Flavonoids with lipase inhibitory activity from lemon squeezing waste: isolation, multispectroscopic and in silico studies.

BACKGROUND: Obesity is recognized as a lifestyle-related disease and the main risk factor for a series of pathological conditions, including cardiovascular diseases, hypertension and type 2 diabetes. Citrus limon is an important medicinal plant, and its fruits are rich in flavonoids investigated for their potential in managing obesity. In the present work, a green extraction applied to lemon squeezing waste (LSW) was optimized to recover pancreatic lipase (PL) inhibitors. RESULTS: The microwave-assisted procedure yielded an extract with higher lipase inhibitory activity than those obtained by maceration and ultrasound. The main compounds present in the extract were identified by high-performance liquid chromatographic-mass spectrometric analysis, and hesperidin, eriocitrin and 4'-methyllucenin II were isolated. The three compounds were evaluated for in vitro PL inhibitory activity, and 4'-methyllucenin II resulted in the most promising inhibitor (IC50 = 12.1 µmol L-1; Ki = 62.2 µmol L-1). Multispectroscopic approaches suggested the three flavonoids act as competitive inhibitors and the binding studies indicated a greater interaction between PL and 4'-methyllucenin II. Docking analysis indicated the significant interactions of the three flavonoids with the PL catalytic site. CONCLUSION: The present work highlights flavonoid glycosides as promising PL inhibitors and proposes LSW as a safe ingredient for the preparation of food supplements for managing obesity. © 2024 Society of Chemical Industry.

Mass Spectrometry Characterization of the SDS-PAGE Protein Profile of Legumins and Vicilins from Chickpea Seed.

Chickpea (Cicer arietinum L.) seed proteins show a lot of functional properties leading this legume to be an interesting component for the development of protein-enriched foods. However, both the in-depth proteomic investigation and structural characterization of chickpea seed proteins are still lacking. In this paper a detailed characterization of chickpea seed protein fraction by means of SDS-PAGE, in-gel protein digestion, high-resolution mass spectrometry, and database searching is reported. Through this approach, twenty SDS gel bands were cut and analyzed. While the majority of the bands and the identified peptides were related to vicilin and legumin storage proteins, metabolic functional proteins were also detected. Legumins, as expected, were revealed at 45-65 kDa, as whole subunits with the α- and ß-chains linked together by a disulphide bond, but also at lower mass ranges (α- and ß-chains migrating alone). Similarly, but not expected, the vicilins were also spread along the mass region between 65 and 23 kDa, with some of them being identified in several bands. An MS structural characterization allowed to determine that, although chickpea vicilins were always described as proteins lacking cysteine residues, they contain this amino acid residue. Moreover, similar to legumins, these storage proteins are firstly synthesized as pre-propolypeptides (Mr 50-80 kDa) that may undergo proteolytic steps that not only cut the signal peptides but also produce different subunits with lower molecular masses. Overall, about 360 different proteins specific of the Cicer arietinum L. species were identified and characterized, a result that, up to the current date, represents the most detailed description of the seed proteome of this legume.

Lipid Nanoparticles Loading Steroidal Alkaloids of Tomatoes Affect Neuroblastoma Cell Viability in an In Vitro Model.

Tomato by-products represent a good source of phytochemical compounds with health properties, such as the steroidal glycoalkaloid α-tomatine (α-TM) and its aglycone tomatidine (TD). Both molecules have numerous beneficial properties, such as potential anticancer activity. Unfortunately, their therapeutic application is limited due to stability and bioavailability issues. Therefore, a valid strategy seems to be their encapsulation into Solid Lipid Nanoparticles (SLN). The nanoformulations containing α-TM (α-TM-SLN) and TD (TD-SLN) were prepared by solvent-diffusion technique and subsequently characterized in terms of technological parameters (particle size, polydispersity index, zeta potential, microscopy, and calorimetric studies). To assess the effect of α-TM and TD on the percentage of cellular viability in Olfactory Ensheathing Cells (OECs), a peculiar glial cell type of the olfactory system used as normal cells, and in SH-SY5Y, a neuroblastoma cancer cell line, an MTT test was performed. In addition, the effects of empty, α-TM-SLN, and TD-SLN were tested. Our results show that the treatment of OECs with blank-SLN, free α-TM (0.25 µg/mL), and TD (0.50 µg/mL) did not induce any significant change in the percentage of cell viability when compared with the control. In contrast, in SH-SY5Y-treated cells, a significant decrease in the percentage of cell viability when compared with the control was found. In particular, the effect appeared more evident when SH-SY5Y cells were exposed to α-TM-SLN and TD-SLN. No significant effect in blank-SLN-treated SH-SY5T cells was observed. Therefore, SLN is a promising approach for the delivery of α-TM and TD.

Count Dracula Resurrected: Proteomic Analysis of Vlad III the Impaler's Documents by EVA Technology and Mass Spectrometry.

