Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain–SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host–virus protein–protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen–host protein–protein interactions.There are an estimated 650,000 protein–protein interactions in a human cell (1). These interactions are integral to cellular function and mediate signaling pathways that are often misregulated in cancer (2) and may be hijacked by viral proteins (3). Commonly, signaling pathways involve moderate affinity interactions between modular domains and short linear motifs (SLiMs; conserved 2- to 10-aa stretches in disordered regions) (4) that are difficult to capture using high-throughput methods, such as yeast two-hybrid (Y2H) or affinity-purification mass spectrometry (AP/MS) but can be identified using peptide arrays, split-protein systems (5, 6), or peptide-phage display (7–10). A major limitation of peptide arrays is coverage, because the number of potential binding peptides in the proteome is orders of magnitude larger than what can be printed on an array. Conventional phage libraries display combinatorially generated peptide sequences that can identify biophysically optimal ligands of modular domains but this approach can exhibit a hydrophobic bias and may not be ideal for detecting natural binders (11). Thus, there is a need for alternative approaches for identification of relevant domain–SLiMs interactions.Here, we report an approach that solves both the problem of coverage and the problem of artificial binders. We take advantage of microarray-based oligonucleotide synthesis to construct custom-made peptide-phage libraries for screening peptide–protein interactions, an approach we call proteomic peptide-phage display (ProP-PD) (Fig. 1). This process is similar in concept to the method for autoantigen discovery recently proposed by Larman et al. (12). In this earlier work, a T7 phage display library comprising 36-residue overlapping peptides covering all ORFs in the human genome was used to develop a phage immunoprecipitation sequencing methodology for the identification of autoantigens. A more general application of the library for the identification of protein–peptide interactions was introduced, but not explored in depth. We here establish that ProP-PD is a straightforward method for the identification of potentially relevant ligands of peptide binding domains. Our approach is based on the filamentous M13 phage, which is highly suited for efficient screening of peptide binding domains (13). The main advantage of our display system is that it is nonlytic and highly validated; random M13 phage-displayed peptide libraries have been used to map binding specificities of hundreds of diverse modular domains (7, 8, 14–16). We showcase our approach by identifying interactions of PSD-95/Dlg/ZO-1 (PDZ) domains.Open in a separate windowFig. 1.Overview of the ProP-PD. The human and viral ProP-PD libraries were designed to contain over 50,000 or 10,000 C-terminal heptapeptides, respectively. Oligonucleotides encoding the sequences were printed on microarray slides, PCR-amplified, and cloned into a phagemid designed for the display of peptides fused to the C terminus of the M13 major coat protein P8. The libraries were used in binding selections with PDZ domains and the selected pools were analyzed by next-generation sequencing on the Illumina platform.The PDZ family is one of the largest domain families in the human proteome, with about 270 members that typically interact with C-terminal peptides (class I binding motif: x-S/T-x-Φ-COO-, class II: x-Φ-x-Φ-COO-) (17) but also with internal peptide stretches and phosphoinositides (18, 19). PDZ–peptide interactions have been extensively analyzed by distinct experimental efforts, such as peptide-phage display (7, 20), peptide arrays (9, 21, 22), and split-ubiquitin membrane Y2H (23), as well as by computational approaches (24–28). Furthermore, the PDZ family has been shown to be the target of viral hijacking, whereby virus proteins mimic the C termini of human proteins to exploit these interactions (29). Thus, the PDZ family offers an excellent model system for validation of the ProP-PD approach.We created ProP-PD libraries displaying all known human and viral C-terminal peptide sequences and used these to identify binding partners for the nine PDZ domains of Densin-180, Erbin, Scribble, and disk large homolog 1 (DLG1) (Fig. 1). These proteins have crucial roles in the postsynaptic density of excitatory neuronal synapses, in the establishment of adherens and tight junctions in epithelial cells, and in the regulation of cell polarity and migration (30–32). Additionally, both Scribble and DLG1 are known targets of viral proteins (33, 34). Using the ProP-PD libraries we identified known and novel human and viral ligands and validated candidates in vivo and in vitro. Our results demonstrate that ProP-PD is a powerful approach for the proteomic screening of human and viral targets. Future studies with larger libraries tiling the complete disordered regions of any proteome can be envisioned, as the technology is highly scalable.     

Psoriatic arthritis: lessons from imaging studies and implications for therapy

Introduction: Modern imaging may aid in the diagnosis, prognosis and monitoring of therapeutic response in psoriatic arthritis (PsA). Detection of osteitis and technical advances like whole body magnetic resonance imaging (MRI) exemplify the value of this technology.

Areas covered: Ultrasound (US) provides a clinic-based tool for evaluating both joint pathologies and extra-articular structures (especially enthesitis) including skin and nail disease. Recent studies have demonstrated subclinical disease in psoriasis without arthritis, as well as in PsA, with implications for diagnosis and treatment classification. Modern imaging can also facilitate decisions on tapering of expensive biologics, though real-world clinical studies are still lacking.

