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.