Tracking chromatin state changes using nanoscale photo-proximity labelling.

Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects1,2. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health1. However, in the complex environment of the nucleus, it is challenging to determine protein-protein interactions owing to low abundance, transient or multivalent binding and a lack of technologies that are able to interrogate these interactions without disrupting the protein-binding surface under study3. Here, we describe a method for the traceless incorporation of iridium-photosensitizers into the nuclear micro-environment using engineered split inteins. These Ir-catalysts can activate diazirine warheads through Dexter energy transfer to form reactive carbenes within an approximately 10 nm radius, cross-linking with proteins in the immediate micro-environment (a process termed µMap) for analysis using quantitative chemoproteomics4. We show that this nanoscale proximity-labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. µMap improves our fundamental understanding of nuclear protein-protein interactions and, in doing so, is expected to have a significant effect on the field of epigenetic drug discovery in both academia and industry.

Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here we deploy proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from premade parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions breakpoint cluster region-Abelson (BCR-ABL) and DNAJ-PKAc where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as new therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.

A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases.

High throughput discovery of functional protein modifications by Hotspot Thermal Profiling.

Mass spectrometry enables global analysis of posttranslationally modified proteoforms from biological samples, yet we still lack methods to systematically predict, or even prioritize, which modification sites may perturb protein function. Here we describe a proteomic method, Hotspot Thermal Profiling, to detect the effects of site-specific protein phosphorylation on the thermal stability of thousands of native proteins in live cells. This massively parallel biophysical assay unveiled shifts in overall protein stability in response to site-specific phosphorylation sites, as well as trends related to protein function and structure. This method can detect intrinsic changes to protein structure as well as extrinsic changes to protein-protein and protein-metabolite interactions resulting from phosphorylation. Finally, we show that functional 'hotspot' protein modification sites can be discovered and prioritized for study in a high-throughput and unbiased fashion. This approach is applicable to diverse organisms, cell types and posttranslational modifications.

Photoproximity Profiling of Protein-Protein Interactions in Cells.

We report a novel photoproximity protein interaction (PhotoPPI) profiling method to map protein-protein interactions in vitro and in live cells. This approach utilizes a bioorthogonal, multifunctional chemical probe that can be targeted to a genetically encoded protein of interest (POI) through a modular SNAP-Tag/benzylguanine covalent interaction. A first generation photoproximity probe, PP1, responds to 365 nm light to simultaneously cleave a central nitroveratryl linker and a peripheral diazirine group, resulting in diffusion of a highly reactive carbene nucleophile away from the POI. We demonstrate facile probe loading, and subsequent interaction- and light-dependent proximal labeling of a model protein-protein interaction (PPI) in vitro. Integration of the PhotoPPI workflow with quantitative LC-MS/MS enabled unbiased interaction mapping for the redox regulated sensor protein, KEAP1, for the first time in live cells. We validated known and novel interactions between KEAP1 and the proteins PGAM5 and HK2, among others, under basal cellular conditions. By contrast, comparison of PhotoPPI profiles in cells experiencing metabolic or redox stress confirmed that KEAP1 sheds many basal interactions and becomes associated with known lysosomal trafficking and proteolytic proteins like SQSTM1, CTSD, and LGMN. Together, these data establish PhotoPPI as a method capable of tracking the dynamic subcellular and protein interaction "social network" of a redox-sensitive protein in cells with high temporal resolution.

Arthroscopic treatment of a one-fourth anteroinferior glenoid comminuted fracture-dislocation with concomitant three-part complex proximal humerus fracture in a middle-aged man: a case report.

INTRODUCTION: Proximal humeral fracture-dislocations can occur in high-energy traumas. This injury can be accompanied by a glenoid fracture; however, it is a rare type of complex injury in patients aged under 60 years. MATERIALS AND METHODS: A 53-year-old man presented with a three-part fracture-dislocation of the proximal humerus and a severely comminuted glenoid fracture. For the glenohumeral dislocation and proximal humeral fracture, we performed closed reduction using a threaded Steinman pin and fixation with percutaneous cannulated screws. Using arthroscopy, while maintaining humeral traction with the Steinman pin, the intra-articular glenoid fragments were reduced and then fixed with a buttressing headless screw and one suture anchor. After a 6-week immobilization with a shoulder spica cast, rehabilitation was initiated. RESULTS: We confirmed bony union of the fracture sites after 6 months post-surgery. The patient showed excellent clinical outcomes with a nearly full range of motion without instability CONCLUSIONS: We reported a successful outcome for a complex proximal humeral fracture involving the glenoid using closed reduction and fixation for the proximal humeral fracture and arthroscopic reduction and fixation for the comminuted anteroinferior glenoid fracture.

