Genome-wide bimolecular fluorescence complementation analysis of SUMO interactome in yeast

The definition of protein–protein interactions (PPIs) in the natural cellular context is essential for properly understanding various biological processes. So far, however, most large-scale PPI analyses have not been performed in the natural cellular context. Here, we describe the construction of a Saccharomyces cerevisiae fusion library in which each endogenous gene is C-terminally tagged with the N-terminal fragment of Venus (VN) for a genome-wide bimolecular fluorescence complementation assay, a powerful technique for identifying PPIs in living cells. We illustrate the utility of the VN fusion library by systematically analyzing the interactome of the small ubiquitin-related modifier (SUMO) and provide previously unavailable information on the subcellular localization, types, and protease dependence of SUMO interactions. Our data set is highly complementary to the existing data sets and represents a useful resource for expanding the understanding of the physiological roles of SUMO. In addition, the VN fusion library provides a useful research tool that makes it feasible to systematically analyze PPIs in the natural cellular context.Most biological processes are mediated by complicated networks of protein–protein interactions (PPIs). Thus, the identification of the occurrence and components of PPIs provides invaluable insights into the cellular functions of proteins. In addition to conventional coimmunoprecipitation techniques, several methods have been developed to study PPIs, including the yeast two-hybrid analysis (Fields and Song 1989), the split ubiquitin system (Johnsson and Varshavsky 1994), fluorescence resonance energy transfer (FRET) assays (Periasamy and Day 1999; Pollok and Heim 1999), tandem affinity purification (TAP) followed by mass spectrometry (MS) analysis (Rigaut et al. 1999), and protein fragment complementation (PFC) assays (Remy and Michnick 1999; Ghosh et al. 2000; Wehrman et al. 2002; Paulmurugan and Gambhir 2003). Recently, the bimolecular fluorescence complementation (BiFC) assay, a specialized form of the PFC assay that uses fluorescent proteins as a reporter, has been developed (Hu et al. 2002). The BiFC assay is based on the formation of a fluorescent complex when two proteins fused to nonfluorescent fragments of a fluorescent protein interact with each other. This approach enables direct visualization of the occurrence and subcellular localization of PPIs with simple equipment.The budding yeast Saccharomyces cerevisiae has been a valuable eukaryotic model system, not only for traditional molecular and cell biology but also for the fields of functional genomics and proteomics. In S. cerevisiae, several large-scale analyses of PPIs have been performed with the yeast two-hybrid method (Uetz et al. 2000; Ito et al. 2001) or TAP-MS analysis (Gavin et al. 2002, 2006; Ho et al. 2002; Krogan et al. 2006). However, these approaches do not measure PPI in the natural cellular context; the yeast two-hybrid method is not appropriate for analyzing the interactions between proteins that cannot be transported to the nucleus or that form interactions only in the presence of other stabilizing interactions, and TAP-MS analysis is not amenable to studying protein complexes that are weakly or transiently formed or that do not survive in vitro purification. Compared with the yeast two-hybrid method and TAP-MS analysis, the FRET, PFC, and BiFC assays have several advantages in that they can detect the interactions between proteins in their natural cellular environment. Recently, a large-scale PPI screen using the PFC assay based on reconstituted dihydrofolate reductase (DHFR) activity has been reported (Tarassov et al. 2008), which is the first example of genome-wide PPI analysis using the PFC assay. However, because positive PPIs are selected by methotrexate resistance in the DHFR PFC assay, it is possible that the addition of methotrexate to the assay medium may perturb cellular physiology and proper PPI networks. In this regard, large-scale PPI screens using the BiFC or FRET assay that do not require any exogenous reagents would provide more accurate information about the structural organization of PPI networks in cells. To date, however, there is no report describing the application of the BiFC or FRET assay to genome-wide PPI analyses, particularly using proteins expressed from their own native promoters.The small ubiquitin-related modifier (SUMO) proteins are ∼10 kDa in size and comprise a family of evolutionarily conserved polypeptides that are post-translationally attached to the lysine residues of target proteins to regulate their subcellular localization, stability, and activity (Kerscher et al. 2006; Geiss-Friedlander and Melchior 2007). SUMO conjugation plays a variety of important roles in diverse eukaryotic cellular processes. SUMO can also mediate the non-covalent interaction of substrate proteins with proteins containing SUMO-interacting motifs and modulate their function (Song et al. 2004). In this regard, the identification of SUMO target proteins is crucial for the elucidation of the function of SUMO. Recent genome-wide PPI screens have identified over 500 putative SUMO conjugates in S. cerevisiae (Panse et al. 2004; Wohlschlegel et al. 2004; Zhou et al. 2004; Denison et al. 2005; Hannich et al. 2005; Wykoff and O''Shea 2005). More recently, an elegant study integrating the information of PPIs and genetic interactions has been conducted and has uncovered novel functional relationships between the SUMO pathway and various biological processes (Makhnevych et al. 2009). These systematic analyses have expanded the pool of SUMO substrates and the understanding of the biological function of sumoylation. However, because SUMO substrates can undergo rapid cycles of modification and demodification, and most target proteins appear to be modified to a small percentage at steady state (Geiss-Friedlander and Melchior 2007), it is likely that many unknown SUMO substrates remain to be discovered. Moreover, previous systematic PPI screens to identify SUMO substrates have primarily been performed with the yeast two-hybrid method and TAP-MS, which do not measure PPIs in the natural cellular context. The low agreement between the data from previous systematic screens demonstrates the limitation of the experimental methods used in those analyses and indicates that no single screen has been comprehensive. For this reason, different systematic approaches, with methods that can detect the interactions between proteins in their natural cellular environment, would greatly contribute to the identification of novel SUMO substrates and thus to the understanding of the function of the SUMO pathway.In the present study, we generated a collection of yeast strains expressing full-length proteins tagged with the N-terminal fragment of Venus (VN), a yellow fluorescent protein variant, from their own native promoters. Through a systematic analysis with the VN fusion library, we identified the interactome of SUMO, comprising 367 proteins, and also obtained previously unavailable information on the subcellular localization, types, and protease dependence of SUMO interactions in living yeast cells. Our data not only highlight a novel relationship between sumoylation and various biological processes but also represent a valuable resource that can be used to study the functional roles of the SUMO pathway. This is the first report that describes the application of the BiFC assay to a genome-wide PPI analysis using proteins expressed from their own native promoters. As demonstrated here, the VN fusion library provides a useful research tool that makes it feasible to systematically analyze PPIs in the natural cellular context.     

