Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

Rab8 Interacts with Distinct Motifs in α2B- and β2-Adrenergic Receptors and Differentially Modulates Their Transport

The molecular mechanism underlying the post-Golgi transport of G protein-coupled receptors (GPCRs) remains poorly understood. Here we determine the role of Rab8 GTPase, which modulates vesicular protein transport between the trans-Golgi network (TGN) and the plasma membrane, in the cell surface targeting of α_2B- and β₂-adrenergic receptors (AR). Transient expression of GDP- and GTP-bound Rab8 mutants and short hairpin RNA-mediated knockdown of Rab8 more potently inhibited the cell surface expression of α_2B-AR than β₂-AR. The GDP-bound Rab8(T22N) mutant attenuated ERK1/2 activation by α_2B-AR, but not β₂-AR, and arrested α_2B-AR in the TGN compartment. Co-immunoprecipitation revealed that both α_2B-AR and β₂-AR physically interacted with Rab8 and glutathione S-transferase fusion protein pulldown assays demonstrated that Rab8 interacted with the C termini of both receptors. Interestingly, mutation of the highly conserved membrane-proximal C terminus dileucine motif selectively blocked β₂-AR interaction with Rab8, whereas mutation of residues Val⁴³¹-Phe⁴³²-Asn⁴³³-Gln⁴³⁴, Pro⁴⁴⁷-Trp⁴⁴⁸, Gln⁴⁵⁰-Thr⁴⁵¹, and Trp⁴⁵³ in the C terminus impaired α_2B-AR interaction with Rab8. Furthermore, transport inhibition by Rab8(T22N) of a chimeric β₂-AR carrying the α_2B-AR C terminus was similar to α_2B-AR. These data provide strong evidence indicating that Rab8 GTPase interacts with distinct motifs in the C termini of α_2B-AR and β₂-AR and differentially modulates their traffic from the TGN to the cell surface.     

Protection of cullin-RING E3 ligases by CSN-UBP12

Neddylation, a process that conjugates the ubiquitin-like polypeptide NEDD8 to cullin proteins, activates cullin-RING ubiquitin ligases (CRLs). Deneddylation, in which the COP9 signalosome (CSN) removes NEDD8 from cullins, inactivates CRLs. However, genetic studies of CSN function conclude that deneddylation also promotes CRL activity. It has been proposed that a cyclic transition through neddylation and deneddylation is required for the regulation of CRL activity in vivo. Recent discoveries suggest that an additional level of complexity exists, whereby CRL components are targets for degradation, mediated either by autocatalytic ubiquitination or by unknown mechanisms. Deneddylation by CSN and deubiquitylation by CSN-associated ubiquitin-specific protease 12 protect CRL components from cellular depletion, thus maintaining the physiological CRL activities.     

Proteome analysis of human lung squamous carcinoma

Few lung cancer-specific molecular markers have been established in regard of "early-stage" diagnosis and prognosis. In this study the proteome analysis of human lung squamous carcinoma (hLSC) was carried out using two strategies to explore the carcinogenic mechanisms and identify its molecular markers more directly and comprehensively. Comparative proteome analysis on 20 hLSC tissues and paired normal bronchial epithelial tissues revealed 76 differential proteins, among which 68 proteins were identified by PMF. The identified proteins fell into three categories: oncoproteins, cell cycle regulators and signaling molecules. To validate the identified differential proteins, the expressions levels of three differential proteins mdm2, c-jun and EGFR were determined by immunohistochemical staining and immunoblots. The results verified proteome analysis results. Serological proteome analysis (SERPA) of ten hLSC tissues was performed to identify the tumor-associated antigens. The results revealed 36 +/- 8 differential proteins reactive with patients' autologous sera, of which 14 proteins were identified. Six of the 14 proteins, alpha enolase, pre-B cell-enhancing factor precursor, triosephosphate isomerase, phosphoglycerate mutase 1, fructose-bisphosphate aldolase A, and guanine nucleotide-binding protein beta subunit-like protein, were also up-regulated in hLSCs in the comparative proteomic study, which suggests potential application of these 6 hLSC-associated antigens in diagnosis and therapy of hLSC.     

Solution structure and dynamics of human metallothionein-3 (MT-3)

Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the alpha-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (beta-domain) and one in the C-terminus (alpha-domain). The overall fold of the alpha-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the beta-domain exhibits similar internal motion to the alpha-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.     

