The role of the cellular microenvironment in enabling metazoan tissue genesis remains obscure. Ctenophora has recently emerged as one of the earliest-branching extant animal phyla, providing a unique opportunity to explore the evolutionary role of the cellular microenvironment in tissue genesis. Here, we characterized the extracellular matrix (ECM), with a focus on collagen IV and its variant, spongin short-chain collagens, of non-bilaterian animal phyla. We identified basement membrane (BM) and collagen IV in Ctenophora, and show that the structural and genomic features of collagen IV are homologous to those of non-bilaterian animal phyla and Bilateria. Yet, ctenophore features are more diverse and distinct, expressing up to twenty genes compared to six in vertebrates. Moreover, collagen IV is absent in unicellular sister-groups. Collectively, we conclude that collagen IV and its variant, spongin, are primordial components of the extracellular microenvironment, and as a component of BM, collagen IV enabled the assembly of a fundamental architectural unit for multicellular tissue genesis.DOI: http://dx.doi.org/10.7554/eLife.24176.001

Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad-a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids-is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N-and C-terminal tags and multiple promoters), is compatible with Gateway TM cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming. Molecular & Cellular Proteomics 9:811-823, 2010.The analysis of protein-protein interactions (PPIs) 1 and protein complexes is of central importance to biological research and facilitates our understanding of how molecular events drive phenotypic outcomes. Moreover, large scale protein interaction data can be used to generate protein interaction networks, which can then be used to predict disease genes and model biology in any living organism.A number of methods (e.g. yeast two-hybrid) have been developed to examine binary protein interactions in a systematic format and applied to model systems (1-8). However, affinity purification (AP) coupled with tandem MS has become the method of choice for the identification of protein complexes (9, 10). Large scale PPI studies using a high throughput and systematic AP-MS approach have been performed for Escherichia coli (11,12) and Saccharomyces cerevisiae (13-15). In fact, large scale efforts using AP-MS have connected an estimated 60% of the yeast proteome, demonstrating the power of coupling systematic biochemical purifications with mass spectrometry (13-16).AP-MS has also been used extensively for purification of mammalian protein complexes (17), but this has been mostly restricted to small scale studies and the use of either cell lines that are easy to transfect or highly validated antibodies against specific targets. For example, Glatter et al. (18) recently developed an integrated workflow where a high density interactome was developed for the protein phosphatase 2A complex. This workflow relies on "flip-in" technology to introduce transgenes into a common genomic site in HEK293 cells and, similar to work by other groups (19,20), utilizes an From the ‡Banting and Best

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We report characterization of a novel member of the short chain dehydrogenase/reductase superfamily. The 1513-base pair cDNA encodes a 319-amino acid protein. The corresponding gene spans over 26 kilobase pairs on chromosome 2 and contains five exons. The recombinant protein produced using the baculovirus system is localized in the microsomal fraction of Sf9 cells and is an integral membrane protein with cytosolic orientation of its catalytic domain. The enzyme exhibits an oxidoreductase activity toward hydroxysteroids with NAD ؉ and NADH as the preferred cofactors. The enzyme is most efficient as a 3␣-hydroxysteroid dehydrogenase, converting 3␣-tetrahydroprogesterone (allopregnanolone) to dihydroprogesterone and 3␣-androstanediol to dihydrotestosterone with similar catalytic efficiency (V max values of 13-14 nmol/min/mg microsomal protein and K m values of 5-7 M). Despite ϳ44 -47% sequence identity with retinol/ 3␣-hydroxysterol dehydrogenases, the enzyme is not active toward retinols. The corresponding message is abundant in human trachea and is present at lower levels in the spinal cord, bone marrow, brain, heart, colon, testis, placenta, lung, and lymph node. Thus, the new short chain dehydrogenase represents a novel type of microsomal NAD ؉

All-trans-retinoic acid is a metabolite of vitaminAll-trans-retinoic acid is a metabolite of vitamin A (all-transretinol) that functions as an activating ligand for a family of nuclear retinoic acid receptors (1). In target tissues, all-transretinoic acid is produced by the oxidation of all-trans-retinaldehyde catalyzed by cytosolic aldehyde dehydrogenases (Fig.

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Non-enzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to αVβ3 integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while non-oxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.