A Nationwide Study on the Incidence of Breast Cancer in Korean Women with Osteoporosis Receiving Raloxifene Treatment

PurposeRaloxifene is a selective estrogen receptor modulator (SERM), and raloxifene treatment for osteoporosis is reimbursable under the Korean National Health Insurance. Evidence suggests that SERMs use reduces the risk of breast cancer in Asian population. Herein, we retrospectively investigated the protective effect of raloxifene on breast cancer rates in Korean population.MethodsUsing the Health Insurance Review and Assessment Service database, we selected women with osteoporosis aged 50 years and above. Patients treated for at least 2 years with raloxifene were assigned to the user group, whereas the remaining patients were assigned to the non-user group. The effect on breast cancer risk was assessed using the Cox proportional-hazards model with a time-dependent covariate to adjust for immortal time bias.ResultsA total of 322,870 women who were registered between 2010 and 2011 were included. The user group comprised 0.7% (n = 2,307) of the total population. The mean age was 65.7 ± 8.0 years and 67.2 ± 8.6 years in the user and non-user groups, respectively (p < 0.001). There was no difference in the previous use of estrogen replacement between the 2 groups (p = 0.087). The incidence of breast cancer per 1,000 person-years was 0.49 (n = 8) and 0.68 (n = 1,714) in the user and non-user groups, respectively (hazard ratio [HR], 0.63, 95% confidence interval [CI], 0.32–1.27). HR decreased with increase in the treatment duration, but this change was not statistically significant (HR, 1.00, 95% CI, 0.32–3.11 in 2–3 years; HR, 0.63, 95% CI, 0.20–1.94 in 3–4 years; and HR, 0.41, 95% CI, 0.10–1.65 in 4–5 years).ConclusionLong-term treatment with raloxifene in women with osteoporosis was not significantly associated with a reduction in breast cancer rates. However, further investigation is required for a conclusive proof.     

