Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling

We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754).Proteins rarely work as monomers to carry out all the biological processes needed for cells to function. An estimate of the total number of protein-protein interactions within the human proteome, based on currently available data sets, is ∼650,000 (1). This is likely an underestimate, given that many proteins form either transient, or weak interactions within intact cells that may not yet have been detected. This suggests that the majority of human proteins can participate in protein complex formation, at least under some conditions. This includes the many well-studied soluble protein complexes in the cytoplasm, exemplified by the proteasome, ribosomes and cytoskeletal network. It also includes many membrane-associated complexes, for example receptor tyrosine kinase signaling complexes, integrin networks and transmembrane transporters (2). To characterize the many roles of multi-protein complexes in biological regulatory mechanisms, it is important to have convenient methods for the rapid and efficient analysis of their composition and dynamics (3). Ideally, such methods should be applicable to system-wide studies and allow the analysis of endogenous proteins, rather than exclusively use tagged and/or over-expressed baits.The methods available for the proteome-wide analysis of protein interactions have developed swiftly over the last ten years. This field is dominated by affinity-enrichment based approaches, using either tagged constructs, or antibodies specific for endogenous proteins. Another approach is in vivo proximity labeling, based, for example, on the exogenous expression of a protein of interest, fused either to a promiscuous biotin-ligase (BioID) (4), or to a peroxidase enzyme that activates biotin-phenol (APEX) (5). While these data sets have proved very useful, there are some downsides. For example, a large expense in terms of both time and money to generate the thousands of individual “bait” proteins required for global interaction analyses. In addition, each of these affinity enrichments will be performed in only one type of buffer system, which is unlikely to be compatible with the maintenance of all protein-protein interactions. Another dimension to the analytical problem is that many proteins are expressed as different sized isoforms and/or in different post-translationally modified forms, resulting in formation of multiple, related, but functionally distinct complexes, with different combinations of interaction partners (6). Using affinity-enrichment/pull-down methods alone makes it difficult to resolve such mixtures of different forms of related protein complexes, complicating a detailed understanding of biological response mechanisms.An alternative strategy involves protein correlation profiling-MS, i.e. correlating similarities in the fractionation profiles of proteins detected by mass spectrometry, assuming that proteins in a common complex will cofractionate. This approach was previously applied to the analysis of subcellular organelle proteomes (7, 8), and subsequently extended to analyze soluble protein complexes. Thus, recent studies have shown that chromatography-based separation of soluble protein complexes, combined with fraction collection and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS)¹, facilitates analysis of many hundreds of soluble complexes from a single experiment (6, 9–11). A limitation of all of these studies, however, is that the native extraction conditions used to preserve protein-protein interactions isolates predominantly stable, soluble complexes. For example, many proteins that are integral to membranes are not recovered (12). Similarly, soluble protein complexes that have weakly bound protein subunits can dissociate upon cell lysis and the inevitable dilution associated with extraction. Thus, the potential value of this approach for the system-wide analysis of protein complexes is limited without a covalent tether to hold protein-protein interactions intact during extraction and subsequent chromatographic separation (13).Covalent protein crosslinking has been used extensively to stabilize protein complexes, cultured cells and tissues for subsequent analysis, either by microscopy, nucleotide sequencing or mass spectrometry. The agents employed to crosslink proteins to each other include various chemical groups able to react with the side-chains of either amino acids, nucleotides, carbohydrates or lipids (14). These crosslinking agents vary in the efficiency with which they perfuse into unbroken cells/tissues and the speed of their reaction when in proximity to a suitable chemical group. One of the most widely used crosslinkers is formaldehyde, which can reversibly form a covalent crosslink to stabilize both protein-protein and protein-nucleotide interactions (15–21). One of the main benefits of using formaldehyde is that because of its small size, it readily permeates intact cells and tissues. Another benefit of using formaldehyde is the easy reversal of the crosslinks by heating and subsequent compatibility with mass spectrometry-based proteome analysis.Here, we describe a mass spectrometry-based proteomic approach for the efficient global analysis of protein complexes, including membrane proteins, using in vivo protein crosslinking combined with denaturing extraction. Using high-resolution, size-exclusion chromatography (SEC) to separate crosslinked complexes under denaturing conditions and MS analysis of fractionated proteins, we could identify membrane bound and membrane associated complexes not accessible in native extracts. We present a detailed comparison of the sets of protein complexes that can be identified using protein correlation profiling MS analysis in conjunction with both formaldehyde crosslinked and native extracts from U2OS cells. We provide access to the entire proteome-wide data sets of both in vivo crosslinked and native U2OScell protein complexes via a searchable online database (http://www.peptracker.com/epd/).     

Effect of Dietary Patterns on Muscle Strength and Physical Performance in the Very Old: Findings from the Newcastle 85+ Study

Background

Healthy diet has been associated with better muscle strength and physical performance in cross-sectional studies of older adults but the effect of dietary patterns (DP) on subsequent decline, particularly in the very old (aged 85+), has not been determined.