An integrated BAC and genome sequence physical map of Phytophthora sojae

Phytophthora spp. are serious pathogens that threaten numerous cultivated crops, trees, and natural vegetation worldwide. The soybean pathogen P. sojae has been developed as a model oomycete. Here, we report a bacterial artificial chromosome (BAC)-based, integrated physical map of the P. sojae genome. We constructed two BAC libraries, digested 8,681 BACs with seven restriction enzymes, end labeled the digested fragments with four dyes, and analyzed them with capillary electrophoresis. Fifteen data sets were constructed from the fingerprints, using individual dyes and all possible combinations, and were evaluated for contig assembly. In all, 257 contigs were assembled from the XhoI data set, collectively spanning approximately 132 Mb in physical length. The BAC contigs were integrated with the draft genome sequence of P. sojae by end sequencing a total of 1,440 BACs that formed a minimal tiling path. This enabled the 257 contigs of the BAC map to be merged with 207 sequence scaffolds to form an integrated map consisting of 79 superscaffolds. The map represents the first genome-wide physical map of a Phytophthora sp. and provides a valuable resource for genomics and molecular biology research in P. sojae and other Phytophthora spp. In one illustration of this value, we have placed the 350 members of a superfamily of putative pathogenicity effector genes onto the map, revealing extensive clustering of these genes.     

Increased HO-1 expression and decreased iNOS expression in the hippocampus from adult spontaneously hypertensive rats

Brain expression of heme oxygenase (HO) and nitric oxide synthase (NOS) in hypertension may participate in the pathogenesis of hypertension-related neuronal disorders, such as vascular dementia. In the present study, expression levels of HO and NOS in spontaneously hypertensive rats (SHR) were investigated using Western immunoblotting assay. Expression level of inducible HO-1 in hippocampus of 4-wk prehypertensive SHR was about twofold of that in age-matched Sprague-Dawley (SD) rats (p<0.01). In 23-wk SHR with fully developed hypertension, hippocampal HO-1 level was significantly greater than that of age-matched SD rats (p<0.05), but not different from 4-wk SHR. There was no difference in expression levels of hippocampal HO-2 between SHR and SD rats at different ages. Total enzymatic activity of hippocampal HO was significantly greater in 23-wk SHR than in age-matched SD rats or 4-wk SD/SHR (p<0.01). Although hippocampal expression of nNOS protein was relatively unchanged, iNOS expression in 23-wk SHR was about fourfold lower than that in age-matched SD rats and 4-wk SD/SHR (p<0.01). Total enzymatic activity of hippocampal NOS was significantly lower in 23-wk SHR than in age-matched SD rats or 4-wk SD/SHR (p<0.01). Significantly suppressed Morris water maze performance was found in 23-wk SHR in comparison with age-matched SD rats. Because SHR has been used as a model of vascular dementia and hippocampus is essential for spatial learning and memory, understanding of altered HO/CO and NOS/NO systems in the hippocampus of adult SHR may shed light on the pathogenic development of memory deficits associated with vascular dementia.     

Protoplast Fusion between Geotrichum candidium and Phanerochaete chrysosporium to Produce Fusants for Corn Stover Fermentation

Lignin impedes the digestion of corn stover when used as an animal feed. Phanerochaete chrysosporium is an efficient lignindegrader. Geotrichum candidum can be used to produce single-cell protein. In this study, protoplasts of the two fungi were prepared and fused. After screening, one of the fusants, Fusant R1, was selected for corn stover fermentation. It decreased lignin from 109 to 54 g/kg and increased protein from 48 to 67 g/kg in corn stover. Comparison with their parental strains indicated that the fusant obtained the lignin-degrading ability from P. chrysosporium and the protein-accumulating ability from G. candidium.     

Epidermal growth factor receptor inhibition promotes desmosome assembly and strengthens intercellular adhesion in squamous cell carcinoma cells

The epidermal growth factor receptor (EGFR) has been proposed as a key modulator of cadherin-containing intercellular junctions, particularly in tumors that overexpress this tyrosine kinase. Here the EGFR tyrosine kinase inhibitor PKI166 and EGFR blocking antibody C225, both of which are used clinically to treat head and neck cancers, were used to determine the effects of EGFR inhibition on intercellular junction assembly and adhesion in oral squamous cell carcinoma cells. EGFR inhibition resulted in a transition from a fibroblastic morphology to a more epithelial phenotype in cells grown in low calcium; under these conditions cadherin-mediated cell-cell adhesion is normally reduced, and desmosomes are absent. The accumulated levels of desmoglein 2 (Dsg2) and desmocollin 2 increased 1.7-2.0-fold, and both desmosomal cadherin and plaque components were recruited to cell-cell borders. This redistribution was paralleled by an increase in Dsg2 and desmoplakin in the Triton-insoluble cell fraction, suggesting that EGFR blockade promotes desmosome assembly. Importantly, E-cadherin expression and solubility were unchanged. Furthermore, PKI166 blocked tyrosine phosphorylation of Dsg2 and plakoglobin following epidermal growth factor stimulation, whereas no change in phosphorylation was detected for E-cadherin and beta-catenin. The increase in Dsg2 protein was in part due to the inhibition of matrix metalloproteinase-dependent proteolysis of this desmosomal cadherin. These morphological and biochemical changes were accompanied by an increase in intercellular adhesion based on functional assays at all calcium concentrations tested. Our results suggest that EGFR inhibition promotes desmosome assembly in oral squamous cell carcinoma cells, resulting in increased cell-cell adhesion.