Chemoproteomic Profiling of Phosphoaspartate Modifications in Prokaryotes.

Phosphorylation at aspartic acid residues represents an abundant and critical post-translational modification (PTM) in prokaryotes. In contrast to most characterized PTMs, such as phosphorylation at serine or threonine, the phosphoaspartate moiety is intrinsically labile, and therefore incompatible with common proteomic profiling methods. Herein, we report a nucleophilic, desthiobiotin-containing hydroxylamine (DBHA) chemical probe that covalently labels modified aspartic acid residues in native proteomes. DBHA treatment coupled with LC-MS/MS analysis enabled detection of known phosphoaspartate modifications, as well as novel aspartic acid sites in the E. coli proteome. Coupled with isotopic labelling, DBHA-dependent proteomic profiling also permitted global quantification of changes in endogenous protein modification status, as demonstrated with the detection of increased E. coli OmpR phosphorylation, but not abundance, in response to changes in osmolarity.

Profiling Reactive Metabolites via Chemical Trapping and Targeted Mass Spectrometry.

Metabolomic profiling studies aim to provide a comprehensive, quantitative, and dynamic portrait of the endogenous metabolites in a biological system. While contemporary technologies permit routine profiling of many metabolites, intrinsically labile metabolites are often improperly measured or omitted from studies due to unwanted chemical transformations that occur during sample preparation or mass spectrometric analysis. The primary glycolytic metabolite 1,3-bisphosphoglyceric acid (1,3-BPG) typifies this class of metabolites, and, despite its central position in metabolism, has largely eluded analysis in profiling studies. Here we take advantage of the reactive acylphosphate group in 1,3-BPG to chemically trap the metabolite with hydroxylamine during metabolite isolation, enabling quantitative analysis by targeted LC-MS/MS. This approach is compatible with complex cellular metabolome, permits specific detection of the reactive (1,3-) instead of nonreactive (2,3-) BPG isomer, and has enabled direct analysis of dynamic 1,3-BPG levels resulting from perturbations to glucose processing. These studies confirmed that standard metabolomic methods misrepresent cellular 1,3-BPG levels in response to altered glucose metabolism and underscore the potential for chemical trapping to be used for other classes of reactive metabolites.

Zeolite Membrane-Based Low-Temperature Dehydrogenation of a Liquid Organic Hydrogen Carrier: A Key Step in the Development of a Hydrogen Economy.

Methylcyclohexane (MCH) dehydrogenation is an equilibrium-limited reaction that requires high temperatures (>300 °C) for complete conversion. However, high-temperature operation can degrade catalytic activity and produce unwanted side products. Thus, a hybrid zeolite membrane (Z) is prepared on the inner surface of a tubular support and used it as a wall in a membrane reactor (MR) configuration. Pt/C catalysts is packed diluted with quartz sand inside the Z-coated tube and applied the MR for MCH dehydrogenation at low temperatures (190-250 °C). Z showed a remarkable H2-permselectivity in the presence of both toluene and MCH, yielding separation factors over 350. The Z-based MR achieved higher MCH conversion (75.3% ± 0.8% at 220 °C) than the conventional packed-bed reactor (56.4% ± 0.3%) and the equilibrium state (53.2%), owing to the selective removal of H2 through Z. In summary, the hybrid zeolite MR enhances MCH dehydrogenation at low temperatures by overcoming thermodynamic limitations and improves the catalytic performance and product selectivity of the reaction.

Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here, we deploy a proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from pre-made parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions BCR/ABL and DNAJB1/PRKACA where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as novel therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.

Knockdown of RON inhibits AP-1 activity and induces apoptosis and cell cycle arrest through the modulation of Akt/FoxO signaling in human colorectal cancer cells.

