生物化学

Plant proteases participate in a wide variety of biological processes, including development, growth, and defense. To date, numerous proteases have been functionally identified through genetic studies. However, redundancy among certain proteases can obscure their roles, as single-gene loss-of-function mutants often exhibit no discernible phenotype, limiting identification through genetic approaches. Here, we describe an efficient system for the identification of target proteases that cleave specific substrates in the Arabidopsis apoplastic fluid. The method involves using Arabidopsis-submerged culture medium, which contains apoplastic proteases, followed by native two-dimensional electrophoresis. Gel fractionation and an in-gel peptide cleavage assay with a fluorescence-quenching peptide substrate are then used to detect specific proteolytic activity. The active fraction is then subjected to mass spectrometry–based proteomics to identify the protease of interest. This method allows for the efficient and comprehensive identification of proteases with specific substrate cleavage activities in the apoplast.

Protein O-GlcNAcylation is a prevalent and dynamic post-translational modification that targets a multitude of nuclear and cytoplasmic proteins. Through the modification of diverse substrates, O-GlcNAcylation plays a pivotal role in essential cellular processes, including transcription, translation, and protein homeostasis. Dysregulation of O-GlcNAc homeostasis has been implicated in a variety of diseases, including cardiovascular diseases, cancer, and neurodegenerative diseases. Studying O-GlcNAcylated proteins in different tissues is crucial to understanding the pathogenesis of these diseases. However, identifying phenotype-relevant candidate substrates in a tissue-specific manner remains unfeasible. We developed a novel tool for the analysis of O-GlcNAcylated proteins, combining a catalytically inactive CpOGA mutant CpOGA^CD and TurboID proximity labeling technology. This tool converts O-GlcNAc modifications into biotin labeling, enabling the enrichment and mass spectrometry (MS) identification of O-GlcNAcylated proteins in specific tissues. Meanwhile, TurboID-CpOGA^DM, which carries two point mutations that inactivate both its catalytic and binding activities toward O-GlcNAc modification, was used as a control to differentiate O-GlcNAc-independent protein–protein interactions. We have successfully used TurboID-CpOGA^CD/DM (TurboID-CpOGA^M) to enrich O-GlcNAc proteins in Drosophila combining the UAS/Gal4 system. Our protocol provides a comprehensive workflow for tissue-specific enrichment of candidate O-GlcNAcylated substrates and offers a valuable tool for dissecting tissue-specific O-GlcNAcylation functions in Drosophila.

Voltage clamp fluorometry (VCF) is a powerful technique in which the voltage of a cell’s membrane is clamped to control voltage-sensitive membrane proteins while simultaneously measuring fluorescent signals from a protein of interest. By combining fluorescence measurements with electrophysiology, VCF provides real-time measurement of a protein’s motions, which gives insight into its function. This protocol describes the use of VCF to study a membrane protein, the voltage-sensing phosphatase (VSP). VSP is a 3 and 5 phosphatidylinositol phosphate (PIP) phosphatase coupled to a voltage sensing domain (VSD). The VSD of VSP is homologous to the VSD of ion channels, with four transmembrane helices (S1–S4). The S4 contains the gating charge arginine residues that sense the membrane’s electric field. Membrane depolarization moves the S4 into a state that activates the cytosolic phosphatase domain. To monitor the movement of S4, the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) is attached extracellularly to the S3-S4 loop. Using VCF, the resulting fluorescence signals from the S4 movement measure the kinetics of activation and repolarization, as well as the voltage dependence of the VSD. This protocol details the steps to express VSP in Xenopus laevis oocytes and then acquire and analyze the resulting VCF data. VCF is advantageous as it provides voltage control of VSP in a native membrane while quantitatively assessing the functional properties of the VSD.

Fatty acid (FA) biosynthesis is a crucial cellular process that converts nutrients into metabolic intermediates necessary for membrane biosynthesis, energy storage, and the production of signaling molecules. Acetyl-CoA carboxylase (ACACA) plays a pivotal catalytic role in both fatty acid synthesis and oxidation. This cytosolic enzyme catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, which represents the first and rate-limiting step in de novo fatty acid biosynthesis. In this study, we developed a rapid and effective purification scheme for separating human ACACA without any exogenous affinity tags, providing researchers with a novel method to obtain human ACACA in its native form.

The Mediator, a multi-subunit protein complex in all eukaryotes, comprises the core mediator (cMED) and the CDK8 kinase module (CKM). As a molecular bridge between transcription factors (TFs) and RNA polymerase II (Pol II), the Mediator plays a critical role in regulating Pol II–dependent transcription. Considering its large size and complex composition, conducting in vitro studies on the Mediator complex is challenging, especially when isolating the intact and homogeneous complex from human cells. Here, we present a method to purify the intact CKM-cMED complex from FreeStyle 293-F cells (293-F cells), which offers advantages for performing large-scale protein purification. To isolate the CKM-bound cMED without the presence of Pol II, FLAG-tagged CDK8, a subunit of the CKM complex, was expressed in 293-F cells for purification, as CKM and Pol II are mutually exclusive in their interaction with cMED. The complex is isolated from nuclear extracts through immunoaffinity purification and further purified by glycerol gradient to enhance its homogeneity. This protocol provides a time- and cost-efficient way to purify the endogenous Mediator complex for structural- and functional-based studies.

