Angelito I. Nepomuceno

Systems Biology of Lignin Biosynthesis in Populus trichocarpa: Heteromeric 4-Coumaric Acid:Coenzyme A Ligase Protein Complex Formation, Regulation, and Numerical Modeling

This work shows that 4CL, an enzyme in monolignol biosynthesis, is found as a heterotetrameric complex of two isoforms in Populus trichocarpa. The activity of the heterotetramer can be described by a mathematical model that explains the effects of each isoform with mixtures of substrates and three types of inhibition, providing insights into the regulation of metabolic flux for this pathway. As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein–protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation.

Accurate identification of deamidated peptides in global proteomics using a quadrupole orbitrap mass spectrometer.

Deamidation of asparagine and glutamine residues is a common post-translational modification. Researchers often rely on mass spectrometric based proteomic techniques for the identification of these post-translational sites. Mass spectral analysis of deamidated peptides is complicated and often misassigned due to overlapping (13)C peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by 19.34 mDa. For proper assignment, it is inherently important to use a mass spectrometer with high mass measurement accuracy and high resolving power. Herein, mouse brain tissue lysate was prepared using filter-aided sample preparation (FASP) method and Stage Tip fractionation followed by analysis on a nanoLC coupled with a quadrupole orbitrap (Q-Exactive) mass spectrometer to accurately identify more than 5400 proteins. Mass spectral data was processed using MASCOT and ProteoIQ for accurate identification of peptides and proteins. MASCOT search values for precursor and MS/MS mass tolerances were investigated, and it was determined that data searched with greater than 5 ppm precursor mass tolerance resulted in the misassignment of deamidated peptides. Peptides that were identified with a mass measurement accuracy of ±5 ppm were correctly assigned.

Comparative Proteomic Analysis and IgE Binding Properties of Peanut Seed and Testa (Skin)

To investigate the protein composition and potential allergenicity of peanut testae or skins, proteome analysis was conducted using nanoLC-MS/MS sequencing. Initial amino acid analysis suggested differences in protein compositions between the blanched seed (skins removed) and skin. Phenolic compounds hindered analysis of proteins in skins when the conventional extraction method was used; therefore, phenol extraction of proteins was necessary. A total of 123 proteins were identified in blanched seed and skins, and 83 of the proteins were common between the two structures. The skins contained all of the known peanut allergens in addition to 38 proteins not identified in the seed. Multiple defense proteins with antifungal activity were identified in the skins. Western blotting using sera from peanut-allergic patients revealed that proteins extracted from both the blanched seed and skin bound significant levels of IgE. However, when phenolic compounds were present in the skin protein extract, no IgE binding was observed. These findings indicate that peanut skins contain potentially allergenic proteins; however, the presence of phenolic compounds may attenuate this effect.

Compartment Proteomics Analysis of White Perch (Morone americana) Ovary Using Support Vector Machines

Compartment proteomics enable broad characterization of target tissues. We employed a simple fractionation method and filter-aided sample preparation (FASP) to characterize the cytosolic and membrane fractions of white perch ovary tissues by semiquantitative tandem mass spectrometry using label-free quantitation based on normalized spectral counts. FASP depletes both low-molecular-weight and high-molecular-weight substances that could interfere with protein digestion and subsequent peptide separation and detection. Membrane proteins are notoriously difficult to characterize due to their amphipathic nature and association with lipids. The simple fractionation we employed effectively revealed an abundance of proteins from mitochondria and other membrane-bounded organelles. We further demonstrate that support vector machines (SVMs) offer categorical classification of proteomics data superior to that of parametric statistical methods such as analysis of variance (ANOVA). Specifically, SVMs were able to perfectly (100% correct) classify samples as either membrane or cytosolic fraction during cross-validation based on the expression of 242 proteins with the highest ANOVA p-values (i.e., those that were not significant for enrichment in either fraction). The white perch ovary cytosolic and membrane proteomes and transcriptome presented in this study can support future investigations into oogenesis and early embryogenesis of white perch and other members of the genus Morone.

C/EBPα regulates CRL4Cdt2-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint

The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4Cdt2. Cdt2 is the substrate recognition subunit of CRL4Cdt2 and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4Cdt2 mediated degradation of p21.

