Ying Wai Lam

Comparative Decellularization and Recellularization of Normal versus Emphysematous Human Lungs

Acellular whole human lung scaffolds represent a unique opportunity for ex vivo tissue engineering. However, it remains unclear whether lungs from individuals with chronic lung diseases such as chronic obstructive pulmonary disease (COPD) can be appropriately decellularized and recellularized. To assess this, cadaveric human lungs from normal (non-smoking) patients and from patients with COPD (smoking history) were decellularized and found by histochemical and immunohistochemical staining, electron microscopy, and mass spectrometry to retain characteristic histological architecture and extracellular matrix components (ECM) reflecting either normal or COPD, particularly emphysematous, origin. Inoculation of human bronchial epithelial cells, endothelial progenitor cells, bone marrow-derived mesenchymal stem cells, and lung fibroblasts via airway or vascular routes into small, excised segments of the decellularized lungs demonstrated that normal lung scaffolds robustly supported initial engraftment and growth of each cell type for up to one month. In contrast, despite initial binding, all cell types inoculated into decellularized emphysematous lungs did not survive beyond one week. However, cell attachment and proliferation on solubilized ECM homogenates of decellularized normal and emphysematous lungs coated onto tissue culture plates was comparable and not impaired, suggesting that the 3-dimensional decellularized emphysematous scaffolds may lack the necessary ECM architecture to support sustained cell growth.

Ribosome-inactivating proteins isolated from dietary bitter melon induce apoptosis and inhibit histone deacetylase-1 selectively in premalignant and malignant prostate cancer cells

Epidemiologic evidence suggests that a diet rich in fruits and vegetables is associated with a reduced risk of prostate cancer (PCa) development. Although several dietary compounds have been tested in preclinical PCa prevention models, no agents have been identified that either prevent the progression of premalignant lesions or treat advanced disease. Momordica charantia, known as bitter melon in English, is a plant that grows in tropical areas worldwide and is both eaten as a vegetable and used for medicinal purposes. We have isolated a protein, designated as MCP30, from bitter melon seeds. The purified fraction was verified by SDS‐PAGE and mass spectrometry to contain only 2 highly related single chain Type I ribosome‐inactivating proteins (RIPs), α‐momorcharin and β‐momorcharin. MCP30 induces apoptosis in PIN and PCa cell lines in vitro and suppresses PC‐3 growth in vivo with no effect on normal prostate cells. Mechanistically, MCP30 inhibits histone deacetylase‐1 (HDAC‐1) activity and promotes histone‐3 and ‐4 protein acetylation. Treatment with MCP30 induces PTEN expression in a prostatic intraepithelial neoplasia (PIN) and PCa cell lines resulting in inhibition of Akt phosphorylation. In addition, MCP30 inhibits Wnt signaling activity through reduction of nuclear accumulation of β‐catenin and decreased levels of c‐Myc and Cyclin‐D1. Our data indicate that MCP30 selectively induces PIN and PCa apoptosis and inhibits HDAC‐1 activity. These results suggest that Type I RIPs derived from plants are HDAC inhibitors that can be utilized in the prevention and treatment of prostate cancer.

Comprehensive Identification and Modified-Site Mapping of S- Nitrosylated Targets in Prostate Epithelial Cells

Background Although overexpression of nitric oxide synthases (NOSs) has been found associated with prostate diseases, the underlying mechanisms for NOS-related prostatic diseases remain unclear. One proposed mechanism is related to the S-nitrosylation of key regulatory proteins in cell-signaling pathways due to elevated levels of NO in the prostate. Thus, our primary objective was to identify S-nitrosylated targets in an immortalized normal prostate epithelial cell line, NPrEC. Methodology/Principal Findings We treated NPrEC with nitroso-cysteine and used the biotin switch technique followed by gel-based separation and mass spectrometry protein identification (using the LTQ-Orbitrap) to discover S-nitrosylated (SNO) proteins in the treated cells. In parallel, we adapted a peptide pull-down methodology to locate the site(s) of S-nitrosylation on the protein SNO targets identified by the first technique. This combined approach identified 116 SNO proteins and determined the sites of modification for 82 of them. Over 60% of these proteins belong to four functional groups: cell structure/cell motility/protein trafficking, protein folding/protein response/protein assembly, mRNA splicing/processing/transcriptional regulation, and metabolism. Western blot analysis validated a subset of targets related to disease development (proliferating cell nuclear antigen, maspin, integrin β4, α-catenin, karyopherin [importin] β1, and elongation factor 1A1). We analyzed the SNO sequences for their primary and secondary structures, solvent accessibility, and three-dimensional structural context. We found that about 80% of the SNO sites that can be mapped into resolved structures are buried, of which approximately half have charged amino acids in their three-dimensional neighborhood, and the other half residing within primarily hydrophobic pockets. Conclusions/Significance We here identified 116 potential SNO targets and mapped their putative SNO sites in NPrEC. Elucidation of how this post-translational modification alters the function of these proteins should shed light on the role of NO in prostate pathologies. To our knowledge, this is the first report identifying SNO targets in prostate epithelial cells.

Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)

Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO(2)) the recalcitrant fused-ring high molecular weight (4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1-Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3-5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493-547 aa) are structurally diverse and novel considering their low overall homology (12-23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.

The effect of age and emphysematous and fibrotic injury on the re-cellularization of de-cellularized lungs.

Use of de-cellularized cadaveric lungs as 3-dimensional scaffolds for ex vivo lung tissue generation offers a new potential therapeutic approach for clinical lung transplantation. However, it is likely that some of the available cadaveric human lungs may be from older donors or from donors with previously existing structural lung diseases such as emphysema or pulmonary fibrosis. It is not known whether these lungs will be suitable for either de-cellularization or re-cellularization. To investigate this, we assessed the effects of advanced age, representative emphysematous and fibrotic injuries, and the combination of advanced age and emphysematous injury and found significant differences both in histologic appearance and in the retention of extracellular matrix (ECM) and other proteins, as assessed by immunohistochemistry and mass spectrometry, between the different conditions. However, despite these differences, binding, retention and growth of bone marrow-derived mesenchymal stromal cells (MSCs) over a 1-month period following intratracheal inoculation were similar between the different experimental conditions. In contrast, significant differences occurred in the growth of C10 mouse lung epithelial cells between the different conditions. Therefore, age, lung injury, and the cell type used for re-cellularization may significantly impact the usefulness of de-cellularized whole lungs for ex vivo lung tissue regeneration.

The effects of storage and sterilization on de-cellularized and re-cellularized whole lung.

Despite growing interest on the potential use of de-cellularized whole lungs as 3-dimensional scaffolds for ex vivo lung tissue generation, optimal processing including sterilization and storage conditions, are not well defined. Further, it is unclear whether lungs need to be obtained immediately or may be usable even if harvested several days post-mortem, a situation mimicking potential procurement of human lungs from autopsy. We therefore assessed effects of delayed necropsy, prolonged storage (3 and 6 months), and of two commonly utilized sterilization approaches: irradiation or final rinse with peracetic acid, on architecture and extracellular matrix (ECM) protein characteristics of de-cellularized mouse lungs. These different approaches resulted in significant differences in both histologic appearance and in retention of ECM and intracellular proteins as assessed by immunohistochemistry and mass spectrometry. Despite these differences, binding and proliferation of bone marrow-derived mesenchymal stromal cells (MSCs) over a one month period following intratracheal inoculation was similar between experimental conditions. In contrast, significant differences occurred with C10 mouse lung epithelial cells between the different conditions. Therefore, delayed necropsy, duration of scaffold storage, sterilization approach, and cell type used for re-cellularization may significantly impact the usefulness of this biological scaffold-based model of ex vivo lung tissue regeneration.

CYP63A2, a Catalytically Versatile Fungal P450 Monooxygenase Capable of Oxidizing Higher-Molecular-Weight Polycyclic Aromatic Hydrocarbons, Alkylphenols, and Alkanes

ABSTRACT Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons, albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbon n-alkanes. A homology-based three-dimensional (3D) model revealed the presence of an extraordinarily large active-site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2, and CYP1B1) and bacterial aliphatic-hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of various ring sizes, including 4 rings (pyrene and fluoranthene), 5 rings [benzo(a)pyrene], and 6 rings [benzo(ghi)perylene], with the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. Recombinant CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane ω-hydroxylase that oxidized n-alkanes with various chain lengths (C9 to C12 and C15 to C19), as well as alkyl side chains (C3 to C9) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long-chain APs and alkanes. To our knowledge, this is the first P450 identified from any of the biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills).

A Fungal P450 (CYP5136A3) Capable of Oxidizing Polycyclic Aromatic Hydrocarbons and Endocrine Disrupting Alkylphenols: Role of Trp129 and Leu324

