High-throughput analysis of peptide-binding modules

Modular protein interaction domains (PIDs) that recognize linear peptide motifs are found in hundreds of proteins within the human genome. Some PIDs such as SH2, 14-3-3, Chromo, and Bromo domains serve to recognize posttranslational modification (PTM) of amino acids (such as phosphorylation, acetylation, methylation, etc.) and translate these into discrete cellular responses. Other modules such as SH3 and PSD-95/Discs-large/ZO-1 (PDZ) domains recognize linear peptide epitopes and serve to organize protein complexes based on localization and regions of elevated concentration. In both cases, the ability to nucleate-specific signaling complexes is in large part dependent on the selectivity of a given protein module for its cognate peptide ligand. High-throughput (HTP) analysis of peptide-binding domains by peptide or protein arrays, phage display, mass spectrometry, or other HTP techniques provides new insight into the potential protein-protein interactions prescribed by individual or even whole families of modules. Systems level analyses have also promoted a deeper understanding of the underlying principles that govern selective protein-protein interactions and how selectivity evolves. Lastly, there is a growing appreciation for the limitations and potential pitfalls associated with HTP analysis of protein-peptide interactomes. This review will examine some of the common approaches utilized for large-scale studies of PIDs and suggest a set of standards for the analysis and validation of datasets from large-scale studies of peptide-binding modules. We will also highlight how data from large-scale studies of modular interaction domain families can provide insight into systems level properties such as the linguistics of selective interactions.     

SRC Homology 2 Domain Binding Sites in Insulin, IGF-1 and FGF receptor mediated signaling networks reveal an extensive potential interactome

ABSTRACT: Specific peptide ligand recognition by modular interaction domains is essential for the fidelity of information flow through the signal transduction networks that control cell behavior in response to extrinsic and intrinsic stimuli. Src homology 2 (SH2) domains recognize distinct phosphotyrosine peptide motifs, but the specific sites that are phosphorylated and the complement of available SH2 domains varies considerably in individual cell types. Such differences are the basis for a wide range of available protein interaction microstates from which signaling can evolve in highly divergent ways. This underlying complexity suggests the need to broadly map the signaling potential of systems as a prerequisite for understanding signaling in specific cell types as well as various pathologies that involve signal transduction such as cancer, developmental defects and metabolic disorders. This report describes interactions between SH2 domains and potential binding partners that comprise initial signaling downstream of activated fibroblast growth factor (FGF), insulin (Ins), and insulin-like growth factor-1 (IGF-1) receptors. A panel of 50 SH2 domains screened against a set of 192 phosphotyrosine peptides defines an extensive potential interactome while demonstrating the selectivity of individual SH2 domains. The interactions described confirm virtually all previously reported associations while describing a large set of potential novel interactions that imply additional complexity in the signaling networks initiated from activated receptors. This study of pTyr ligand binding by SH2 domains provides valuable insight into the selectivity that underpins complex signaling networks that are assembled using modular protein interaction domains.     

Metabolic and ventilatory physiology of the Barrow Island golden bandicoot (Isoodon auratus barrowensis) and the northern brown bandicoot (Isoodon macrourus)

Metabolic and ventilatory parameters were measured for the smallest and largest Isoodon bandicoots; the arid-adapted Barrow Island golden bandicoot (Isoodon auratus barrowensis) and the tropical northern brown bandicoot (Isoodon macrourus). I. a. barrowensis has a number of physiological characteristics that aid its tolerance of high T_a and survival in a hot and dry climate, including a low and labile body temperature, a very low basal metabolic rate, low total evaporative water loss, and an effective panting mechanism. I. macrourus generally has an “average” physiology for a bandicoot despite its size, although a number of its physiological characteristics aid survival in (sub)tropical conditions. These include a low body temperature, low total evaporative water loss and minute ventilation at high ambient temperatures, and an average thermal conductance. These data support the theory that phylogeny is a more important predictor of bandicoot physiology than habitat/distribution.     

Genetic evidence for the origins of range disjunctions in the Australian dry sclerophyll plant Hardenbergia violacea

Aim The distribution of genetic variation in the Australian dry sclerophyll plant Hardenbergia violacea (Fabaceae) is examined in the context of Pleistocene climate change in order to identify likely refugia. Particular consideration is given to the origin of range disjunctions in South Australia and Tasmania, and to determining whether the Tasmanian population is indigenous or recently introduced from mainland Australia. Location Southeastern Australian mainland and Tasmania. Methods A combination of chloroplast polymerase chain reaction–restriction fragment length polymorphism and genomic amplified fragment length polymorphism (AFLP) marker systems was used to examine the genetic structure of 292 individuals from 13 populations across the range of H. violacea in southeastern Australia. Results Hardenbergia violacea populations in Tasmania and southern Victoria were characterized by low, almost monotypic chloroplast diversity. New South Wales showed higher haplotype diversity and haplotype sharing among widely distributed populations. Principal coordinates analysis (PCoA) of the AFLP data found a strong latitudinal cline in AFLP variation from northern New South Wales south to Tasmania. The Tasmanian population formed an isolated and somewhat disjunct genetic cluster at one end of this cline. However, the South Australian population was an exception to the clinal variation shown by all other populations, forming a highly disjunct cluster in the PCoA. Within‐population genetic diversity was low in both disjunct populations. Main conclusions The genetic evidence indicates that the Tasmanian population is likely to be indigenous and probably the product of vicariance, which was followed by range contraction at the Last Glacial Maximum or an earlier glacial event. The deep phylogenetic disjunction in South Australia is evidence of a much earlier separation on mainland Australia. The chloroplast structure indicates that, during the Pleistocene, H. violacea underwent broad‐scale recolonization in southern Victoria and Tasmania, possibly from a large continental refugium in eastern New South Wales. We conclude that H. violacea, and presumably the sclerophyll communities in which it occurs, have undergone multiple range contractions to large continental refugia during different Pleistocene glaciations in southeastern Australia.     