BACKGROUND/AIMS: Altered Recepteur d'Origine nantais (RON) expression transduces signals inducting invasive growth phenotype that includes cell proliferation, migration, matrix invasion, and protection of apoptosis in human cancer cells. The aims of the current study were to evaluate whether RON affects tumor cell behavior and cellular signaling pathways including activator protein-1 (AP-1) and Akt/forkhead box O (FoxO) in human colorectal cancer cells. METHODS: To study the biological role of RON on tumor cell behavior and cellular signaling pathways in human colorectal cancer, we used small interfering RNA (siRNA) to knockdown RON gene expression in human colorectal cancer cell line, DKO-1. RESULTS: Knockdown of RON diminished migration, invasion, and proliferation of human colorectal cancer cells. Knockdown of RON decreased AP-1 transcriptional activity and expression of AP-1 target genes. Knockdown of RON activated cleaved caspase-3, -7, -9, and PARP, and down-regulated the expression of Mcl-1, survivin and XIAP, leading to induction of apoptosis. Knockdown of RON induced cell cycle arrest in the G2/M phase of cancer cells by an increase of p27 and a decrease of cyclin D3. Knockdown of RON inhibited the phosphorylation of Akt/FoxO signaling proteins such as Ser473 and Thr308 of Akt and FoxO1/3a. CONCLUSIONS: These results indicate that knockdown of RON inhibits AP-1 activity and induces apoptosis and cell cycle arrest through the modulation of Akt/FoxO signaling in human colorectal cancer cells.

Black tea extract prevents lipopolysaccharide-induced NF-κB signaling and attenuates dextran sulfate sodium-induced experimental colitis.

BACKGROUND: Black tea has been shown to elicit anti-oxidant, anti-carcinogenic, anti-inflammatory and anti-mutagenic properties. In this study, we investigated the impact of black tea extract (BTE) on lipopolysaccharide (LPS)-induced NF-κB signaling in bone marrow derived-macrophages (BMM) and determined the therapeutic efficacy of this extract on colon inflammation. METHODS: The effect of BTE on LPS-induced NF-κB signaling and pro-inflammatory gene expression was evaluated by RT-PCR, Western blotting, immunofluorescence and electrophoretic mobility shift assay (EMSA). The in vivo efficacy of BTE was assessed in mice with 3% dextran sulfate sodium (DSS)-induced colitis. The severity of colitis was measured by weight loss, colon length and histologic scores. RESULTS: LPS-induced IL-12p40, IL-23p19, IL-6 and IL-1ß mRNA expressions were inhibited by BTE. LPS-induced IκBα phosphorylation/degradation and nuclear translocation of NF-κB/p65 were blocked by BTE. BTE treatment blocked LPS-induced DNA-binding activity of NF-κB. BTE-fed, DSS-exposed mice showed the less weight loss, longer colon length and lower histologic score compared to control diet-fed, DSS-exposed mice. DSS-induced IκBα phosphorylation/degradation and phosphorylation of NF-κB/p65 were blocked by BTE. An increase of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) in DSS-exposed mice was blocked by BTE. CONCLUSIONS: These results indicate that BTE attenuates colon inflammation through the blockage of NF-κB signaling and apoptosis in DSS-induced experimental colitis model.

Comparison of survival analysis between surgical and non-surgical treatments in Duchenne muscular dystrophy scoliosis.

