Beyond hairballs: The use of quantitative mass spectrometry data to understand protein-protein interactions

The past 10years have witnessed a dramatic proliferation in the availability of protein interaction data. However, for interaction mapping based on affinity purification coupled with mass spectrometry (AP-MS), there is a wealth of information present in the datasets that often goes unrecorded in public repositories, and as such remains largely unexplored. Further, how this type of data is represented and used by bioinformaticians has not been well established. Here, we point out some common mistakes in how AP-MS data are handled, and describe how protein complex organization and interaction dynamics can be inferred using quantitative AP-MS approaches.     

Histone recognition and large-scale structural analysis of the human bromodomain family

Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of?more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.     

Backbone and side chain 1H, 13C and 15N resonance assignments of the catalytic domain of diphtheria toxin

Diphtheria is a serious upper respiratory tract disease caused by the diphtheria toxin (DT) secreted from the bacteria Corynebacterium diphtheriae. Vaccination is the best way to protect against this infectious disease. Diphtheria vaccines are prepared by isolating, purifying and chemically deactivating DT. Although toxoids have been used for decades in immunization, there is still little understanding at the molecular level of the process of toxoid preparation, and how chemical treatment enhances their immunogenicity. We have undertaken an NMR study of the catalytic domain as a first step in understanding the molecular details involved in vaccine antigen preparation. Here we report a near complete assignment for the backbone and side chain resonances of the diphtheria toxin catalytic domain.     

Seawater temperature, Gambierdiscus spp. variability and incidence of ciguatera poisoning in French Polynesia

In the context of global warming and climate change, ciguatera disease is put forward as an indicator of environmental disturbance. However, to validate this indicator, some unknown parameters such as the delay between environmental perturbation and outbreaks of ciguatera need to be investigated. The main goal of this study was to investigate the temporal link between the growth of Gambierdiscus spp., and one of its influencing factors and the declared cases of ciguatera disease in humans. Algal cell density and seawater temperature (SWT) were recorded monthly from February 1993 to December 2001 on the Atimaono barrier reef of Tahiti Island. Reports of ciguatera cases were obtained from three community health clinics near the study sites. The autoregressive integrated moving average model (ARIMA) shows: (1) SWT were positively associated with Gambierdiscus spp. growth at a lagtime of 13 and 17 months (p < 0.001); (2) Gambierdiscus spp. growth measured at a given time is related to a peak number of cases of ciguatera recorded 3 months after peak densities of this dinoflagellate (p < 0.001). These results allow the construction of a predictive model of the temporal link between ciguatera disease in humans and its etiologic agent: Gambierdiscus spp. This model constructed by using 1993–1999 data, then validated by 2000–2001 data, demonstrates an appreciable ability to predict changes in the incidence of ciguatera disease following algae blooms.     

Endocrine and exocrine secretion of leptin by the gastric mucosa.

Leptin is a hormone that plays important roles in nutritional status and in obesity. By means of immunocytochemistry, two populations of leptin-secreting cells were found in the lower half of the gastric mucosa. One consists of numerous large cells located around the gastric pits, the Chief epithelial cells, whereas the second refers to much smaller cells, strongly stained, few in number, and scattered between the gastric pits, the endocrine cells. By double immunostaining, leptin and pepsinogen were colocalized in the Chief cells, whereas the endocrine cells were positive only for leptin. Immunoelectron microscopy showed that leptin is present along the rough endoplasmic reticulum-Golgi-granules secretory pathways of the Chief and endocrine cells. On the other hand, leptin-receptor (long and short forms) immunolabelings, although absent in the gastric epithelial cell plasma membranes, were present in enterocytes at the level of their apical and basolateral membranes. Duodenal, jejunal, and ileal enterocytes displayed similar labelings for the leptin receptor. Thus, exocrine and endocrine secretions of leptin together with the presence of leptin receptors on enterocyte plasma membranes constitute a gastroenteric axis that coordinates the role played by leptin in the digestive tract.     

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.     

NMR assignment of the N-terminal TRAF-like RING zinc finger domain of human FLN29

Resistance of cancer cells to oncotherapeutics designed to trigger programmed cell death (a.k.a. apoptosis) greatly limits clinical efficacy. The human FLN29 protein may play a role in this process via protein-protein interactions. Here we report the NMR spectral assignment of the N-terminal TRAF2/6-RING-zinc finger-like domain of this protein.     

NMR assignment of human granulocyte-macrophage colony-stimulating factor

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF), also known as sargramostim or molgramostin, is a cytokine that functions as a hematopoietic cell growth factor. Here we report a near complete assignment for the backbone and side chain resonances for the mature polypeptide.