Yield and Nutritional Quality with Transpiration Response to Vapor Pressure Deficit in Lentil

High temperature and water deficit are among the major limitations reducing lentil (Lens culinaris Medik.) yield in many growing regions. In addition, increasing atmospheric vapor pressure deficit (VPD) due to global warming causes a severe challenge by influencing the water balance of the plants, thus also affecting growth and yield. In the present study, we evaluated 20 lentil genotypes under field conditions and controlled environments with the following objectives: (i) to investigate the impact of temperature stress and combined temperature-drought stress on traits related to phenology, grain yield, nutritional quality, and canopy temperature under field conditions, and (ii) to examine the genotypic variability for limited transpiration (TRlim) trait in response to increased VPD under controlled conditions. The field experiment results revealed that high-temperature stress significantly affected all parameters compared to normal conditions. The protein content ranged from 23.4 to 31.9%, while the range of grain zinc and iron content varied from 33.1 to 64.4 and 62.3 to 99.3 mg kg−1 , respectively, under normal conditions. The grain protein content, zinc and iron decreased significantly by 15, 14 and 15% under high-temperature stress, respectively. However, the impact was more severe under combined temperature-drought stress with a reduction of 53% in protein content, 18% in zinc and 20% in iron. Grain yield declined significantly by 43% in temperature stress and by 49% in the combined temperature-drought stress. The results from the controlled conditions showed a wide variation in TR among studied lentil genotypes. Nine genotypes displayed TRlim at 2.76 to 3.51 kPa, with the genotypes ILL 7833 and ILL 7835 exhibiting the lowest breakpoint. Genotypes with low breakpoints had the ability to conserve water, allowing it to be used at later stages for increased yield. Our results identified promising genotypes including ILL 7835, ILL 7814 and ILL 4605 (Bakria) that could be of great interest in breeding for high yields, protein and micronutrient contents under high-temperature and drought stress. In addition, it was found that the TRlim trait has the potential to select for increased lentil yields under field water-deficit environment

Experimental on-farm trials data of faba bean and wheat intercropping field validation in Lebanon and Morocco

This data paper describes the content of four datasets col- lected by the International Center for Agricultural Research in the Dry Areas (ICARDA) as a partner in the project “Designing InnoVative plant teams for Ecosystem Resilience and agricultural Sustainability (DIVERSify)”with the objec- tive of assessing the feasibility of faba bean-wheat mix- ture in Mediterranean environments under diverse rainfed conditions. Data was collected during the trials conducted in Kfardan-Lebanon during 2017/2018 where 40 faba bean varieties were evaluated as sole and as mixture with 2 wheat cultivars ‘Margherita’ and ‘Miki’ and during 2018/2019 where 40 faba bean varieties and one durum wheat cultivar ‘Margherita’ were evaluated under low rainfall environments. Trials were also conducted in Tal Amara-Lebanon during 2019/2020 where 20 faba bean lines and one durum wheat cultivar ‘Margherita’ were evaluated under high rainfall en- vironments and in Marchouch-Morocco during 2019/2020 where 7 faba bean lines with 3 cultivars and one durum wheat cultivar ‘Margherita’ were evaluated under extremely low rainfall environments. A detailed list of the different bi- ological traits collected for wheat and faba bean is found in the specification table in this article. The Kfardan 2018/ 2019, Tal Amara and Marchouch data is related to the conference paper “Performance of faba bean-wheat mixture under di- verse Mediterranean environments”

Evaluation of performance and stability of new sources for tolerance to post-emergence herbicides in lentil (Lens culinaris ssp. culinaris Medik.)