BACKGROUND CONTEXT: There are still controversies about the effects of spinal surgeries for Duchenne muscular dystrophy (DMD) scoliosis on functional outcome, respiratory function, and the survival rate. PURPOSE: The purpose of this retrospective investigation was to compare the clinical course over time between the patients who were treated surgically and those who were treated nonsurgically. Through this comparison, we tried to determine how surgical treatment could affect the functional status, pulmonary function, and survival rate in patients with DMD scoliosis. STUDY DESIGN/SETTING: Single-center retrospective cohort study. PATIENT SAMPLE: We reviewed the clinical data of 199 male patients with DMD scoliosis who were followed up at our center for an average of 6.4 years between 2003 and 2017. OUTCOME MEASURES: The basic radiologic parameters evaluated include the Cobb angle and pelvic obliquity on a whole spine X-ray. Further, the Swinyard scale for functional status, forced vital capacity (FVC) for respiratory function, and mortality were compared between the surgical group and nonsurgical group. METHODS: The radiologic parameters and Swinyard scale stage were compared between the surgical group and nonsurgical group at baseline and 2, 5, and 10 years. For the FVC, serial changes every year were investigated in both groups. Mortality was surveyed between the surgical group and nonsurgical group. RESULTS: Of the 199 patients, 99 patients underwent the instrumented spinal fusion surgery and 100 patients in the nonsurgical group opted for conservative management. Radiologic results of the two groups were not different at baseline, but during the follow-up periods, the surgical group demonstrated better Cobb angles and pelvic obliquities. The surgical group showed a better functional status than did the nonsurgical group (6.7±0.9 versus [vs.] 7.2±0.7, p<.001). These functional differences between the groups were continuously observed during the follow-up period. Similarly, the FVC at baseline was higher in the surgical group than in the nonsurgical group (1005.7±421.4 mL vs. 787.3±574.1 mL, p=.005). Although FVC in the nonsurgical group consistently decreased during the follow-up (4.8% decrease/year), FVC in the surgical group increased up to the 2-year follow-up period compared with the baseline value and decreased during the follow-up period (2.8% decrease/year). Mortality was higher in the nonsurgical group than in the surgical group (n=22/100, 22.0% vs. n=8/99, 8.1%; p<.001) during an average follow-up duration of 6.4 years. Mean survival was longer in the surgical group than in the nonsurgical group (12.2 years vs. 8.3 years, hazard ratio=2.43, p=.02). CONCLUSIONS: Spinal surgery for DMD scoliosis improved the FVC for approximately 2 years postoperatively compared to non-surgical treatment. The surgical group had a better functional status and FVC at baseline than the non-surgical group. The positive effect of surgical treatment on the FVC is owing to scoliosis correction, which delayed the decrease of FVC and consequently extended the survival rate of the patients with DMD scoliosis.

Evolving artificial metalloenzymes via random mutagenesis.

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.

Expression of survivin in gastric cancer and its relationship with tumor angiogenesis.

BACKGROUND/AIMS: Survivin, a member of inhibitors of apoptosis, has been found in various human cancers. Its expression is associated with tumor progression and adverse outcome. Angiogenesis is an essential process for the primary tumor to grow and invade the adjacent normal structures. Angiogenic factors such as vascular endothelial growth factor induce survivin expression in endothelial cells. The current study was designed to investigate the possible role of survivin and vascular endothelial growth factor status for angiogenesis in human gastric cancer. METHODS: In this study, we conducted an immunohistochemical investigation of survivin and vascular endothelial growth factor expression in 106 tissue samples obtained from gastric cancer patients undergoing surgical treatment. To assess tumor angiogenesis, microvessel density was counted by staining endothelial cells immunohistochemically using anti-CD34 monoclonal antibody. RESULTS: The positive expression of survivin and vascular endothelial growth factor in gastric cancer tissues was demonstrated in 50.0 and 69.8% of cases, respectively. The expression of survivin did not associate with vascular endothelial growth factor expression. Expression of survivin was significantly associated with tumor size, depth of invasion, lymph node metastasis, tumor stage and poor survival (P=0.011, 0.004, 0.020, 0.002, 0.046, respectively). High microvessel density was significantly associated with lymph node metastasis and poor survival (P=0.006 and 0.017, respectively). The mean microvessel density value of survivin positive tumors was 87.4+/-34.4 and significantly higher than that of survivin negative tumors (P=0.016). The mean microvessel density value of vascular endothelial growth factor positive tumors was 98.7+/-37.0 and significantly higher than that of vascular endothelial growth factor negative tumors (P=0.001). A combined analysis of survivin and vascular endothelial growth factor status showed that the mean microvessel density value of both positive tumors was 103.7+/-33.1 and significantly higher than that of both negative tumors (P<0.001). CONCLUSION: These results suggest that survivin may play an important role in carcinogenesis by stimulating tumor angiogenesis in human gastric cancer.

Expression of RON in colorectal cancer and its relationships with tumor cell behavior and prognosis.

