Functional analysis tools for post‐translational modification: a post‐translational modification database for analysis of proteins and metabolic pathways

Post‐translational modifications (PTMs) are critical regulators of protein function, and nearly 200 different types of PTM have been identified. Advances in high‐resolution mass spectrometry have led to the identification of an unprecedented number of PTM sites in numerous organisms, potentially facilitating a more complete understanding of how PTMs regulate cellular behavior. While databases have been created to house the resulting data, most of these resources focus on individual types of PTM, do not consider quantitative PTM analyses or do not provide tools for the visualization and analysis of PTM data. Here, we describe the Functional Analysis Tools for Post‐Translational Modifications (FAT‐PTM) database ( https://bioinformatics.cse.unr.edu/fat-ptm/ ), which currently supports eight different types of PTM and over 49 000 PTM sites identified in large‐scale proteomic surveys of the model organism Arabidopsis thaliana. The FAT‐PTM database currently supports tools to visualize protein‐centric PTM networks, quantitative phosphorylation site data from over 10 different quantitative phosphoproteomic studies, PTM information displayed in protein‐centric metabolic pathways and groups of proteins that are co‐modified by multiple PTMs. Overall, the FAT‐PTM database provides users with a robust platform to share and visualize experimentally supported PTM data, develop hypotheses related to target proteins or identify emergent patterns in PTM data for signaling and metabolic pathways.     

Prospects for seascape repair: Three case studies from eastern Australia

Three case studies spanning tropical, subtropical and temperate environments highlight the minimum potential benefits of investing in repair of coastal seascapes. Fisheries, a market benefit indicator readily understood by a range of stakeholders from policymakers to community advocates, were used as a surrogate for ecosystem services generated through seascape habitat restoration. For each case study, while recognising that biological information will always remain imperfect, the prospects for seascape repair are compelling.     

Purification and properties of NAD(P)-independent polyol dehydrogenase complex from the plasma membrane of Gluconobacter oxydans

Gluconobacter oxydans rapidly oxidizes many different polyhydroxy alcohols (polyols). Polyol oxidations are catalyzed by constitutively synthesized membrane-bound dehydrogenases directly linked to the electron transport chain. A polyol-oxidizing enzyme was isolated from the membranes of G. oxydans and tested for its ability to oxidize various substrates. The enzyme was composed of three subunits: a 67 kDa catalytic unit, a 46 kDa c-type cytochrome, and a 15 kDa subunit. The enzyme oxidized compounds containing three or more hydroxyl groups but did not oxidize mono-, di-, or cyclic alcohols; aldehydes; carboxylic acids; or mono- or di-saccharides. Therefore, we propose this enzyme be considered a polyol dehydrogenase.     

The role of the fibronectin IGD motif in stimulating fibroblast migration

The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 10(6) times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN.     

MVB-12, a fourth subunit of metazoan ESCRT-I, functions in receptor downregulation

After ligand binding and endocytosis, cell surface receptors can continue to signal from endosomal compartments until sequestered from the cytoplasm. An important mechanism for receptor downregulation in vivo is via the inward budding of receptors into intralumenal vesicles to form specialized endosomes called multivesicular bodies (MVBs) that subsequently fuse with lysosomes, degrading their cargo. This process requires four heterooligomeric protein complexes collectively termed the ESCRT machinery. In yeast, ESCRT-I is a heterotetrameric complex comprised of three conserved subunits and a fourth subunit for which identifiable metazoan homologs were lacking. Using C. elegans, we identify MVB-12, a fourth metazoan ESCRT-I subunit. Depletion of MVB-12 slows the kinetics of receptor downregulation in vivo, but to a lesser extent than inhibition of other ESCRT-I subunits. Consistent with these findings, targeting of MVB-12 to membranes requires the other ESCRT-I subunits, but MVB-12 is not required to target the remaining ESCRT-I components. Both endogenous and recombinant ESCRT-I are stable complexes with a 1:1:1:1 subunit stoichiometry. MVB-12 has two human homologs that co-localize and co-immunoprecipitate with the ESCRT-I component TSG101. Thus, MVB-12 is a conserved core component of metazoan ESCRT-I that regulates its activity during MVB biogenesis.     

Potential for clinical ex vivo expansion of cord blood haemopoietic stem cells using non-haemopoietic factor supplements

The establishment of culture systems that promote haemopoietic stem cell (HSC) self-renewal and expansion ex vivo will increase the clinical potential of umbilical cord blood (CB) HSC transplantation. Studies defining key signalling pathways that regulate development and expansion of HSC in vivo have greatly facilitated development of protocols for expanding HSC in ex vivo culture. Recently a number of soluble factors with novel stem cell expansion activity have been identified as part of pathways associated with mesodermal induction, or as factors produced by supportive stroma. These have been reported to support, to varying degrees, HSC self-renewal under in vitro conditions. Here we review the activities of these new factors and consider their future potential as components in ex vivo expansion culture for CB HSC. Finally we discuss the challenges associated with applying these factors to clinically relevant culture systems.     

Public proteomic MS repositories and pipelines: available tools and biological applications

As proteomic MS has increased in throughput, so has the demand to catalogue the increasing number of peptides and proteins observed by the community using this technique. As in other 'omics' fields, this brings obvious scientific benefits such as sharing of results and prevention of unnecessary repetition, but also provides technical insights, such as the ability to compare proteome coverage between different laboratories, or between different proteomic platforms. Journals are also moving towards mandating that proteomics data be submitted to public repositories upon publication. In response to these demands, several web-based repositories have been established to store protein and peptide identifications derived from MS data, and a similar number of peptide identification software pipelines have emerged to deliver identifications to these repositories. This paper reviews the latest developments in public domain peptide and protein identification databases and describes the analysis pipelines that feed them. Recent applications of the tools to pertinent biological problems are examined, and through comparing and contrasting the capabilities of each system, the issues facing research users of web-based repositories are explored. Future developments and mechanisms to enhance system functionality and user-interfacing opportunities are also suggested.