Low-molecular-weight thiols: identification of novel thiol compounds and applications in winemaking processes

Thiols are reduced sulphur molecules that occur both in plants and animals with relevant roles. The thiol group is strongly nucleophilic, thus participating in a lot of biological redox processes, as for example the modulation of oxidative stress and participation to enzymatic reactions. Low-molecular-weight (LMW) thiols are a class of highly reactive compounds mainly involved in the maintenance of the redox homeostasis in the cells, thanks to the reactivity of their nucleophilic sulfur groups. In plants, they are implicated in the responses to almost all stress factors, as well as in the regulation of cell metabolism. Moreover, they can conjugate or make complexes with xenobiotics and toxic compounds (detoxification processes) and deactivate them, and they can post-translationally modify regulatory enzymes. They also have important implications in food quality and safety, as well as in human health. The most studied LMW thiols are glutathione and its biosynthetically related compounds (cysteine, gamma-glutamylcysteine and cysteinylglycine). Other LMW thiols are described in the literature, such as cysteamine, homocysteine, and many species-specific volatile thiols but less is known about them. Research shows that exists a huge amount of thiols in plants, but many species-specific and organ-specific thiols remain to be identified. Some of these unknown LMW thiols have light dependence, suggesting that they could be related to the photosynthesis processes. Recent advances in technology should help in this challenging work, helping to know the physiological and metabolic function in plants. However, their identification remains challenging due to their low concentration in plant tissues. In order to discover new unidentified thiols, in this work it was carried out a derivatization with SBD-F (ammonium 7-fluoro 2,1,3-benzooxadiazole-4-sulfonate) of the plant extracts, and then, SBD-derivatives were analyzed by HPLC-fluorescence and LC-MS/MS in negative mode using an ion trap (Varian 500 MS), to obtain their fragmentation pattern. Known LMW thiols such as cysteine, homocysteine, glutathione, cysteamine, gamma-glutamylcysteine, N-acetylcysteine and cysteinylglycine were used as reference compounds and their fragmentation pattern was first studied in order to highlight a fragmentation rule and molecular markers to systematically identify the unknown LMW thiols. Also high resolution measurements were obtained on a Xevo G-2 Q-TOF mass spectrometer (Waters). After the derivatization with the fluorophore, thiols can be easily recognized in fragmentation spectra due to the presence of a clear signal arising from the SBD-S fragment (m/z 231). This fragment corresponds to the fluorophore attached to the sulphur group from the LMW thiol. This signal was then used as a marker to confirm the presence of thiol groups in unknown molecules. In this way, some molecules could be identified and further confirmed by Q-TOF analysis; as for example the presence of thioglucose and glutathione containing derivatives. This protocol now opens the way to the identification of unknown thiol molecules. In winemaking processes, LMW thiols and specifically GSH, have an antioxidant function, which is present in grapes, must and wine. They help contrasting the oxidative browning by protecting grapes, and also the must during fermentation and the wine during the aging processes. They have a key role in the antioxidant activity by protecting wines, mostly white ones, from the oxidative process during aging. Due to this fact, in this study it was also tried to develop and optimize an easy and fast method to quantify the amount of LMW thiols in several German grape varieties (white and red). These compounds were extracted and analyzed using the fluorescent dye SBD-F and HPLC-FL separation. Also using HPLC, the sugars and organic acids were also quantified. The results of this quantification show that there is a very good reproducibility either in sampling or in the measurement of these compounds in the grape berries. This method can be also applied in must, wine and yeast. The method allowed not only the quantification of GSH, but also of its related compounds: cysteine, gamma-glutamylcysteine and cysteinylglycine in the same chromatogram, showing also the correlation between them. This study on German grape varieties is then showing that GSH is the most important LMW thiol in grapes, whose content is largely depending on the variety. Given their role as an antioxidant and possible beneficial effects during the winemaking processes, GSH and related LMW thiols are an important factor to consider in the evaluation of the grapes used for making wine

