Nedd4-2 E3 ligase regulates Na+ homeostasis by ubiquitinating various channels and membrane transporters, including the epithelial sodium channel ENaC. In turn, Nedd4-2 dysregulation leads to various conditions, including electrolytic imbalance, respiratory distress, hypertension, and kidney diseases. However, Nedd4-2 regulation remains mostly unclear. The present study aims at elucidating Nedd4-2 regulation by structurally characterizing Nedd4-2 and its complexes using several biophysical techniques. Our cryo-EM reconstruction shows that the C2 domain blocks the E2-binding surface of the HECT domain. This blockage, ubiquitin-binding exosite masking by the WW1 domain, catalytic C922 blockage and HECT domain stabilization provide the structural basis for Nedd4-2 autoinhibition. Furthermore, Ca2+-dependent C2 membrane binding disrupts C2/HECT interactions, but not Ca2+ alone, whereas 14-3-3 protein binds to a flexible region of Nedd4-2 containing the WW2 and WW3 domains, thereby inhibiting its catalytic activity and membrane binding. Overall, our data provide key mechanistic insights into Nedd4-2 regulation toward fostering the development of strategies targeting Nedd4-2 function.

• MeSH
• Cryoelectron Microscopy MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Domains MeSH
• 14-3-3 Proteins * metabolism   chemistry   MeSH
• Ubiquitination MeSH
• Nedd4 Ubiquitin Protein Ligases * metabolism   chemistry   genetics   ultrastructure   MeSH
• Calcium * metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nedd4 protein, human MeSH Browser
• Nedd4L protein, human MeSH Browser
• 14-3-3 Proteins * MeSH
• Nedd4 Ubiquitin Protein Ligases * MeSH
• Calcium * MeSH

In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shield's limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG super-family, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat.

• MeSH
• Humans MeSH
• Membrane Glycoproteins metabolism   MeSH
• Protozoan Proteins metabolism   MeSH
• Variant Surface Glycoproteins, Trypanosoma * metabolism   MeSH
• Trypanosomiasis, African * parasitology   immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Glycoproteins MeSH
• Protozoan Proteins MeSH
• Variant Surface Glycoproteins, Trypanosoma * MeSH

Ca2+ /CaM-dependent protein kinase kinases 1 and 2 (CaMKK1 and CaMKK2) phosphorylate and enhance the catalytic activity of downstream kinases CaMKI, CaMKIV, and protein kinase B. Accordingly, CaMKK1 and CaMKK2 regulate key physiological and pathological processes, such as tumorigenesis, neuronal morphogenesis, synaptic plasticity, transcription factor activation, and cellular energy homeostasis, and promote cell survival. Both CaMKKs are partly inhibited by phosphorylation, which in turn triggers adaptor and scaffolding protein 14-3-3 binding. However, 14-3-3 binding only significantly affects CaMKK1 function. CaMKK2 activity remains almost unchanged after complex formation for reasons still unclear. Here, we aim at structurally characterizing CaMKK1:14-3-3 and CaMKK2:14-3-3 complexes by SAXS, H/D exchange coupled to MS, and fluorescence spectroscopy. The results revealed that complex formation suppresses the interaction of both phosphorylated CaMKKs with Ca2+ /CaM and affects the structure of their kinase domains and autoinhibitory segments. But these effects are much stronger on CaMKK1 than on CaMKK2 because the CaMKK1:14-3-3γ complex has a more compact and rigid structure in which the active site of the kinase domain directly interacts with the last two C-terminal helices of the 14-3-3γ protein, thereby inhibiting CaMKK1. In contrast, the CaMKK2:14-3-3 complex has a looser and more flexible structure, so 14-3-3 binding only negligibly affects the catalytic activity of CaMKK2. Therefore, Ca2+ /CaM binding suppression and the interaction of the kinase active site of CaMKK1 with the last two C-terminal helices of 14-3-3γ protein provide the structural basis for 14-3-3-mediated CaMKK1 inhibition.

