MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin β1 Interactomes

Importin β1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin β1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin β1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.     

Deciphering Spatial Protein–Protein Interactions in Brain Using Proximity Labeling

Cellular biomolecular complexes including protein–protein, protein–RNA, and protein–DNA interactions regulate and execute most biological functions. In particular in brain, protein–protein interactions (PPIs) mediate or regulate virtually all nerve cell functions, such as neurotransmission, cell–cell communication, neurogenesis, synaptogenesis, and synaptic plasticity. Perturbations of PPIs in specific subsets of neurons and glia are thought to underly a majority of neurobiological disorders. Therefore, understanding biological functions at a cellular level requires a reasonably complete catalog of all physical interactions between proteins. An enzyme-catalyzed method to biotinylate proximal interacting proteins within 10 to 300 nm of each other is being increasingly used to characterize the spatiotemporal features of complex PPIs in brain. Thus, proximity labeling has emerged recently as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and PPIs in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte–neuron junctions. In this review, we summarize recent advances in proximity labeling methods and their application to neurobiology.     

When corridors collide: Road-related disturbance in commuting bats

As an increasingly dominant feature in the landscape, transportation corridors are becoming a major concern for bats. Although wildlife–vehicle collisions are considered to be a major source of mortality, other negative implications of roads on bat populations are just now being realized. Recent studies have revealed that bats, like many other wildlife species, will avoid roads rather than cross them. The consequence is that roads act as barriers or filters to movement, restricting bats from accessing critical resources. Our objective was to assess specific features along the commuting route, road, or surrounding landscape (alone or in combination) that exacerbated or alleviated the likelihood of a commuting bat exhibiting an avoidance behavior in response to an approaching vehicle. At 5 frequently used commuting routes bisected by roads, we collected data on vehicles travelling along the roads (such as visibility and audibility), commuting bats (such as height), and composition of the commuting route. We revealed that commuting route structure dictated the frequency at which bats turned back along their commuting routes and avoided the road. We found that gaps (>2 m) in commuting routes, such as the road itself, caused bats to turn away just before they reached the road. Furthermore, we found that turning frequencies of bats increased with vehicle noise levels and the locations at which bats responded to vehicles corresponded with areas where noise levels were greatest, including gaps <2 m. This suggested that bats had a disturbance threshold, and only reacted to vehicles when associated noise reached a certain level. We found that threshold levels for our study species were approximately 88 dB, but this value was likely to vary among species. Thus, our findings indicate that restoring (e.g., replanting native trees and shrubs in gaps) and establishing commuting routes (such as planting tree-lines and wooded hedgerows), as well as creating road-crossing opportunities (such as interlinking canopies) will improve the permeability of a road-dominated landscape to bats. Furthermore, our study highlights the influence of the soundscape. We recommend that effective management and mitigation strategies should take into account the ecological design of the acoustic environment. © 2012 The Wildlife Society.     

A procedure for quantification of cytokinins as free bases involving scintillation proximity immunoassay

Cytokinins occur in a diversity of forms and determination of their individual levels requires extensive purification. However, determination of the total level of each major base in free, riboside and nucleotide forms would often be adequate. Hence, a methanolysis procedure which releases cytokinin bases from 9-ribosyl derivatives was developed and applied to plant extracts. A simple procedure, involving low pressure column chromatography, for purification of the cytokinin bases in treated extracts, and a scintillation proximity immunoassay for their quantification, were developed. The total level of each cytokinin base [N⁶-(2-isopentenyl)adenine, zeatin and dihydrozeatin] in free and ribosylated forms determined by these methods is reported for several plant tissues and the results are compared with those obtained after additional purification by HPLC. Values for zeatin were not changed by HPLC but isopentenyl-adenine and dihydrozeatin levels were usually reduced indicating the presence of unknown compounds which cross-react in the immunoassay. Modifications to the above purification method to quantify O-glucosyl cytokinins are also described.
The methods described facilitate the quantification of the total amount of each cytokinin base in forms closely associated with cytokinin action, and the detection of cytokinin biosynthesis by labelled precursor incorporation.     

Effect of genetic and environmental factors on protein biomarkers for common non-communicable disease and use of personally normalized plasma protein profiles (PNPPP)

Abstract

Objective: To study the impact of genetic and lifestyle factors on protein biomarkers and develop personally normalized plasma protein profiles (PNPPP) controlling for non-disease-related variance.

Materials and methods: Proximity extension assays were used to measure 145 proteins in 632 controls and 344 cases with non-communicable diseases.

Results: Genetic and lifestyle factors explained 20–88% of the variation in healthy controls. Adjusting for these factors reduced the number of candidate biomarkers by 63%.

Conclusion: PNPPP efficiently controls for non-disease-related variance, allowing both for efficient discovery of novel biomarkers and for covariate-independent linear cut-offs suitable for clinical use.     

融于自然、展现风景——美国国家公园道路系统建设历程与经验启示

在中国国家公园体制试点工作不断推进的当下,探讨国家公园内基础设施的建设显得敏感而有必要。在梳理美国国家公园道路系统发展建设历程的基础上,总结提炼出其在前期论证、建设理念以及多部门合作等方面的经验。同时就如何统筹交通游赏需求与资源保护之间的关系也做了有益的探讨,对中国未来国家公园的发展建设乃至自然资源的保护和利用都具有较为重要的参考和借鉴意义。