Objective

We investigated the association between previously established DP and decline in muscle strength and physical performance in the very old.

Design

791 participants (61.8% women) from the Newcastle 85+ Study were followed-up for change in hand grip strength (HGS) and Timed Up-and Go (TUG) test over 5 years (four waves 1.5 years apart). Mixed models were used to determine the effects of DP on muscle strength and physical performance in the entire cohort and separately by sex.

Results

Previously we have established three DP that varied in intake of red meats, potato, gravy and butter and differed with key health and social factors. HGS declined linearly by 1.59 kg_F in men and 1.08 kg_F in women (both p<0.001), and TUG slowed by 0.13 log₁₀-transformed seconds (log₁₀-s) in men and 0.11 log₁₀-s in women per wave after adjusting for important covariates (both p<0.001), and also showed a nonlinear change (p<0.001). Men in DP1 (‘High Red Meat’) had worse overall HGS (β = -1.70, p = 0.05), but men in DP3 (‘High Butter’) had a steeper decline (β = -0.63, p = 0.05) than men in DP2 (‘Low Meat’). Men in DP1 and women in DP3 also had overall slower TUG than those in DP2 (β = 0.08, p = 0.001 and β = 0.06, p = 0.01, respectively), but similar rate of decline after adjusting for sociodemographic, lifestyle, health, and functioning factors. The results for HGS and TUG were not affected by participants’ cognitive status.

Conclusions

DP high in red meats, potato and gravy (DP1), or butter (DP3) may adversely affect muscle strength and physical performance in later life, independently of important covariates and cognitive status.     

Human IgG and IgM monoclonal antibodies against autologous melanoma produced by Epstein-Barr-virus-transformed B lymphocytes

Summary The serum antibody response to human melanoma has prognostic and potential physiological consequences. The specificity of the host B cell antibody response may be an important determinant of disease outcome. We have utilized Epstein-Barr virus (EBV) transformation to analyze the repertory of the host B cell response to melanoma. Production of antibody that binds selectively to autologous (eight cases) or allogeneic (four cases) short-term-cultured melanoma cells was assessed from EBV-transformed B lymphoblastoid cells. Forty-two cultures of EBV-transformed B cells that secreted IgM and 23 that secreted IgG antibodies gave patterns of differential reactivity with autologous or allogeneic melanoma. Antibodyforming B cells persisted in producing melanoma-reactive IgG and IgM for 8–21 weeks. Preselection of B cells by adsorption to tumor cell antigens before transformation enhanced the frequency of antibody secretion. The specificity of the antibody produced by the longest-producing culture appears to be restricted to a subset of melanomas. The patient from whom this tumor-restricted IgG-producing B cell was retrieved was unusual, having had a transient serum IgG of similar specificity, and having manifest a syndrome of vitiligo at the time of her development of serum antimelanoma antibody, followed by disease-free survival of resected recurrent metastatic melanoma to the present (more than 6 years). This study has given support to findings of conventional serology, revealing the production of melanoma-reactive antibody from B cells of patients who have demonstrable serological response to tumor.Supported by the American Cancer Society grant IM 433     

A mutation in aspergillus nidulans that blocks the transition from interphase to prophase

In order to develop a method for obtaining mitotic synchrony in aspergillus nidulans, we have characterized previously isolated heat-sensitive nim mutations that block the nuclear division cycle in interphase at restrictive temperature. After 3.5 h at restrictive temperature the mitotic index of a strain carrying one of these mutations, nimA5, was 0, but when this strain was subsequently shifted from restrictive to permissive temperature the mitotic index increased rapidly, reaching a maximum of 78 percent after 7.5 min. When this strain was examined electron-microscopically, mitotic spindles were absent at restrictive temperature. From these data we conclude that at restrictive temperature nimA5 blocks the nuclear division cycle at a point immediately preceding the initiation of chromosomal condensation and mitotic microtubule assembly, and upon shifting to permissive control over the initiation of microtubule assembly and chromosomal condensation in vivo through a simple temperature shift and, consequently, nimA5 should be a powerful tool for studying these processes. Electron-microscopic examination of spindles of material synchronized in this manner reveals that spindle formation, although very rapid, is gradual in the sense that spindle microtubule numbers increase as spindle formation proceeds.     

Theories of cell ageing: a case of mistaken identity

In social insects which reproduce by colony fission there is often only one queen in each swarm (e.g. honeybees, army ants) and the number of males greatly exceeds the number of new queens produced at swarming time. This seems to contradict Fisher's principle that there should be equal investment in male and female reproductives. Hamilton 1975 has suggested that the principle can be saved by counting the investment in the swarm as part of the investment in female reproductives. Craig 1980, on the other hand, argues that few queens are produced because any further investment in queens would be wasted since a queen without a swarm is valueless; on this view the sex ratio is male-biased because of local competition between queens for swarms. The present paper investigates from first principles how many males should be produced by a species which reproduces by colony fission and how the workers should divide themselves between the new colonies. The results of the analysis do not support Hamilton's conjecture but show that a number of factors are involved in the evolution of male production, including kin selection and the relationship between the “fitness” of a colony and the number of workers in it.     