Bilateral variations of musculocutaneous nerves with rare pattern in a Korean female cadaver

Bilateral variations in the formation and branching of brachial plexus are rare. Variations between median and musculocutaneous nerves were observed on both sides during the dissection of an 87-year-old Korean female cadaver, whose cause of death was cholangiocarcinoma. The variations found were bilateral, in which each musculocutaneous nerve did not pierce the coracobrachialis muscle. The musculocutaneous nerve was rudimentary in the right arm and all branches arose from the median nerve separately, which corresponds to previous classification type 0-2. In the case of the left arm, the musculocutaneous nerve originated from the lateral cord, but had connections between median and musculocutaneous nerves below the coracobrachialis muscle, which corresponds to previous classification type 1-B-2. To the best of our knowledge, the bilateral variations between median and musculocutaneous nerves in this case have different features from other previous reports. Awareness of the possible variations between median and musculocutaneous nerves is important to both anatomists and clinicians.     

A simple fluorescence assay for trypsin through a protamine-induced carbon quantum dot-quenching aggregation platform

The development of a simple detection strategy for trypsin (Try) is urgent, and is ascribed to the diagnostic value of Try in several diseases. Herein, a facile but effective fluorescence strategy for Try was developed based on the protamine (Pro)-induced aggregation of carbon quantum dots (CQDs). The fluorescence of negatively charged CQDs was quenched with Pro due to the assembly of CQDs and Pro (CQDs/Pro) through electrostatic interaction. However, the highly positively charged Pro, which is rich in basic arginine residues, was preferred to be hydrolyzed by Try. Try can induce the deaggregation of CQDs/Pro, thereby enabling the release of CQDs to restore the fluorescence intensity. Thus, the use of CQDs/Pro as a testing platform will be employed as a “turn-on” method for Try. In addition, the fluorescence-resuming response was proportional to Try, ranging from 25 ng mL⁻¹ to 500 ng mL⁻¹ with a limit of detection (LOD) of 8.08 ng mL⁻¹. This “turn-on” fluorescence assay for Try was label-free, convenient, and relatively free of interference from coexisting substances. Actual applications for Try monitoring and trypsin inhibitor screening also illustrated the considerable prospect of CQDs in the clinical field, combined with the superiority of the simple mixing operation.

In this work, a simple melting method was developed for carbon quantum dot fabrication to integrate with protamine as an effective signal-on fluorescence strategy for trypsin detection.

With increasing attention being paid to human health for disease diagnosis and treatment, increasing efforts have been spent on the development of new analytical methods toward biological macromolecule detection.^1,2 Trypsin (Try) is one of the most vital digestive proteases produced in pancreatic acinar cells, and can catalyze the hydrolysis of peptides mainly found at the C-terminus of lysine and arginine residues.^3–5 Abnormal Try activity will be reflected in pancreatic function and the corresponding pathological changes of the human body. In general, Try is a biomarker for several diseases, such as acute pancreatitis, cystic fibrosis, and pancreatic cancer.^5–7 Therefore, accurate monitoring for Try levels through simple and economical assays has attracted increasing interest in disease diagnosis and treatment.At present, considerable efforts have been devoted to developing efficient and reliable methods for Try detection based on various strategies, including colorimetry,⁸ electrochemistry,⁹ and fluorescence methods.¹⁰ Fluorescent approaches have been widely used due to their characteristics of simplicity, rapidity, sensitivity, and realizing real-time detection.^11,12 Fluorescent methods for Try have mainly been developed based on the hydrolytic effect of Try using peptide chains or specific proteins as substrates,^7,10,13,14 and using fluorescent dots, like CdTe QDs.¹⁴ Although current fluorescent methods are accurate and reliable, a simple and easy sensing system that detects Try with a controllable response integration modulated by Try through a biocompatible probe is still lacking. Given its special structural properties, protamine (Pro) is a highly cationic peptide under physiological conditions that possesses unique physiological function and substrates for enzymatic catalysis.⁸ The arginine-rich structure of protamine can also be catalyzed by Try as an ideal substrate for Try monitoring.^8,15,16 Hence, the interaction system containing Pro and a fluorescence probe, such as AIEgen, can be applied for the analytical assembly of Try.¹⁷ With regard to the relatively complicated preparation of AIE, other fluorescent probes for Try detection are desirable and urgently needed.Among the current fluorescent nanomaterials, fluorescent carbon quantum dots (CQDs) have been used to construct biosensors due to their safety, unique optical properties, and ease of modification.^18–20 Changing the structure or the surface properties of CQDs can result in a difference in the interaction and function between CQDs and external substances.^21,22 Thus, various fluorescent sensors based on CQDs have been established to detect specific targets varying from ions, nucleic acids, and drugs.^22–25 Positively and negatively charged carbon dots with controlled N-doping play opposite adsorption interaction with ssDNA in different applications.^21–23 Hence, a series of different application systems can be proposed to establish by utilizing the different surface properties of CQDs.Herein, negatively charged CQDs, fabricated from a simple melting operation, were applied as the sensing probe for Try detection without any complex surface functionalization or conjugation and compared with other reported quantum dots,²⁶ as shown in Scheme 1. Using Pro as the selective enzymatic substrate for Try, the integration of the prepared CQDs and Pro induce them to assemble and form an aggregation-caused quenching system of CQDs and Pro (CQDs/Pro) via electrostatic interaction. Under the strong and specific hydrolysis of Try to Pro, Pro loses its function with CQDs, and then the CQDs/Pro disaggregates to recover the fluorescence intensity. This facile system using Pro as the medium for inducing CQD aggregation provided novel insights into the design of some quantum dots with the appropriate surface property for the fabrication of simple, selective, and effective fluorescence assays for bioanalysis without a complicated surface functionalization.Open in a separate windowScheme 1The fabrication of CQDs and its electrostatic interaction with protamine as a simple quenching platform for trypsin detection.     