Yield losses in lentil (Lens culinaris ssp. culinaris Medik.) caused by weeds are estimated at 20–80%. In the absence of effective broad-spectrum herbicides, the menace of weed has emerged as a serious yield constraint in lentil. Aims. Identification of tolerance to two broad-spectrum post emergence herbicides, imazethapyr and metribuzin. Methods. 221 lentil accessions were screened at the experimentalresearch station of the International Centerfor Agricultural Research in the Dry Areas, Terbol (Lebanon) over four crop seasons (2014/15–2018/19). During the 2014/15 season, a preliminary screening experiment was conducted with 221 lentil accessions in an alpha lattice design with two replications, where accessions were treated at the pre-flowering stage at 150% of the recommended dose of imazethapyr (112.5 g active ingredient/ha) or metribuzin (315 g active ingredient/ha). Based on the preliminary results, 38 accessions were selected for further evaluation. In subsequent testing, the selected accessions were screened at 100% and 150% of the recommended doses of imazethapyr or metribuzin. Key results. Herbicide treatments delayed the flowering time and maturation of tested lentil accessions. The plant height of selected tolerant accessions was reduced by 15% and 8% in the plots treated by imazethapyr and metribuzin, respectively. Reduction index (RIsy) was an effective tool to select for herbicide tolerance. Stability analysis indicated that two accessions (IG4400 and IG5722) performed under high rainfall environments while two other accessions (IG323 and IG4605) were adapted to low rainfall environments. Conclusions. Four accessions (IG323, IG5722, IG4400, IG4605) were identified as independently tolerant to metribuzin and imazethapyr. Implications. The identified herbicide-tolerant accessions can be used to develop herbicide-tolerant cultivars

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow site-specific introduction of ubiquitin (Ub) chains to a specific substrate. Here, we describe chemical and semisynthetic strategies for site-specific incorporation of K48-linked di- or tetra-Ub chains onto the side chain of Lys12 of α-Synuclein (α-Syn). These advances provided unique opportunities to elucidate the role of ubiquitination and Ub chain length in regulating α-Syn stability, aggregation, phosphorylation, and clearance. In addition, we investigated the cross-talk between phosphorylation and ubiquitination, the two most common α-Syn pathological modifications identified within Lewy bodies and Parkinson disease. Our results suggest that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications

The Size of the Proteasomal Substrate Determines Whether Its Degradation Will Be Mediated by Mono- or Polyubiquitylation

A polyubiquitin chain anchored to the substrate has been the hallmark of proteasomal recognition. However, the degradation signal appears to be more complex and to contain also a substrate's unstructured region. Recent reports have shown that the proteasome can degrade also monoubiquitylated proteins, which adds an additional layer of complexity to the signal. Here, we demonstrate that the size of the substrate is an important determinant in its extent of ubiquitylation: a single ubiquitin moiety fused to a tail of up to ∼150 residues derived from either short artificial repeats or from naturally occurring proteins, is sufficient to target them for proteasomal degradation. Importantly, chemically synthesized adducts, where ubiquitin is attached to the substrate via a naturally occurring isopeptide bond, display similar characteristics. Taken together, these findings suggest that the ubiquitin proteasomal signal is adaptive, and is not always made of a long polyubiquitin chain

Effect of High Temperature Stress During the Reproductive Stage on Grain Yield and Nutritional Quality of Lentil (Lens culinaris Medikus)

High temperature during the reproductive stage limits the growth and development of lentil (Lens culinaris Medikus). The reproductive and seed filling periods are the most sensitive to heat stress, resulting in limited yield and nutritional quality. Climate change causes frequent incidents of heat stress for global food crop production. This study aimed to assess the impact of high temperature during the reproductive stage of lentil on grain yield, nutritional value, and cooking quality. Thirty-six lentil genotypes were evaluated under controlled conditions for their high temperature response. Genotypic variation was significant (p < 0.001) for all the traits under study. High temperature-induced conditions reduced protein, iron (Fe) and zinc (Zn) concentrations in lentils. Under heat stress conditions, mineral concentrations among lentil genotypes varied from 6.0 to 8.8 mg/100 g for Fe and from 4.9 to 6.6 mg/100 g for Zn. Protein ranged from 21.9 to 24.3 g/100 g. Cooking time was significantly reduced due to high temperature treatment; the range was 3–11 min, while under no stress conditions, cooking time variation was from 5 to 14 min. Phytic acid variation was 0.5–1.2 g/100 g under no stress conditions, while under heat stress conditions, phytic acid ranged from 0.4 to 1.4 g/100 g. All genotypes had highly significant bioavailable Fe and moderately bioavailable Zn under no stress conditions. Whereas under heat stress conditions, Fe and Zn bioavailability was reduced due to increased phytic acid levels. Our results will greatly benefit the development of biofortified lentil cultivars for global breeding programs to generate promising genotypes with low phytic acid and phytic acid/micronutrient ratio to combat micronutrient malnutrition