The model white rot fungus Phanerochaete chrysosporium, which is known for its versatile pollutant-biodegradation ability, possesses an extraordinarily large repertoire of P450 monooxygenases in its genome. However, the majority of these P450s have hitherto unknown function. Our initial studies using a genome-wide gene induction strategy revealed multiple P450s responsive to individual classes of xenobiotics. Here we report functional characterization of a cytochrome P450 monooxygenase, CYP5136A3 that showed common responsiveness and catalytic versatility towards endocrine-disrupting alkylphenols (APs) and mutagenic/carcinogenic polycyclic aromatic hydrocarbons (PAHs). Using recombinant CYP5136A3, we demonstrated its oxidation activity towards APs with varying alkyl side-chain length (C3-C9), in addition to PAHs (3–4 ring size). AP oxidation involves hydroxylation at the terminal carbon of the alkyl side-chain (ω-oxidation). Structure-activity analysis based on a 3D model indicated a potential role of Trp129 and Leu324 in the oxidation mechanism of CYP5136A3. Replacing Trp129 with Leu (W129L) and Phe (W129F) significantly diminished oxidation of both PAHs and APs. The W129L mutation caused greater reduction in phenanthrene oxidation (80%) as compared to W129F which caused greater reduction in pyrene oxidation (88%). Almost complete loss of oxidation of C3-C8 APs (83–90%) was observed for the W129L mutation as compared to W129F (28–41%). However, the two mutations showed a comparable loss (60–67%) in C9-AP oxidation. Replacement of Leu324 with Gly (L324G) caused 42% and 54% decrease in oxidation activity towards phenanthrene and pyrene, respectively. This mutation also caused loss of activity towards C3-C8 APs (20–58%), and complete loss of activity toward nonylphenol (C9-AP). Collectively, the results suggest that Trp129 and Leu324 are critical in substrate recognition and/or regio-selective oxidation of PAHs and APs. To our knowledge, this is the first report on an AP-oxidizing P450 from fungi and on structure-activity relationship of a eukaryotic P450 for fused-ring PAHs (phenanthrene and pyrene) and AP substrates.

Characterization of the bovine milk proteome in early-lactation Holstein and Jersey breeds of dairy cows

UNLABELLED Milk is a highly nutritious natural product that provides not only a rich source of amino acids to the consumer but also hundreds of bioactive peptides and proteins known to elicit health-benefitting activities. We investigated the milk protein profile produced by Holstein and Jersey dairy cows maintained under the same diet, management and environmental conditions using proteomic approaches that optimize protein extraction and characterization of the low abundance proteins within the skim milk fraction of bovine milk. In total, 935 low abundance proteins were identified. Gene ontology classified all proteins identified into various cellular localization and function categories. A total of 43 low abundance proteins were differentially expressed between the two dairy breeds. Bioactive proteins involved in host-defense, including lactotransferrin (P=0.0026) and complement C2 protein (P=0.0001), were differentially expressed by the two breeds, whereas others such as osteopontin (P=0.1788) and lactoperoxidase (P=0.2973) were not. This work is the first to outline the protein profile produced by two important breeds of dairy cattle maintained under the same diet, environment and management conditions in order to observe likely true breed differences. This research now allows us to better understand and contrast further research examining the bovine proteome that includes these different breeds. BIOLOGICAL SIGNIFICANCE Within the last decade, the amount of research characterizing the bovine milk proteome has increased due to growing interest in the bioactive proteins that are present in milk. Proteomic analysis of low abundance whey proteins has mainly focused on human breast milk; however, previous research has highlighted the presence of bioactive proteins in bovine milk. Recent publications outlining the cross-reactivity of bovine bioactive proteins on human biological function highlight the need for further investigation into the bovine milk proteome. The rationale behind this study is to characterize and compare the low abundance protein profile in the skim milk fraction produced from Holstein and Jersey breeds of dairy cattle, which are two major dairy cattle breeds in the USA. A combination of fractionation strategies was used to efficiently enrich the low abundance proteins from bovine skim milk for proteomic profiling. A total of 935 low abundance proteins were identified and compared between the two bovine breeds. The results from this study provide insight into breed differences and similarities in the milk proteome profile produced by two breeds of dairy cattle.

Phosphorylation of human estrogen receptor-beta at serine 105 inhibits breast cancer cell migration and invasion

Multiple phosphorylation sites on the human estrogen receptor (hER)α were identified and shown to influence mammary carcinogenesis. In contrast, functional phosphorylation sites of hERβ have yet to be experimentally identified and validated. Here, using mass spectrometry, we uncovered three serines (S75, S87, and S105) in the N-terminus of hERβ as targets of ERK1/2 and p38 kinases. We raised a specific antibody against phosphorylated S105 (pS105) and demonstrated that this site was endogenously phosphorylated in MDA-MB-231 and BT-474 cells. A phospho-mimetic mutant generated from hERβ1 was found to exhibit higher transactivation activity than hERβ1. Ectopic expression of this mutant inhibited cell migration and invasion, but did not affect cell growth and cell-cycle progression in these cell models. In breast cancer specimens, pS105-hERβ immunoreactivity was detected with a higher prevalence and intensity than that of hERβ1. These results underscore the functional importance of the first experimentally identified hERβ-phosphorylation site in breast cancer.