Proteome readjustments in the apoplastic space of Arabidopsis thaliana ggt1 mutant leaves exposed to UV-B radiation

Ultraviolet-B radiation acts as an environmental stimulus, but in high doses it has detrimental effects on plant metabolism. Plasma membranes represent a major target for ROS generated by this harmful radiation. Oxidative reactions occurring in the apoplastic space are counteracted by antioxidative systems mainly involving ascorbate and, to some extent, glutathione. The occurrence of the latter and its exact role in the extracellular space are not well documented, however. In Arabidopsis thaliana, the gamma-glutamyl transferase isoform GGT1 bound to the cell wall takes part in the so-called gamma-glutamyl cycle for extracellular glutathione degradation and recovery, and may be implicated in redox sensing and balance. In this work, oxidative conditions were imposed with UV-B and studied in redox altered ggt1 mutants. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in extracellular glutathione and ascorbate content and their redox state, and in apoplastic protein composition. Our results show that, on UV-B exposure, soluble antioxidants respond to the oxidative conditions in both genotypes. Rearrangements occur in their apoplastic protein composition, suggesting an involvement of H2O2, which may ultimately act as a signal. Other important changes relating to hormonal effects, cell wall remodeling, and redox activities are discussed. We argue that oxidative stress conditions imposed by UV-B and disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Data are available via ProteomeXchange with identifier PXD001807

Stroke genetics informs drug discovery and risk prediction across ancestries

Previous genome-wide association studies (GWASs) of stroke — the second leading cause of death worldwide — were conducted predominantly in populations of European ancestry1,2. Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis3, and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach4, we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry5. Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries

Low-molecular-weight thiols: identification of novel thiol compounds and applications in winemaking processes