Regioselective C–H sulfenylation of N-sulfonyl protected 7-azaindoles promoted by TBAI: a rapid synthesis of 3-thio-7-azaindoles

This paper describes the regioselective C-3 sulfenylation of N-sulfonyl protected 7-azaindoles with sulfonyl chlorides. In this transformation, dual roles of TBAI serving as both promoter and desulfonylation reagent have been demonstrated. The reaction proceeded smoothly under simple conditions to afford 3-thio-7-azaindoles in moderate to good yields with broad substrate scopes. This protocol refrains from using transition-metal catalysts, strong oxidants or bases, and shows its practical synthetic value in organic synthesis.

A novel, practical and highly regioselective TBAI promoted C-3 sulfenylation reaction of N-sulfonyl protected 7-azaindoles with sulfonyl chlorides is presented here.     

Complete recovery of herpes zoster radiculopathy based on electrodiagnostic study: A case report

BACKGROUNDHerpes zoster is a painful infectious disease caused by the varicella zoster virus. Herpes zoster radiculopathy, which is a type of segmental zoster paresis, can complicate the disease and cause motor weakness. This complication should be considered when a patient with a rash complains of acute-onset motor weakness, and the diagnosis can be verified via electrodiagnostic study. CASE SUMMARYA 64-year-old female with a history of asthma presented to the emergency department with stabbing pain, an itching sensation, and a rash on the right anterior shoulder that had begun 5 d prior. Physical examination revealed multiple erythematous grouped vesicles in the right C4-5 and T1 dermatome regions. Because herpes zoster was suspected, the patient immediately received intravenous acyclovir. On the third hospital day, she complained of motor weakness in the right upper extremity. Magnetic resonance imaging of the cervical spine revealed mild intervertebral disc herniation at C4-C5 without evidence of nerve root compression. On the 12^th hospital day, electrodiagnostic study revealed right cervical radiculopathy, mainly in the C5/6 roots. Six months later, monoparesis resolved, and follow-up electrodiagnostic study was normal. CONCLUSIONThis case emphasizes that clinicians should consider the possibility of post-herpetic paresis, such as herpes zoster radiculopathy, and that electrodiagnostic study is useful for diagnosis and follow-up.     

Halitosis: prevalence,risk factors,sources, measurement and treatment – a review of the literature

Halitosis, an offensive breath odour, has multiple sources and negative impacts on people’s social interactions and quality of life. It is important for health care professionals, including general physicians and dental professionals, to understand its aetiology and risk factors in order to diagnose and treat patients appropriately. In this study, we have reviewed the current literature on halitosis regarding its prevalence, classification, risk factors, sources, measurement and treatment.