Expert commentary: The increase in novel PsA therapies should increase the utilization of modern imaging, providing both increased validation of imaging biomarkers as well as responsive outcome measures.     

Do systolic murmurs predict mortality in the elderly? A 15-year longitudinal population study of 70-year-olds

Systolic murmurs are common in the elderly but there is a striking paucity regarding published reports on their clinical significance and relation with mortality. This study describes prevalence of systolic murmurs in the elderly and cardiovascular diseases in 70-year-olds with or without systolic murmurs, and investigates the relation between systolic murmurs at age 70 and 15-year mortality. This cohort study is based on 973 (449 males and 524 females) 70-year-olds from G?teborg, Sweden who were examined in 1971/1972 at the Department of Geriatric Medicine, G?teborg University, and was followed-up to the year 2001. The prevalence of systolic murmur was 31% (females 36.4%, males 23.9%). Among subjects with systolic murmurs the prevalence of coronary heart disease (CHD) and hypertension was significantly higher in both sexes and congestive heart failure (CHF) in females only. Systolic murmur was a predictor for mortality in females (RR 1.49, 95% CI 1.17-1.91) but not in males (RR 1.14, 95% CI 0.89-1.49). Diagnosis of a cardiovascular disease was a significant predictor in both sexes for mortality irrespective of having systolic murmurs. In conclusion, there is a significant positive association of cardiovascular diseases with systolic murmurs in the elderly. The increased risk for mortality due to the presence of systolic murmur at age 70 is mediated through cardiovascular diseases.     

3D Cryo‐Imaging: A Very High‐Resolution View of the Whole Mouse

We developed the Case Cryo‐imaging system that provides information rich, very high‐resolution, color brightfield, and molecular fluorescence images of a whole mouse using a section‐and‐image block‐face imaging technology. The system consists of a mouse‐sized, motorized cryo‐microtome with special features for imaging, a modified, brightfield/fluorescence microscope, and a robotic xyz imaging system positioner, all of which is fully automated by a control system. Using the robotic system, we acquired microscopic tiled images at a pixel size of 15.6 μm over the block face of a whole mouse sectioned at 40 μm, with a total data volume of 55 GB. Viewing 2D images at multiple resolutions, we identified small structures such as cardiac vessels, muscle layers, villi of the small intestine, the optic nerve, and layers of the eye. Cryo‐imaging was also suitable for imaging embryo mutants in 3D. A mouse, in which enhanced green fluorescent protein was expressed under gamma actin promoter in smooth muscle cells, gave clear 3D views of smooth muscle in the urogenital and gastrointestinal tracts. With cryo‐imaging, we could obtain 3D vasculature down to 10 μm, over very large regions of mouse brain. Software is fully automated with fully programmable imaging/sectioning protocols, email notifications, and automatic volume visualization. With a unique combination of field‐of‐view, depth of field, contrast, and resolution, the Case Cryo‐imaging system fills the gap between whole animal in vivo imaging and histology. histology. Anat Rec, 292:342–351, 2009. © 2009 Wiley‐Liss, Inc.     

Efficacy and safety of colchicine for the treatment of osteoarthritis: a systematic review and meta-analysis of intervention trials

Clinical Rheumatology - Colchicine, an approved treatment for gout, has been trialed in many diseases including osteoarthritis (OA) due to its anti-inflammatory effects. However, its efficacy and...     

Recent advances in the management of Takayasu arteritis

Takayasu arteritis (TA) is a challenging large vessel vasculitis to treat. Distinguishing disease activity from vascular damage is difficult, often relying on clinician judgement aided by composite clinical disease activity indices with angiographic evidence of vessel wall thickening or vessel wall hypermetabolism demonstrable on positron emission tomography computerized tomography (PET CT). Glucocorticoids form the mainstay of remission induction. While other conventional disease modifying anti‐rheumatic drugs (cDMARDs) or biologic DMARDs (bDMARDs) are commonly used, evidence supporting their usefulness is sparse and generally of low quality. The only two randomized controlled trials (RCT) of a DMARD in TA failed to show efficacy of abatacept in reducing relapses of TA, however, tocilizumab showed a trend towards reduction in time to relapses. Of the cDMARDs, methotrexate, azathioprine, mycophenolate mofetil (MMF), leflunomide and cyclophosphamide have shown clinical efficacy in case series, with some evidence that methotrexate, azathioprine and MMF might retard angiographic progression. Among bDMARDs, anti‐tumor necrosis factor alpha agents and tocilizumab may be useful in patients refractory to cDMARDs with retardation of angiographic progression, based on evidence derived from mostly retrospective case series, whereas the role of rituximab and ustekinumab needs further elucidation. Revascularization, either surgical or endovascular, is the treatment of choice to relieve critical, symptomatic stenoses and are best undertaken during inactive disease. Emerging evidence suggests that patients with TA also have increased cardiovascular risk and this requires appropriate management. Large studies involving multiple centers are the need of the hour to appropriately evaluate utility of currently available immunosuppressive therapy in TA.