Thiols are reduced sulphur molecules that occur both in plants and animals with relevant roles. The thiol group is strongly nucleophilic, thus participating in a lot of biological redox processes, as for example the modulation of oxidative stress and participation to enzymatic reactions. Low-molecular-weight (LMW) thiols are a class of highly reactive compounds mainly involved in the maintenance of the redox homeostasis in the cells, thanks to the reactivity of their nucleophilic sulfur groups. In plants, they are implicated in the responses to almost all stress factors, as well as in the regulation of cell metabolism. Moreover, they can conjugate or make complexes with xenobiotics and toxic compounds (detoxification processes) and deactivate them, and they can post-translationally modify regulatory enzymes. They also have important implications in food quality and safety, as well as in human health. The most studied LMW thiols are glutathione and its biosynthetically related compounds (cysteine, gamma-glutamylcysteine and cysteinylglycine). Other LMW thiols are described in the literature, such as cysteamine, homocysteine, and many species-specific volatile thiols but less is known about them. Research shows that exists a huge amount of thiols in plants, but many species-specific and organ-specific thiols remain to be identified. Some of these unknown LMW thiols have light dependence, suggesting that they could be related to the photosynthesis processes. Recent advances in technology should help in this challenging work, helping to know the physiological and metabolic function in plants. However, their identification remains challenging due to their low concentration in plant tissues. In order to discover new unidentified thiols, in this work it was carried out a derivatization with SBD-F (ammonium 7-fluoro 2,1,3-benzooxadiazole-4-sulfonate) of the plant extracts, and then, SBD-derivatives were analyzed by HPLC-fluorescence and LC-MS/MS in negative mode using an ion trap (Varian 500 MS), to obtain their fragmentation pattern. Known LMW thiols such as cysteine, homocysteine, glutathione, cysteamine, gamma-glutamylcysteine, N-acetylcysteine and cysteinylglycine were used as reference compounds and their fragmentation pattern was first studied in order to highlight a fragmentation rule and molecular markers to systematically identify the unknown LMW thiols. Also high resolution measurements were obtained on a Xevo G-2 Q-TOF mass spectrometer (Waters). After the derivatization with the fluorophore, thiols can be easily recognized in fragmentation spectra due to the presence of a clear signal arising from the SBD-S fragment (m/z 231). This fragment corresponds to the fluorophore attached to the sulphur group from the LMW thiol. This signal was then used as a marker to confirm the presence of thiol groups in unknown molecules. In this way, some molecules could be identified and further confirmed by Q-TOF analysis; as for example the presence of thioglucose and glutathione containing derivatives. This protocol now opens the way to the identification of unknown thiol molecules. In winemaking processes, LMW thiols and specifically GSH, have an antioxidant function, which is present in grapes, must and wine. They help contrasting the oxidative browning by protecting grapes, and also the must during fermentation and the wine during the aging processes. They have a key role in the antioxidant activity by protecting wines, mostly white ones, from the oxidative process during aging. Due to this fact, in this study it was also tried to develop and optimize an easy and fast method to quantify the amount of LMW thiols in several German grape varieties (white and red). These compounds were extracted and analyzed using the fluorescent dye SBD-F and HPLC-FL separation. Also using HPLC, the sugars and organic acids were also quantified. The results of this quantification show that there is a very good reproducibility either in sampling or in the measurement of these compounds in the grape berries. This method can be also applied in must, wine and yeast. The method allowed not only the quantification of GSH, but also of its related compounds: cysteine, gamma-glutamylcysteine and cysteinylglycine in the same chromatogram, showing also the correlation between them. This study on German grape varieties is then showing that GSH is the most important LMW thiol in grapes, whose content is largely depending on the variety. Given their role as an antioxidant and possible beneficial effects during the winemaking processes, GSH and related LMW thiols are an important factor to consider in the evaluation of the grapes used for making wine.I tioli sono composti ridotti dello zolfo che svolgono importanti funzioni in animali e piante. Il gruppo -SH è fortemente nucleofilo, per tale motivo queste molecole sono spesso coinvolte in processi biologici di ossidoriduzione, come ad esempio la modulazione degli stress ossidativi e la partecipazione a varie reazioni enzimatiche. I tioli a basso peso molecolare (LMW) sono una classe di composti coinvolti principalmente nel mantenimento dell’omeostasi ossidoriduttiva nella cellula, tale caratteristica si deve alla reattività dei loro gruppi tiolici nucleofili. Nelle piante sono coinvolti nella risposta a quasi tutti i fattori di stress e nella regolazione del metabolismo cellulare. I tioli LMW possono legarsi o creare complessi