19 research outputs found
An apoplastic fluid extraction method for the characterization of grapevine leaves proteome and metabolome from a single sample
The analysis of complex biological systems keeps challenging
researchers. The main goal of systems biology is to decipher interactions
within cells, by integrating datasets from large scale analytical
approaches including transcriptomics, proteomics and metabolomics
andmore specialized âOMICSâ such as epigenomics and lipidomics. Studying
different cellular compartments allows a broader understanding of cell
dynamics. Plant apoplast, the cellular compartment external to the plasma
membrane including the cell wall, is particularly demanding to analyze.
Despite our knowledge on apoplast involvement on several processes from
cell growth to stress responses, its dynamics is still poorly known due to the
lack of efficient extraction processes adequate to each plant system.Analyzing
woody plants such as grapevine raises even more challenges. Grapevine is
among the most important fruit crops worldwide and awider characterization
of its apoplast is essential for a deeper understanding of its physiology and cellular
mechanisms. Here, we describe, for the first time, a vacuum-infiltrationcentrifugationmethod
that allows a simultaneous extraction of grapevine apoplastic
proteins and metabolites from leaves on a single sample, compatible
with high-throughput mass spectrometry analyses. The extracted apoplast
from two grapevine cultivars, Vitis vinifera cv âTrincadeiraâ and âRegentâ, was
directly used for proteomics and metabolomics analysis. The proteome was
analyzed by nanoLC-MS/MS and more than 700 common proteinswere identified,
with highly diverse biological functions. The metabolome profile
through FT-ICR-MS allowed the identification of 514 unique putative compounds
revealing a broad spectrum of molecular classesinfo:eu-repo/semantics/publishedVersio
3D Profile-Based Approach to Proteome-Wide Discovery of Novel Human Chemokines
Chemokines are small secreted proteins with important roles in immune responses. They consist of a conserved three-dimensional (3D) structure, so-called IL8-like chemokine fold, which is supported by disulfide bridges characteristic of this protein family. Sequence- and profile-based computational methods have been proficient in discovering novel chemokines by making use of their sequence-conserved cysteine patterns. However, it has been recently shown that some chemokines escaped annotation by these methods due to low sequence similarity to known chemokines and to different arrangement of cysteines in sequence and in 3D. Innovative methods overcoming the limitations of current techniques may allow the discovery of new remote homologs in the still functionally uncharacterized fraction of the human genome. We report a novel computational approach for proteome-wide identification of remote homologs of the chemokine family that uses fold recognition techniques in combination with a scaffold-based automatic mapping of disulfide bonds to define a 3D profile of the chemokine protein family. By applying our methodology to all currently uncharacterized human protein sequences, we have discovered two novel proteins that, without having significant sequence similarity to known chemokines or characteristic cysteine patterns, show strong structural resemblance to known anti-HIV chemokines. Detailed computational analysis and experimental structural investigations based on mass spectrometry and circular dichroism support our structural predictions and highlight several other chemokine-like features. The results obtained support their functional annotation as putative novel chemokines and encourage further experimental characterization. The identification of remote homologs of human chemokines may provide new insights into the molecular mechanisms causing pathologies such as cancer or AIDS, and may contribute to the development of novel treatments. Besides, the genome-wide applicability of our methodology based on 3D protein family profiles may open up new possibilities for improving and accelerating protein function annotation processes
Splicing factors stimulate polyadenylation via USEs at non-canonical 3Ⲡend formation signals
The prothrombin (F2) 3Ⲡend formation signal is highly susceptible to thrombophilia-associated gain-of-function mutations. In its unusual architecture, the F2 3ⲠUTR contains an upstream sequence element (USE) that compensates for weak activities of the non-canonical cleavage site and the downstream U-rich element. Here, we address the mechanism of USE function. We show that the F2 USE contains a highly conserved nonameric core sequence, which promotes 3Ⲡend formation in a position- and sequence-dependent manner. We identify proteins that specifically interact with the USE, and demonstrate their function as trans-acting factors that promote 3Ⲡend formation. Interestingly, these include the splicing factors U2AF35, U2AF65 and hnRNPI. We show that these splicing factors not only modulate 3Ⲡend formation via the USEs contained in the F2 and the complement C2 mRNAs, but also in the biocomputationally identified BCL2L2, IVNS and ACTR mRNAs, suggesting a broader functional role. These data uncover a novel mechanism that functionally links the splicing and 3Ⲡend formation machineries of multiple cellular mRNAs in an USE-dependent manner