AIMS AND BACKGROUND: The aims of the current study were to evaluate whether recepteur d'origine nantais (RON) affects tumor cell behavior and oncogenic signaling pathways in colorectal cancer, and to examine the relationship of its expression with various clinicopathological parameters and patient survival. METHODS: Immunohistochemistry, Western blot and RT-PCR were used to detect the expression of the RON gene in human colorectal cancer tissue. To study the biological role of RON in tumor cell behavior and cellular signaling pathways, we used small interfering RNA (siRNA) to knock down RON gene expression in human colorectal cancer cell lines. RESULTS: Knockdown of RON inhibited the induction of the invasive growth phenotype and the activation of oncogenic signaling pathways including Akt, MAPK and ß-catenin. RON overexpression was associated with tumor size, lymphovascular invasion, depth of invasion, lymph node metastasis, distant metastasis, tumor stage and poor survival. CONCLUSIONS: These results suggest that RON overexpression may help in predicting poor clinical outcomes in colorectal cancer.

Association between single nucleotide polymorphisms of the transient receptor potential vanilloid 1 (TRPV-1) gene and patients with irritable bowel syndrome in Korean populations.

BACKGROUND AND STUDY AIMS: Transient receptor potential vanilloid type 1 (TRPV1) plays a crucial role in pain perception and its expression is up-regulated in patients with irritable bowel syndrome (IBS). The aim of this study was to investigate the potential association between Single nucleotide polymorphism (SNPs) of the TRPV-1 gene and patients with IBS. PATIENTS AND METHODS: We chose to focus on three SNPs in the human TRPV1 coding region (rs222749, rs9894618 and rs222747) in 80 healthy controls and 103 IBS patients. We developed the high resolution melting (HRM) method to determine the genotyping of rs222747 and rs9894618 and the genotyping of rs222749 was also determined by direct sequencing method. RESULTS: The CG genotype of rs222747 was 58.8% in controls and 45.6% in the IBS group. The GG genotype of rs222747 was 15.0% in controls and 20.4% in the IBS group. The CT genotype of rs222749 was 313% in controls and 32.0% in the IBS group. The CC genotype of rs9894618 was 98.8% in controls and 100.0% in the IBS group. There was no significant difference in allele frequency of these three SNPs of the TRPV1 gene between controls and the IBS group. Also, no significant difference was observed between the IBS subtypes. CONCLUSIONS: These results suggest that the SNPs of the TRPV1 gene may not be associated with IBS in Korean populations. Further studies with large cases are needed to validate the results of the present study.

Small interfering RNA-directed targeting of RON alters invasive and oncogenic phenotypes of human hepatocellular carcinoma cells.

The recepteur d'origine nantais (RON) receptor tyrosine kinase is highly expressed in various cancers including human hepatocellular carcinoma (HCC) and involved in tumor progression. The aims of the current study were to evaluate whether RON affects tumor cell behavior and oncogenic signaling cascades in HCC cells. We investigated the biologic role of RON on tumor cell behavior and oncogenic signaling cascades including Akt, c-Raf and extracellular signal-regulated kinase (ERK) by using the small interfering RNA (siRNA) in HCC cell lines, chang, HepG2 and Huh7. Knockdown of RON suppressed tumor cell migration and invasion in all tested HCC cell lines. The proportion of apoptotic cells induced by knockdown of RON was greater than that induced by transfection of the scramble siRNA in all tested HCC cell lines. Knockdown of RON resulted in cell cycle arrest in the G2/M phase of chang and Huh7 cells, and sub G1 phase of HepG2 cells. Knockdown of RON activated cleaved caspase-3 and PARP, and down-regulated the expression of Bcl-2, Bcl-xL and survivin, leading to induction of apoptosis in all tested cell lines. Knockdown of RON negatively regulates the progression of the cell cycle by decreasing cyclin D1 and D3, and increasing p21 and p27 in all tested cell lines. The phosphorylation of Akt, c-Raf and ERK1/2 signal proteins was significantly blocked by knockdown of RON in all tested cell lines. These results suggest that RON is associated with invasive and oncogenic phenotypes such as tumor cell migration, invasion, resistance to apoptosis and cell cycle arrest through the modulation of Akt, c-Raf and ERK signaling cascades in HCC cells.