The interest of scientists in analyzing items of World Cultural Heritage has been exponentially increasing since the beginning of the new millennium. These studies have grown considerably in tandem with the development and use of sophisticated and sensitive technologies such as high-resolution mass spectrometry (MS) and the non-invasive and non-damaging technique, known under the acronym EVA (ethylene-vinyl acetate). Here, we report the results of the MS characterization of the peptides and proteins harvested by the EVA technology applied to three letters written in 1457 and 1475 by the voivode of Wallachia, Vlad III, also known as Vlad the Impaler, or Vlad Dracula. The discrimination of the "original" endogenous peptides from contaminant ones was obtained by monitoring their different levels of deamidation and of other diagenetic chemical modifications. The characterization of the ancient proteins extracted from these documents allowed us to explore the environmental conditions, in the second half of the 15th century, of the Wallachia, a region considered as a meeting point for soldiers, migrants, and travelers that probably carried not only trade goods and cultural traditions but also diseases and epidemics. In addition, the identification of many human peptides and proteins harvested from the letters allowed us to uncover more about Vlad Dracula the Impaler. Particularly, the experimental data show that he probably suffered from inflammatory processes of the respiratory tract and/or of the skin. In addition, proteomics data, although not exhaustive, suggest that, according to some stories, he might also have suffered from a pathological condition called hemolacria, that is, he could shed tears admixed with blood. It is worth noting that more medieval people may have touched these documents, which cannot be denied, but it is also presumable that the most prominent ancient proteins should be related to Prince Vlad the Impaler, who wrote and signed these letters. The data have been deposited to the ProteomeXchange with the identifier ⟨PXD041350⟩.

The TriMet_DB: A Manually Curated Database of the Metabolic Proteins of Triticum aestivum.

Mass-spectrometry-based wheat proteomics is challenging because the current interpretation of mass spectrometry data relies on public databases that are not exhaustive (UniProtKB/Swiss-Prot) or contain many redundant and poor or un-annotated entries (UniProtKB/TrEMBL). Here, we report the development of a manually curated database of the metabolic proteins of Triticum aestivum (hexaploid wheat), named TriMet_DB (Triticum aestivum Metabolic Proteins DataBase). The manually curated TriMet_DB was generated in FASTA format so that it can be read directly by programs used to interpret the mass spectrometry data. Furthermore, the complete list of entries included in the TriMet_DB is reported in a freely available resource, which includes for each protein the description, the gene code, the protein family, and the allergen name (if any). To evaluate its performance, the TriMet_DB was used to interpret the MS data acquired on the metabolic protein fraction extracted from the cultivar MEC of Triticum aestivum. Data are available via ProteomeXchange with identifier PXD037709.

Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry.

Damage induced by oxidative stress is a key driver of the selective motor neuron death in amyotrophic lateral sclerosis (ALS). Mitochondria are among the main producers of ROS, but they also suffer particularly from their harmful effects. Voltage-dependent anion-selective channels (VDACs) are the most represented proteins of the outer mitochondrial membrane where they form pores controlling the permeation of metabolites responsible for mitochondrial functions. For these reasons, VDACs contribute to mitochondrial quality control and the entire energy metabolism of the cell. In this work we assessed in an ALS cell model whether disease-related oxidative stress induces post-translational modifications (PTMs) in VDAC3, a member of the VDAC family of outer mitochondrial membrane channel proteins, known for its role in redox signaling. At this end, protein samples enriched in VDACs were prepared from mitochondria of an ALS model cell line, NSC34 expressing human SOD1G93A, and analyzed by nUHPLC/High-Resolution nESI-MS/MS. Specific over-oxidation, deamidation, succination events were found in VDAC3 from ALS-related NSC34-SOD1G93A but not in non-ALS cell lines. Additionally, we report evidence that some PTMs may affect VDAC3 functionality. In particular, deamidation of Asn215 alone alters single channel behavior in artificial membranes. Overall, our results suggest modifications of VDAC3 that can impact its protective role against ROS, which is particularly important in the ALS context. Data are available via ProteomeXchange with identifier PXD036728.

Meta-proteomic analysis of two mammoth's trunks by EVA technology and high-resolution mass spectrometry for an indirect picture of their habitat and the characterization of the collagen type I, alpha-1 and alpha-2 sequence.

