Proteomic Analysis of the Endosperm Ontogeny of Jatropha curcas L. Seeds

Seeds of Jatropha curcas L. represent a potential source of raw material for the production of biodiesel. However, this use is hampered by the lack of basic information on the biosynthetic pathways associated with synthesis of toxic diterpenes, fatty acids, and triacylglycerols, as well as the pattern of deposition of storage proteins during seed development. In this study, we performed an in-depth proteome analysis of the endosperm isolated from five developmental stages which resulted in the identification of 1517, 1256, 1033, 752, and 307 proteins, respectively, summing up 1760 different proteins. Proteins with similar label free quantitation expression pattern were grouped into five clusters. The biological significance of these identifications is discussed with special focus on the analysis of seed storage proteins, proteins involved in the metabolism of fatty acids, carbohydrates, toxic components and proteolytic processing. Although several enzymes belonging to the biosynthesis of diterpenoid precursors were identified, we were unable to find any terpene synthase/cyclase, indicating that the synthesis of phorbol esters, the main toxic diterpenes, does not occur in seeds. The strategy used enabled us to provide a first in depth proteome analysis of the developing endosperm of this biodiesel plant, providing an important glimpse into the enzymatic machinery devoted to the production of C and N sources to sustain seed development

Table_2_Proteome level analysis of drug-resistant Prevotella melaninogenica for the identification of novel therapeutic candidates.DOCX

The management of infectious diseases has become more critical due to the development of novel pathogenic strains with enhanced resistance. Prevotella melaninogenica, a gram-negative bacterium, was found to be involved in various infections of the respiratory tract, aerodigestive tract, and gastrointestinal tract. The need to explore novel drug and vaccine targets against this pathogen was triggered by the emergence of antimicrobial resistance against reported antibiotics to combat P. melaninogenica infections. The study involves core genes acquired from 14 complete P. melaninogenica strain genome sequences, where promiscuous drug and vaccine candidates were explored by state-of-the-art subtractive proteomics and reverse vaccinology approaches. A stringent bioinformatics analysis enlisted 18 targets as novel, essential, and non-homologous to humans and having druggability potential. Moreover, the extracellular and outer membrane proteins were subjected to antigenicity, allergenicity, and physicochemical analysis for the identification of the candidate proteins to design multi-epitope vaccines. Two candidate proteins (ADK95685.1 and ADK97014.1) were selected as the best target for the designing of a vaccine construct. Lead B- and T-cell overlapped epitopes were joined to generate potential chimeric vaccine constructs in combination with adjuvants and linkers. Finally, a prioritized vaccine construct was found to have stable interactions with the human immune cell receptors as confirmed by molecular docking and MD simulation studies. The vaccine construct was found to have cloning and expression ability in the bacterial cloning system. Immune simulation ensured the elicitation of significant immune responses against the designed vaccine. In conclusion, our study reported novel drug and vaccine targets and designed a multi-epitope vaccine against the P. melaninogenica infection. Further experimental validation will help open new avenues in the treatment of this multi-drug-resistant pathogen.</p

Table_1_Proteome level analysis of drug-resistant Prevotella melaninogenica for the identification of novel therapeutic candidates.DOCX

The management of infectious diseases has become more critical due to the development of novel pathogenic strains with enhanced resistance. Prevotella melaninogenica, a gram-negative bacterium, was found to be involved in various infections of the respiratory tract, aerodigestive tract, and gastrointestinal tract. The need to explore novel drug and vaccine targets against this pathogen was triggered by the emergence of antimicrobial resistance against reported antibiotics to combat P. melaninogenica infections. The study involves core genes acquired from 14 complete P. melaninogenica strain genome sequences, where promiscuous drug and vaccine candidates were explored by state-of-the-art subtractive proteomics and reverse vaccinology approaches. A stringent bioinformatics analysis enlisted 18 targets as novel, essential, and non-homologous to humans and having druggability potential. Moreover, the extracellular and outer membrane proteins were subjected to antigenicity, allergenicity, and physicochemical analysis for the identification of the candidate proteins to design multi-epitope vaccines. Two candidate proteins (ADK95685.1 and ADK97014.1) were selected as the best target for the designing of a vaccine construct. Lead B- and T-cell overlapped epitopes were joined to generate potential chimeric vaccine constructs in combination with adjuvants and linkers. Finally, a prioritized vaccine construct was found to have stable interactions with the human immune cell receptors as confirmed by molecular docking and MD simulation studies. The vaccine construct was found to have cloning and expression ability in the bacterial cloning system. Immune simulation ensured the elicitation of significant immune responses against the designed vaccine. In conclusion, our study reported novel drug and vaccine targets and designed a multi-epitope vaccine against the P. melaninogenica infection. Further experimental validation will help open new avenues in the treatment of this multi-drug-resistant pathogen.</p

