Filtering high-throughput protein-protein interaction data using a combination of genomic features

Background  

Protein-protein interaction data used in the creation or prediction of molecular networks is usually obtained from large scale or high-throughput experiments. This experimental data is liable to contain a large number of spurious interactions. Hence, there is a need to validate the interactions and filter out the incorrect data before using them in prediction studies.     

The enzymatic activities of the Werner syndrome protein are disabled by the amino acid polymorphism R834C

The Werner syndrome protein, WRN, is a member of the RecQ family of DNA helicases. It possesses both 3'-->5' DNA helicase and 3'-->5' DNA exonuclease activities. Mutations in WRN are causally associated with a rare, recessive disorder, Werner syndrome (WS), distinguished by premature aging and genomic instability; all are reported to result in loss of protein expression. In addition to WS-linked mutations, single nucleotide polymorphisms, with frequencies that exceed those of WS-associated mutations, are also present in WRN. We have initiated studies to determine if six of these polymorphisms affect the enzymatic activities of WRN. We show that two common polymorphisms, F1074L and C1367R, and two infrequent polymorphisms, Q724L and S1079L, exhibit little change in activity relative to wild-type WRN; the polymorphism, T172P, shows a small but consistent reduction of activity. However, an infrequent polymorphism, R834C, located in the helicase domain dramatically reduces WRN helicase and helicase-coupled exonuclease activity. The structure of the E. coli helicase core suggests that R834 may be involved in interactions with ATP. As predicted, substitution of Arg with Cys interferes with ATP hydrolysis that is absolutely required for unwinding DNA. R834C thus represents the first missense amino acid polymorphism in WRN that nearly abolishes enzymatic activity while leaving expression largely unaffected.     

DDB1 maintains genome integrity through regulation of Cdt1

DDB1, a component of a Cul4A ubiquitin ligase complex, promotes nucleotide excision repair (NER) and regulates DNA replication. We have investigated the role of human DDB1 in maintaining genome stability. DDB1-depleted cells accumulate DNA double-strand breaks in widely dispersed regions throughout the genome and have activated ATM and ATR cell cycle checkpoints. Depletion of Cul4A yields similar phenotypes, indicating that an E3 ligase function of DDB1 is important for genome maintenance. In contrast, depletion of DDB2, XPA, or XPC does not cause activation of DNA damage checkpoints, indicating that defects in NER are not involved. One substrate of DDB1-Cul4A that is crucial for preventing genome instability is Cdt1. DDB1-depleted cells exhibit increased levels of Cdt1 protein and rereplication, despite containing other Cdt1 regulatory mechanisms. The rereplication, accumulation of DNA damage, and activation of checkpoint responses in DDB1-depleted cells require entry into S phase and are partially, but not completely, suppressed by codepletion of Cdt1. Therefore, DDB1 prevents DNA lesions from accumulating in replicating human cells, in part by regulating Cdt1 degradation.     

Cationic amino acid transport across the blood-brain barrier is mediated exclusively by system y+

Cationic amino acid (CAA) transport is brought about by two families of proteins that are found in various tissues: Cat (CAA transporter), referred to as system y+, and Bat [broad-scope amino acid (AA) transporter], which comprises systems b0,+, B0,+, and y+L. CAA traverse the blood-brain barrier (BBB), but experiments done in vivo have only been able to examine the BBB from the luminal (blood-facing) side. In the present study, plasma membranes isolated from bovine brain microvessels were used to identify and characterize the CAA transporter(s) on both sides of the BBB. From these studies, it was concluded that system y+ was the only transporter present, with a prevalence of activity on the abluminal membrane. System y+ was voltage dependent and had a Km of 470 +/- 106 microM (SE) for lysine, a Ki of 34 microM for arginine, and a Ki of 290 microM for ornithine. In the presence of Na+, system y+ was inhibited by several essential neutral AAs. The Ki values were 3-10 times the plasma concentrations, suggesting that system y+ was not as important a point of access for these AAs as system L1. Several small nonessential AAs (serine, glutamine, alanine,and glycine) inhibited system y+ with Ki values similar to their plasma concentrations, suggesting that system y+ may account for the permeability of the BBB to these AAs. System y+ may be important in the provision of arginine for NO synthesis. Real-time PCR and Western blotting techniques established the presence of the three known nitric oxide synthases in cerebral endothelial cells: NOS-1 (neuronal), NOS-2 (inducible), and NOS-3 (endothelial). These results confirm that system y+ is the only CAA transporter in the BBB and suggest that NO can be produced in brain endothelial cells.     

Mycobacterium tuberculosis cells growing in macrophages are filamentous and deficient in FtsZ rings

FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing FtsZ(TB) tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported FtsZ(TB) inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.     

Calcium/calmodulin stimulates the autophosphorylation of elongation factor 2 kinase on Thr-348 and Ser-500 to regulate its activity and calcium dependence

Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca(2+)/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca(2+)/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM.     

A rat model of central venous catheter to study establishment of long-term bacterial biofilm and related acute and chronic infections

Formation of resilient biofilms on medical devices colonized by pathogenic microorganisms is a major cause of health-care associated infection. While in vitro biofilm analyses led to promising anti-biofilm approaches, little is known about their translation to in vivo situations and on host contribution to the in vivo dynamics of infections on medical devices. Here we have developed an in vivo model of long-term bacterial biofilm infections in a pediatric totally implantable venous access port (TIVAP) surgically placed in adult rats. Using non-invasive and quantitative bioluminescence, we studied TIVAP contamination by clinically relevant pathogens, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis, and we demonstrated that TIVAP bacterial populations display typical biofilm phenotypes. In our study, we showed that immunocompetent rats were able to control the colonization and clear the bloodstream infection except for up to 30% that suffered systemic infection and death whereas none of the immunosuppressed rats survived the infection. Besides, we mimicked some clinically relevant TIVAP associated complications such as port-pocket infection and hematogenous route of colonization. Finally, by assessing an optimized antibiotic lock therapy, we established that our in vivo model enables to assess innovative therapeutic strategies against bacterial biofilm infections.