The recent paleoproteomic studies, including paleo-metaproteomic analyses, improved our understanding of the dietary of ancient populations, the characterization of past human diseases, the reconstruction of the habitat of ancient species, but also provided new insights into the phylogenetic relationships between extant and extinct species. In this respect, the present work reports the results of the metaproteomic analysis performed on the middle part of a trunk, and on the portion of a trunk tip tissue of two different woolly mammoths some 30,000 years old. In particular, proteins were extracted by applying EVA (Ethylene-Vinyl Acetate studded with hydrophilic and hydrophobic resins) films to the surface of these tissues belonging to two Mammuthus primigenus specimens, discovered in two regions located in the Russian Far East, and then investigated via a shotgun MS-based approach. This approach allowed to obtain two interesting results: (i) an indirect description of the habitat of these two mammoths, and (ii) an improved characterization of the collagen type I, alpha-1 and alpha-2 chains (col1a1 and col1a2). Sequence characterization of the col1a1 and col1a2 highlighted some differences between M. primigenius and other Proboscidea together with the identification of three (two for col1a1, and one for col1a2) potentially diagnostic amino acidic mutations that could be used to reliably distinguish the Mammuthus primigenius with respect to the other two genera of elephantids (i.e., Elephas and Loxodonta), and the extinct American mastodon (i.e., Mammut americanum). The results were validated through the level of deamidation and other diagenetic chemical modifications of the sample peptides, which were used to discriminate the "original" endogenous peptides from contaminant ones. The data have been deposited to the ProteomeXchange with identifier < PXD029558 > .

VDACs Post-Translational Modifications Discovery by Mass Spectrometry: Impact on Their Hub Function.

VDAC (voltage-dependent anion selective channel) proteins, also known as mitochondrial porins, are the most abundant proteins of the outer mitochondrial membrane (OMM), where they play a vital role in various cellular processes, in the regulation of metabolism, and in survival pathways. There is increasing consensus about their function as a cellular hub, connecting bioenergetics functions to the rest of the cell. The structural characterization of VDACs presents challenging issues due to their very high hydrophobicity, low solubility, the difficulty to separate them from other mitochondrial proteins of similar hydrophobicity and the practical impossibility to isolate each single isoform. Consequently, it is necessary to analyze them as components of a relatively complex mixture. Due to the experimental difficulties in their structural characterization, post-translational modifications (PTMs) of VDAC proteins represent a little explored field. Only in recent years, the increasing number of tools aimed at identifying and quantifying PTMs has allowed to increase our knowledge in this field and in the mechanisms that regulate functions and interactions of mitochondrial porins. In particular, the development of nano-reversed phase ultra-high performance liquid chromatography (nanoRP-UHPLC) and ultra-sensitive high-resolution mass spectrometry (HRMS) methods has played a key role in this field. The findings obtained on VDAC PTMs using such methodologies, which permitted an in-depth characterization of these very hydrophobic trans-membrane pore proteins, are summarized in this review.

Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation.

Mitochondria from affected tissues of amyotrophic lateral sclerosis (ALS) patients show morphological and biochemical abnormalities. Mitochondrial dysfunction causes oxidative damage and the accumulation of ROS, and represents one of the major triggers of selective death of motor neurons in ALS. We aimed to assess whether oxidative stress in ALS induces post-translational modifications (PTMs) in VDAC1, the main protein of the outer mitochondrial membrane and known to interact with SOD1 mutants related to ALS. In this work, specific PTMs of the VDAC1 protein purified by hydroxyapatite from mitochondria of a NSC34 cell line expressing human SOD1G93A, a suitable ALS motor neuron model, were analyzed by tryptic and chymotryptic proteolysis and UHPLC/High-Resolution ESI-MS/MS. We found selective deamidations of asparagine and glutamine of VDAC1 in ALS-related NSC34-SOD1G93A cells but not in NSC34-SOD1WT or NSC34 cells. In addition, we identified differences in the over-oxidation of methionine and cysteines between VDAC1 purified from ALS model or non-ALS NSC34 cells. The specific range of PTMs identified exclusively in VDAC1 from NSC34-SOD1G93A cells but not from NSC34 control lines, suggests the appearance of important changes to the structure of the VDAC1 channel and therefore to the bioenergetics metabolism of ALS motor neurons. Data are available via ProteomeXchange with identifier .

Cysteine Oxidations in Mitochondrial Membrane Proteins: The Case of VDAC Isoforms in Mammals.

Cysteine residues are reactive amino acids that can undergo several modifications driven by redox reagents. Mitochondria are the source of an abundant production of radical species, and it is surprising that such a large availability of highly reactive chemicals is compatible with viable and active organelles, needed for the cell functions. In this work, we review the results highlighting the modifications of cysteines in the most abundant proteins of the outer mitochondrial membrane (OMM), that is, the voltage-dependent anion selective channel (VDAC) isoforms. This interesting protein family carries several cysteines exposed to the oxidative intermembrane space (IMS). Through mass spectrometry (MS) analysis, cysteine posttranslational modifications (PTMs) were precisely determined, and it was discovered that such cysteines can be subject to several oxidization degrees, ranging from the disulfide bridge to the most oxidized, the sulfonic acid, one. The large spectra of VDAC cysteine oxidations, which is unique for OMM proteins, indicate that they have both a regulative function and a buffering capacity able to counteract excess of mitochondrial reactive oxygen species (ROS) load. The consequence of these peculiar cysteine PTMs is discussed.