Table_4_Proteome level analysis of drug-resistant Prevotella melaninogenica for the identification of novel therapeutic candidates.DOCX

The management of infectious diseases has become more critical due to the development of novel pathogenic strains with enhanced resistance. Prevotella melaninogenica, a gram-negative bacterium, was found to be involved in various infections of the respiratory tract, aerodigestive tract, and gastrointestinal tract. The need to explore novel drug and vaccine targets against this pathogen was triggered by the emergence of antimicrobial resistance against reported antibiotics to combat P. melaninogenica infections. The study involves core genes acquired from 14 complete P. melaninogenica strain genome sequences, where promiscuous drug and vaccine candidates were explored by state-of-the-art subtractive proteomics and reverse vaccinology approaches. A stringent bioinformatics analysis enlisted 18 targets as novel, essential, and non-homologous to humans and having druggability potential. Moreover, the extracellular and outer membrane proteins were subjected to antigenicity, allergenicity, and physicochemical analysis for the identification of the candidate proteins to design multi-epitope vaccines. Two candidate proteins (ADK95685.1 and ADK97014.1) were selected as the best target for the designing of a vaccine construct. Lead B- and T-cell overlapped epitopes were joined to generate potential chimeric vaccine constructs in combination with adjuvants and linkers. Finally, a prioritized vaccine construct was found to have stable interactions with the human immune cell receptors as confirmed by molecular docking and MD simulation studies. The vaccine construct was found to have cloning and expression ability in the bacterial cloning system. Immune simulation ensured the elicitation of significant immune responses against the designed vaccine. In conclusion, our study reported novel drug and vaccine targets and designed a multi-epitope vaccine against the P. melaninogenica infection. Further experimental validation will help open new avenues in the treatment of this multi-drug-resistant pathogen.</p

Proteome Analysis of the Inner Integument from Developing <i>Jatropha curcas</i> L. Seeds

In this study, we performed a systematic proteomic analysis of the inner integument from developing seeds of <i>Jatropha curcas</i> and further explored the protein machinery responsible for generating the carbon and nitrogen sources to feed the growing embryo and endosperm. The inner integument of developing seeds was dissected into two sections called distal and proximal, and proteins were extracted from these sections and from the whole integument and analyzed using an EASY-nanoLC system coupled to an ESI-LTQ-Orbitrap Velos mass spectrometer. We identified 1526, 1192, and 1062 proteins from the proximal, distal, and whole inner integuments, respectively. The identifications include those of peptidases and other hydrolytic enzymes that play a key role in developmental programmed cell death and proteins associated with the cell-wall architecture and modification. Because many of these proteins are differentially expressed within the integument cell layers, these findings suggest that the cells mobilize an array of hydrolases to produce carbon and nitrogen sources from proteins, carbohydrates, and lipids available within the cells. Not least, the identification of several classes of seed storage proteins in the inner integument provides additional evidence of the role of the seed coat as a transient source of reserves for the growing embryo and endosperm

Table_3_Proteome level analysis of drug-resistant Prevotella melaninogenica for the identification of novel therapeutic candidates.DOCX

The management of infectious diseases has become more critical due to the development of novel pathogenic strains with enhanced resistance. Prevotella melaninogenica, a gram-negative bacterium, was found to be involved in various infections of the respiratory tract, aerodigestive tract, and gastrointestinal tract. The need to explore novel drug and vaccine targets against this pathogen was triggered by the emergence of antimicrobial resistance against reported antibiotics to combat P. melaninogenica infections. The study involves core genes acquired from 14 complete P. melaninogenica strain genome sequences, where promiscuous drug and vaccine candidates were explored by state-of-the-art subtractive proteomics and reverse vaccinology approaches. A stringent bioinformatics analysis enlisted 18 targets as novel, essential, and non-homologous to humans and having druggability potential. Moreover, the extracellular and outer membrane proteins were subjected to antigenicity, allergenicity, and physicochemical analysis for the identification of the candidate proteins to design multi-epitope vaccines. Two candidate proteins (ADK95685.1 and ADK97014.1) were selected as the best target for the designing of a vaccine construct. Lead B- and T-cell overlapped epitopes were joined to generate potential chimeric vaccine constructs in combination with adjuvants and linkers. Finally, a prioritized vaccine construct was found to have stable interactions with the human immune cell receptors as confirmed by molecular docking and MD simulation studies. The vaccine construct was found to have cloning and expression ability in the bacterial cloning system. Immune simulation ensured the elicitation of significant immune responses against the designed vaccine. In conclusion, our study reported novel drug and vaccine targets and designed a multi-epitope vaccine against the P. melaninogenica infection. Further experimental validation will help open new avenues in the treatment of this multi-drug-resistant pathogen.</p