Machine learning reveals sex-specific 17β-estradiol-responsive expression patterns in white perch (Morone americana) plasma proteins

With growing abundance and awareness of endocrine disrupting compounds (EDCs) in the environment, there is a need for accurate and reliable detection of EDC exposure. Our objective in the present study was to observe differences within and between the global plasma proteomes of sexually mature male and female white perch (Morone americana) before (Initial Control, IC) and after 17β‐estradiol (E2) induction. Semiquantitative nanoLC‐MS/MS data were analyzed by machine learning support vector machines (SVMs) and by two‐way ANOVA. By ANOVA, the expression levels of 44, 77, and 57 proteins varied significantly by gender, treatment, and the interaction of gender and treatment, respectively. SVMs perfectly classified male and female perch IC and E2‐induced plasma samples using the protein expression data. E2‐induced male and female perch plasma proteomes contained significantly higher levels of the yolk precursors vitellogenin Aa and Ab (VtgAa, VtgAb), as well as latrophilin and seven transmembrane domain‐containing protein 1 (Eltd1) and kininogen 1 (Kng1). This is the first report that Eltd1 and Kng1 may be E2‐responsive proteins in fishes and therefore may be useful indicators of estrogen induction.

In-depth LC-MS/MS analysis of the chicken ovarian cancer proteome reveals conserved and novel differentially regulated proteins in humans

AbstractOvarian cancer (OVC) remains the most lethal gynecological malignancy in the world due to the combined lack of early-stage diagnostics and effective therapeutic strategies. The development and application of advanced proteomics technology and new experimental models has created unique opportunities for translational studies. In this study, we investigated the ovarian cancer proteome of the chicken, an emerging experimental model of OVC that develops ovarian tumors spontaneously. Matched plasma, ovary, and oviduct tissue biospecimens derived from healthy, early-stage OVC, and late-stage OVC birds were quantitatively characterized by label-free proteomics. Over 2600 proteins were identified in this study, 348 of which were differentially expressed by more than twofold (p ≤ 0.05) in early- and late-stage ovarian tumor tissue specimens relative to healthy ovarian tissues. Several of the 348 proteins are known to be differentially regulated in human cancers including B2M, CLDN3, EPCAM, PIGR, S100A6, S100A9, S100A11, and TPD52. Of particular interest was ovostatin 2 (OVOS2), a novel 165-kDa protease inhibitor found to be strongly upregulated in chicken ovarian tumors (p = 0.0005) and matched plasma (p = 0.003). Indeed, RT-quantitative PCR and Western blot analysis demonstrated that OVOS2 mRNA and protein were also upregulated in multiple human OVC cell lines compared to normal ovarian epithelia (NOE) cells and immunohistochemical staining confirmed overexpression of OVOS2 in primary human ovarian cancers relative to non-cancerous tissues. Collectively, these data provide the first evidence for involvement of OVOS2 in the pathogenesis of both chicken and human ovarian cancer. Graphical AbstractTranslational workflow for the LC-MS/MS identification of novel differentially expressed proteins such as ovostatin 2 (OVOS2) in the chicken followed by targeted analysis in humans

Global Proteomic Analysis of Functional Compartments in Immature Avian Follicles Using Laser Microdissection Coupled to LC-MS/MS

Laser microdissection (LMD) was utilized for the separation of the yolk, follicular wall (granulosa and theca), and surrounding stromal cells of small white follicles (SWF) obtained from reproductively active domestic fowl. Herein, we provide an in situ proteomics-based approach to studying follicular development through the use of LMD and mass spectrometry. This study resulted in a total of 2889 proteins identified from the three specific isolated compartments. White yolk from the smallest avian follicles resulted in the identification of 1984 proteins, while isolated follicular wall and ovarian stroma yielded 2470 and 2456 proteins, respectively. GO annotations highlighted the functional differences between the compartments. Among the three compartments examined, the relative abundance of vitellogenins, steroidogenic enzymes, anti-Mullerian hormone, transcription factors, and proteins involved in retinoic acid receptors/retinoic acid synthesis, transcription factors, and cell surface receptors such as EGFR and their associated signaling pathways reflected known cellular function of the ovary. This study has provided a global proteome for SWF, white yolk, and ovarian stroma of the avian ovary that can be used as a baseline for future studies and verifies that the coupling of LMD with proteomic analysis can be used to evaluate proteins from small, physiologically functional compartments of complex tissue.