Characterization and Regional Distribution of Peptides Derived from the Vasoactive Intestinal Peptide Precursor in the Normal Human Brain

To study the biosynthetic processing of the precursor for vasoactive intestinal peptide (prepro-VIP) in the human brain, we have developed antisera against the five functional domains of the precursor molecule: prepro-VIP 22-79, peptide histidine methionine (PHM), prepro-VIP 111-122, VIP, and prepro-VIP 156-170. The antisera were used in radioimmunoassays in combination with HPLC to identify and quantify the peptides in regions of the human brain. All five peptides were expressed, but mainly in nonequimolar ratios. In only three regions were the same amounts of VIP and PHM found; in the remaining areas the concentration of PHM was two-thirds that of VIP. The concentrations of prepro-VIP 22-79, prepro-VIP 111-122, and prepro-VIP 156-170 were considerably lower than the corresponding VIP concentrations, and the relative concentration of prepro-VIP 111-122 differed between cortical and subcortical areas. A small proportion of the VIP precursor followed a pathway in which the dibasic conversion site after PHM is not cleaved, as evidenced by the presence of a C-terminally extended form of PHM. Finally, it was found that the C-terminal lysine residue of prepro-VIP is not removed during processing. The findings indicate that differences in the posttranslational processing of prepro-VIP exist in subpopulations of neurons in the human brain.     

Thermoregulatory physiology of the Crested Pigeon<Emphasis Type="Italic"> Ocyphaps lophotes</Emphasis> and the Brush Bronzewing<Emphasis Type="Italic"> Phaps elegans</Emphasis>

The metabolic physiology of the Crested Pigeon (Ocyphaps lophotes) and the Brush Bronzewing (Phaps elegans) is generally similar to that expected for birds of their size, but the Crested Pigeon has a number of characteristics which would aid survival in hot and dry regions. Body temperature increased similarly for the Crested Pigeon (from 38.8 degrees C to 41.5 degrees C) and the Brush Bronzewing (39.3 degrees C to 41.4 degrees C) over ambient temperatures (T(a)s) from 10 degrees C to 35 degrees C. Both species became hyperthermic (body temperature, T(b)>42 degrees C) at T(a)=45 degrees C. Basal metabolic rate of the Crested Pigeon (0.65 ml O(2) g(-1) h(-1) at 40 degrees C) was approximately 71% of that predicted for a columbid bird, while BMR of the Brush Bronzewing (0.87 ml O(2) g(-1) h(-1) at 20 degrees C to 40 degrees C) was approximately 102% of predicted. Total evaporative water loss increased exponentially with T(a) for both species, from <1 mg H(2)O g(-1) h(-1) at 10 degrees C to >12 mg H(2)O g(-1) h(-1) at 45 degrees C. It was similar and low for both species at T(a)<30 degrees C, but was higher for the Brush Bronzewing than the Crested Pigeon at T(a)>30 degrees C. Ventilatory minute volume matched oxygen consumption, such that oxygen extraction efficiency did not change with T(a) and was similar for both species (approximately 20%). Expired air temperature was considerably lower than T(b) for both species at T(a)<35 degrees C, potentially reducing respiratory water loss by approximately 65% at T(a)=10 degrees C to approximately 30% at T(a)=35 degrees C. Cutaneous evaporative cooling was significant for both species, with skin resistance decreasing as T(a) increased. The Crested Pigeon had a lower skin resistance than the Brush Bronzewing at T(a)=45 degrees C. The Brush Bronzewing had apparently reached its maximum cutaneous water loss at 30 degrees C and relied on panting to cool at higher T(a).     

Molecular Characterization of Caveolin-induced Membrane Curvature

The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however, the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis. Cryoelectron tomography reveals the core membrane-juxtaposed domain to be sufficient to maintain oligomerization as defined by polyhedral distortion of the caveolar membrane. Through mutagenesis we demonstrate the importance of the membrane association of the oligomerization domain/CSD for defined caveola biogenesis and furthermore, highlight the functional significance of the intramembrane domain and the CSD for defined caveolin-induced membrane deformation. Finally, we define the core structural domain of Cav1, constituting only 66 amino acids and of great potential to nanoengineering applications, which is required for caveolin-induced vesicle formation in a bacterial system. These results have significant implications for understanding the role of Cav1 in caveola formation and in regulating cellular signaling events.     

High-throughput phosphotyrosine profiling using SH2 domains

Protein tyrosine phosphorylation controls many aspects of signaling in multicellular organisms. One of the major consequences of tyrosine phosphorylation is the creation of binding sites for proteins containing Src homology 2 (SH2) domains. To profile the global tyrosine phosphorylation state of the cell, we have developed proteomic binding assays encompassing nearly the full complement of human SH2 domains. Here we provide a global view of SH2 domain binding to cellular proteins based on large-scale far-western analyses. We also use reverse-phase protein arrays to generate comprehensive, quantitative SH2 binding profiles for phosphopeptides, recombinant proteins, and entire proteomes. As an example, we profiled the adhesion-dependent SH2 binding interactions in fibroblasts and identified specific focal adhesion complex proteins whose tyrosine phosphorylation and binding to SH2 domains are modulated by adhesion. These results demonstrate that high-throughput comprehensive SH2 profiling provides valuable mechanistic insights into tyrosine kinase signaling pathways.