con composti tossici disattivandoli (detossificazione), inoltre possono modificare, dopo la traduzione, enzimi regolatori. Queste molecole sono implicate nella qualità e salubrità degli alimenti e anche nella salute umana. I tioli LMW più studiati sono il glutatione e i suoi composti derivati (cisteina, gamma-glutamil-cisteina e cisteinil-glicina). In letteratura sonostati descritti altri tioli LMW come la cisteammina, l’omocisteina e molti altri tioli volatili specie-specifici di cui però poco è conosciuto. In particolare, nelle piante è dimostrata la presenza di moltissimi tioli specie-specifici e organo-specifici molti dei quali però non sono ancora stati identificati. Alcuni di questi tioli LMW sconosciuti sono luce-dipendenti e ciò suggerisce un loro coinvolgimento nel processo della fotosintesi. I miglioramenti nella tecnologia potrebbero aiutare lo studio e la conoscenza della funzione fisiologica e metabolica di questi composti. Tuttavia la loro identificazione è resa ardua dalla loro bassa concentrazione nei tessuti vegetali. In questo lavoro, allo scopo di scoprire nuovi tioli non ancora identificati, sono stati derivatizati con SBD-F degli estratti di piante e in seguito i derivatizzati sono stati sottoposti ad analisi HPLC a fluorescenza e LC-MS/MS in modalità negativa usando una trappola ionica (Varian 500 MS) per ottenere la frammentazione. Sono stati usati come riferimento i tioli LMW già noti come la cisteina, l’omocisteina, il glutatione, la cisteammina, la gamma-glutamil-cisteina, l’N-acetilcisteina e la cisteinil-glicina, i cui modelli di frammentazione sono stati inizialmente studiati per evidenziare la modalità di frammentazione e i marcatori molecolari che hanno consentito la identificazione sistematica di tioli LMW sconosciuti. Inoltre è stata ottenuta la misurazione ad alta risoluzione su spettrometro di massa Q-ToF (Waters) su Xevo G-2. Dopo la derivatizzazione con fluoroforo i tioli possono essere facilmente riconosciuti dallo spettro di frammentazione per la presenza di un chiaro segnale dato dal frammento SBD-S (m/z 231). Questo frammento corrisponde al fluoroforo legato al gruppo tiolico, ed è stato usato per marcare e confermare la presenza del gruppo tiolico nelle molecole sconosciute. In questo modo le molecole possono essere identificatee poi confermate dall’analisi Q-ToF come nel caso del tioglucosio e di derivati contenenti glutatione. Il protocollo che è stato definito con il presente lavoro permette ora l’identificazione di nuovi composti dello zolfo al momento sconosciuti. Nel processo di produzione del vino, i tioli LMW e il glutatione in particolare, hanno una importante funzione antiossidante che si manifesta nell’uva, nel mosto e nel vino. I tioli contribuiscono a contrastare l’imbrunimento ossidativo delle proteine nell’uva e soprattutto nel mosto durante la fermentazione e del vino nei diversi processi di lavorazione. I tioli giocano perciò un ruolo chiave nella conservazione del vino con la loro attività antiossidante, in particolar modo nei vini bianchi. Per questa ragione è stato condotto uno studio volto a sviluppare una metodologia semplice e rapida per la quantificazione dei tioli LWM totali. In tale studio sono state poste a confronto diverse varietà tedesche di uva da vino. I composti dello zolfo sono stati estratti ed analizzati utilizzando un colorante fluorescente SBD-F e separazione HPLC-FL. Sono stati quantificati anche gli zuccheri e gli acidi organici tramite HPLC. I risultati di questa quantificazione mostrano un’elevata riproducibilità tra i campioni e tra le misurazioni di questi composti nelle bacche. Il metodo è utilizzabile anche su mosto, vino e lieviti. Il metodo permette non solo la quantificazione del glutatione ma anche di altri composti relativi nello stesso cromatogramma e mostra una correlazione tra di essi. Il confronto di varietà differenti mostra la presenza di GSH nella maggior parte dei tioli dell’uva, mentre il loro contenuto è molto influenzato dalla varietà. In considerazione del loro ruolo di antiossidanti, il GSH e i tioli LMW possono avere un ruolo favorevole nella vinificazione e sono perciò un fattore fondamentale che deve essere preso in considerazione nelle decisioni che riguardano il processo di produzione del vino

Low-molecular-weight thiols in plants: Functional and analytical implications

Low-molecular-weight (LMW) thiols are a class of highly reactive compounds massively involved in the maintenance of cellular redox homeostasis. They are implicated in plant responses to almost all stress factors, as well as in the regulation of cellular metabolism. The most studied LMW thiols are glutathione and its biosynthetically related compounds (cysteine, c-glutamylcysteine, cysteinylglycine, and phytochelatins). Other LMW thiols are described in the literature, such as thiocysteine, cysteamine, homocysteine, lipoic acid, and many species-specific volatile thiols. Here, we review the known LMW thiols in plants, briefly describing their physico-chemical properties, their relevance in post-translational protein modification, and recently-developed thiol detection methods. Current research points to a huge thiol biodiversity in plants and many species-specific and organ-specific thiols remain to be identified. Recent advances in technology should help researchers in this very challenging task, helping us to decipher the roles of thiols in plant metabolism