Predictions of the somatic mutation and mortalization theories of cellular ageing are contrary to experimental observations

An accumulation of recessive lethal somatic mutations has often been proposed as a basis of cellular ageing. We have developed a mathematical model for the somatic mutation theory as applied to the finite in vitro lifespan of diploid fibroblast populations. Provided the mutation rate is sufficiently high, the model readily explains the cessation of proliferation of fibroblast cultures, but it predicts a much lower proportion of viable cells than is observed experimentally and also requires an unrealistically short cell division time. It is noted that the somatic mutation model is formally quite similar to the “mortalization” theory of Shall & Stein (1979), and that the mortalization theory is also incompatible with the same experimental data. We conclude that neither the somatic mutation theory nor the mortalization theory can explain the observed features of the growth of fibroblast populations in vitro. We discuss the possibility that deleterious mutations become important in the terminal stages of the lifespan, when they may accumulate as an indirect result of a general breakdown in information transfer between macromolecules.     

Genetic deletion of apolipoprotein A-I increases airway hyperresponsiveness,inflammation, and collagen deposition in the lung

The relationship between high-density lipoprotein and pulmonary function is unclear. To determine mechanistic relationships we investigated the effects of genetic deletion of apolipoprotein A-I (apoA-I) on plasma lipids, paraoxonase (PON1), pro-inflammatory HDL (p-HDL), vasodilatation, airway hyperresponsiveness and pulmonary oxidative stress, and inflammation. ApoA-I null (apoA-I^−/−) mice had reduced total and HDL cholesterol but increased pro-inflammatory HDL compared with C57BL/6J mice. Although PON1 protein was increased in apoA-I^−/− mice, PON1 activity was decreased. ApoA-I deficiency did not alter vasodilatation of facialis arteries, but it did alter relaxation responses of pulmonary arteries. Central airway resistance was unaltered. However, airway resistance mediated by tissue dampening and elastance were increased in apoA-I^−/− mice, a finding also confirmed by positive end-expiratory pressure (PEEP) studies. Inflammatory cells, collagen deposition, 3-nitrotyrosine, and 4-hydroxy-2-nonenal were increased in apoA-I^−/− lungs but not oxidized phospholipids. Colocalization of 4-hydroxy-2-nonenal with transforming growth factor β-1 (TGFβ-1 was increased in apoA-I^−/− lungs. Xanthine oxidase, myeloperoxidase and endothelial nitric oxide synthase were increased in apoA-I^−/− lungs. Dichlorodihydrofluorescein-detectable oxidants were increased in bronchoalveolar lavage fluid (BALF) in apoA-I^−/− mice. In contrast, BALF nitrite+nitrate levels were decreased in apoA-I^−/− mice. These data demonstrate that apoA-I plays important roles in limiting pulmonary inflammation and oxidative stress, which if not prevented, will decrease pulmonary artery vasodilatation and increase airway hyperresponsiveness.     

Error propagation in intracellular information transfer

The translation of genetic information from polynucleotides to proteins is mediated by proteins themselves. The cyclic nature of this process admits the possibility of a feedback of errors which may become lethal to the cell. During ageing, it is known that cells in some organisms show increased levels of altered or defective protein, and it has been suggested that the propagation of macromolecular errors may play a causative role in the progressive loss of homeostasis with increasing age. Experimental studies of this hypothesis have so far been inconclusive, and it is shown that theoretical models of intracellular error propagation throw important light on the determinants of stability within the translation apparatus and can improve the design of future experiments, as well as aid in their interpretation.Critical features of any model are its assumptions about the amino acid sequence changes required for a component of the translation apparatus to become error-prone and about the magnitude of any resultant change in activity. Existing models, which differ in these respects, are critically compared, and one is shown to be more flexible than the rest. In common with others, this model predicts that a normally stable translation apparatus has a threshold error level above which stability cannot be regained. The risk of crossing onto an irreversible path to cell death is determined by the distance between the stable and threshold error levels, and experiments to estimate this “safety margin” are suggested. Evolutionary modification of translational stability is also discussed.     

Energy requirements of captive cotton-top tamarins (Saguinus oedipus oedipus)

Food intake of captive cotton-top tamarins was measured at various stages of the life cycle and energy intake was calculated from the results. The intake of adults for maintenance was found to vary around 500 kJ X kg-3/4 X day-1 and to decrease with age. During the last 8 weeks of pregnancy, females increased in weight by about 2 g X day-1 but energy intake did not show a marked rise above the maintenance range. During lactation, their energy intake appeared to double. Measurements of intake at various stages of growth were compared with a model of the energy requirements for growth which predicted intake quite closely. Compared with non-primate mammals of similar size, the growth rate of this species is slow, and because of this, the energy intake during growth and lactation is also comparatively low.