Time-resolved cryo-EM (TRCEM) makes it possible to provide structural and kinetic information on a reaction of biomolecules before the equilibrium is reached. Several TRCEM methods have been developed in the past to obtain key insights into the mechanism of action of molecules and molecular machines on the time scale of tens to hundreds of milliseconds, which is unattainable by the normal blotting method. Here, we present our TRCEM setup utilizing a polydimethylsiloxane (PDMS)-based microfluidics chip assembly, comprising three components: a PDMS-based, internally SiO₂-coated micromixer, a glass-capillary microreactor, and a PDMS-based microsprayer for depositing the reaction product onto the EM grid. As we have demonstrated in recent experiments, this setup is capable of addressing problems of severe sample adsorption and ineffective mixing of fluids and leads to highly reproducible results in applications to the study of translation. As an example, we used our TRCEM sample preparation method to investigate the molecular mechanism of ribosome recycling mediated by High frequency of lysogenization X (HflX), which demonstrated the efficacy of the TRCEM device and its capability to yield biologically significant, reproducible information. This protocol has the promise to provide structural and kinetic information on pre-equilibrium intermediates in the 10–1,000 ms time range in applications to many other biological systems.

The physiological role of a-synuclein (a-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, a-syn will self-assemble into β-sheet-rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient-derived brain tissues have concluded that these inclusions are associated with Parkinson’s disease, the second most common neurodegenerative disorder, and other synuclein-related diseases called synucleinopathies. In addition, repetitions of specific mutations to the SNCA gene, the gene that encodes a-syn, result in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of a-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 Å. Here, we provide a protocol for a-syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step-by-step protocol for high-resolution cryo-EM structure determination of a-syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high-resolution cryo-EM map. This workflow resulted in a 2.04 Å structure of a-syn fibrils with excellent resolution of residues 36–97 and an additional island of density for residues 15–22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of a-syn and other amyloid fibrils.

Different research methods aim to clarify the intracellular trafficking of target proteins or unknown pathways. Currently, existing methods are mostly complex and expensive, requiring expert knowledge. Detailed microscopy for protein co-localization detection or omic technologies, which provide holistic network data, are elaborate, mostly complex, and expensive to apply. Our protocol illustrates a method to track a target protein by detecting expression changes of user-selected marker proteins that directly or indirectly interact with the target. Modulation of protein expression indicates interactions between the target and marker protein. Even without co-localization analysis, the results of the protein expression change are the first insights into the target's fate. Moreover, the use of the cell-sonar is straightforward and affordable, and the results are rapidly available. Furthermore, this method could also be used to determine if and how pathways are affected by compounds added to the cells. In conclusion, our method is adaptable to a wide range of proteins, easy to apply, inexpensive, and expandable with substances that affect proteins.

Myosin-5a (Myo5a) is an actin-dependent molecular motor that recognizes a diverse range of cargo proteins through its tail domain, playing a crucial role in the transport and localization of various organelles within the cell. We have identified a new interaction between Myo5a and its cargo protein melanophilin (Mlph), i.e., the interaction between the middle tail domain of Myo5a (Myo5a-MTD) and the actin-binding domain of Mlph (Mlph-ABD), by GST pulldown assay. We then intend to obtain the dissociation constant between Myo5a-MTD and Mlph-ABD using isothermal titration calorimetry (ITC) or microscale thermophoresis (MST), both of which are two commonly used methods for determining quantitative data on protein interactions. The advantages of MST over ITC include less protein usage, shorter operation time, and higher sensitivity. In this protocol, we present a method for using MST to determine the dissociation constants of Myo5a-MTD and Mlph-ABD, which were purified through overexpression in bacteria using affinity chromatography. The dissociation constant values obtained directly reflect the binding strength between these two proteins and provide a foundation for the isolation and purification of the complex in the future.

Amylin is an amyloidogenic neuroendocrine hormone co-synthesized and co-secreted with insulin from the pancreas. It readily crosses the blood–brain barrier and synergistically forms mixed amyloid plaques with β-amyloid (Aβ) in brain parenchyma. Parenchymal amylin-Aβ plaques are found in both sporadic and early-onset familial Alzheimer’s disease (AD), yet their (patho)physiological role remains elusive, particularly due to a lack of detection modalities for these mixed plaques. Previously, we developed an enzyme-linked immunosorbent assay (ELISA) capable of detecting amylin-Aβ hetero-oligomers in brain lysate and blood using a polyclonal anti-amylin antibody to capture hetero-oligomers and a monoclonal anti-Aβ mid-domain detection antibody combination. This combination allows for the recognition of distinct amylin epitopes, which remain accessible after amylin-Aβ oligomerization has begun, and precise detection of Aβ epitopes available after oligomer formation. The utility of this assay is evidenced in our previous report, wherein differences in hetero-oligomer content in brain tissue from patients with and without AD and patients with and without diabetes were distinguished. Additionally, using AD model rats, we provided evidence that our assay can be employed for the detection of amylin-Aβ in blood. This assay and protocol are important innovations in the field of AD research because they meet an unmet need to detect mixed amyloid plaques that, if targeted therapeutically, could reduce AD progression and severity.