Image_2_Proteome level analysis of drug-resistant Prevotella melaninogenica for the identification of novel therapeutic candidates.JPEG

The management of infectious diseases has become more critical due to the development of novel pathogenic strains with enhanced resistance. Prevotella melaninogenica, a gram-negative bacterium, was found to be involved in various infections of the respiratory tract, aerodigestive tract, and gastrointestinal tract. The need to explore novel drug and vaccine targets against this pathogen was triggered by the emergence of antimicrobial resistance against reported antibiotics to combat P. melaninogenica infections. The study involves core genes acquired from 14 complete P. melaninogenica strain genome sequences, where promiscuous drug and vaccine candidates were explored by state-of-the-art subtractive proteomics and reverse vaccinology approaches. A stringent bioinformatics analysis enlisted 18 targets as novel, essential, and non-homologous to humans and having druggability potential. Moreover, the extracellular and outer membrane proteins were subjected to antigenicity, allergenicity, and physicochemical analysis for the identification of the candidate proteins to design multi-epitope vaccines. Two candidate proteins (ADK95685.1 and ADK97014.1) were selected as the best target for the designing of a vaccine construct. Lead B- and T-cell overlapped epitopes were joined to generate potential chimeric vaccine constructs in combination with adjuvants and linkers. Finally, a prioritized vaccine construct was found to have stable interactions with the human immune cell receptors as confirmed by molecular docking and MD simulation studies. The vaccine construct was found to have cloning and expression ability in the bacterial cloning system. Immune simulation ensured the elicitation of significant immune responses against the designed vaccine. In conclusion, our study reported novel drug and vaccine targets and designed a multi-epitope vaccine against the P. melaninogenica infection. Further experimental validation will help open new avenues in the treatment of this multi-drug-resistant pathogen.</p

Image_1_Proteome level analysis of drug-resistant Prevotella melaninogenica for the identification of novel therapeutic candidates.JPEG

The management of infectious diseases has become more critical due to the development of novel pathogenic strains with enhanced resistance. Prevotella melaninogenica, a gram-negative bacterium, was found to be involved in various infections of the respiratory tract, aerodigestive tract, and gastrointestinal tract. The need to explore novel drug and vaccine targets against this pathogen was triggered by the emergence of antimicrobial resistance against reported antibiotics to combat P. melaninogenica infections. The study involves core genes acquired from 14 complete P. melaninogenica strain genome sequences, where promiscuous drug and vaccine candidates were explored by state-of-the-art subtractive proteomics and reverse vaccinology approaches. A stringent bioinformatics analysis enlisted 18 targets as novel, essential, and non-homologous to humans and having druggability potential. Moreover, the extracellular and outer membrane proteins were subjected to antigenicity, allergenicity, and physicochemical analysis for the identification of the candidate proteins to design multi-epitope vaccines. Two candidate proteins (ADK95685.1 and ADK97014.1) were selected as the best target for the designing of a vaccine construct. Lead B- and T-cell overlapped epitopes were joined to generate potential chimeric vaccine constructs in combination with adjuvants and linkers. Finally, a prioritized vaccine construct was found to have stable interactions with the human immune cell receptors as confirmed by molecular docking and MD simulation studies. The vaccine construct was found to have cloning and expression ability in the bacterial cloning system. Immune simulation ensured the elicitation of significant immune responses against the designed vaccine. In conclusion, our study reported novel drug and vaccine targets and designed a multi-epitope vaccine against the P. melaninogenica infection. Further experimental validation will help open new avenues in the treatment of this multi-drug-resistant pathogen.</p

Proteome Analysis of Plastids from Developing Seeds of <i>Jatropha curcas</i> L.

In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing <i>Jatropha curcas</i> seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled inline to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 proteins were considered as true identifications, and this considerably expands the repertoire of <i>J. curcas</i> proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in PPDB, SUBA, or PlProt databases while homologues for 13 proteins were not found in any of the three plastid proteins databases but were marked as plastidial by at least one of the three prediction programs used. Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in the plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO₂ as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, our study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level

Proteome Analysis of Plastids from Developing Seeds of <i>Jatropha curcas</i> L.

In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing <i>Jatropha curcas</i> seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled inline to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 proteins were considered as true identifications, and this considerably expands the repertoire of <i>J. curcas</i> proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in PPDB, SUBA, or PlProt databases while homologues for 13 proteins were not found in any of the three plastid proteins databases but were marked as plastidial by at least one of the three prediction programs used. Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in the plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO₂ as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, our study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level