• Keywords
• 14-3-3 proteins, CaMKK, SAXS, calcium/calmodulin-dependent protein kinase, fluorescence spectroscopy, hydrogen/deuterium exchange coupled to MS, protein-protein interaction,
• MeSH
• X-Ray Diffraction MeSH
• Phosphorylation MeSH
• Catalytic Domain MeSH
• Calcium-Calmodulin-Dependent Protein Kinase Kinase * chemistry   metabolism   MeSH
• Scattering, Small Angle MeSH
• 14-3-3 Proteins * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Calcium-Calmodulin-Dependent Protein Kinase Kinase * MeSH
• 14-3-3 Proteins * MeSH

Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.

• MeSH
• Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase MeSH
• Glycoproteins * analysis   MeSH
• Glycosylation MeSH
• Polysaccharides * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase MeSH
• Glycoproteins * MeSH
• Polysaccharides * MeSH

Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

• Keywords
• Antibody, Biosimilars, Hydrogen/deuterium exchange, Mass spectrometry, Protein structure and dynamics, Proteolysis,
• MeSH
• Deuterium MeSH
• Mass Spectrometry MeSH
• Antibodies * MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry * MeSH
• Hydrogen MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Antibodies * MeSH
• Hydrogen MeSH

Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no limitations in terms of protein size, flexibility, or reaction conditions and can thus be performed in solution at different pH values and temperatures under controlled redox conditions. Thanks to its coupling with mass spectrometry (MS), it is also straightforward to perform and has relatively high throughput, making it an excellent complement to the high-resolution methods of structural biology. Given the recent expansion of artificial intelligence-aided protein structure modeling, there is considerable demand for techniques allowing fast and unambiguous validation of in silico predictions; HDX-MS is well-placed to meet this demand. Here we present a protocol for HDX-MS and illustrate its use in characterizing the dynamics and structural changes of a dimeric heme-containing oxygen sensor protein as it responds to changes in its coordination and redox state. This allowed us to propose a mechanism by which the signal (oxygen binding to the heme iron in the sensing domain) is transduced to the protein's functional domain.

• Keywords
• Globin-coupled histidine kinase, Heme-containing oxygen sensors, Hydrogen/deuterium exchange, Ligand binding, Mass spectrometry, Protein conformational dynamics, Signal transduction,
• MeSH
• Deuterium MeSH
• Heme chemistry   MeSH
• Hemeproteins * MeSH
• Mass Spectrometry methods   MeSH
• Oxygen metabolism   MeSH
• Artificial Intelligence MeSH
• Deuterium Exchange Measurement methods   MeSH
• Hydrogen chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Heme MeSH
• Hemeproteins * MeSH
• Oxygen MeSH
• Hydrogen MeSH

Intrinsic protein dynamics contribute to their biological functions. Rational engineering of protein dynamics is extremely challenging with only a handful of successful examples. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) represents a powerful technique for quantitative analysis of protein dynamics. Here we provide a detailed description of the preparation of protein samples, collection of high-quality data, and their in-depth analysis using various computational tools. We illustrate the application of HDX-MS for the study of protein dynamics in the rational engineering of flexible loops in the reconstructed ancestor of haloalkane dehalogenase and Renilla luciferase. These experiments provided unique and valuable data rigorously describing the modification of protein dynamics upon grafting of the loop-helix element. Tips and tricks are provided to stimulate the wider use of HDX-MS to study and engineer protein dynamics.

• Keywords
• Ancestral luciferase, Hydrogen/deuterium exchange, LoopGrafter, Mass spectrometry, Protein dynamics, Protein engineering,
• MeSH
• Deuterium chemistry   MeSH
• Mass Spectrometry methods   MeSH
• Protein Conformation MeSH
• Deuterium Exchange Measurement * methods   MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry * MeSH
• Hydrogen chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Hydrogen MeSH

Identification of a protein minimal fragment amenable to crystallisation can be time- and labour intensive especially if large amounts are required and the protein has a complex fold and functionally important post-translational modifications. In addition, a lack of homologues and structural information can further complicate the design of a minimal expression construct. Recombinant expression in E. coli promises high yields, low costs and fast turnover times, but falls short for many extracellular, eukaryotic proteins. Eukaryotic expression systems provide an alternative but are costly, slow and require special handling and equipment. Using a member of a structurally uncharacterized, eukaryotic receptor family as an example we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) guided construct design in conjunction with truncation scanning and targeted expression host switching to identify a minimal expression construct that can be produced with high yields and moderate costs.

• MeSH
• Escherichia coli genetics   MeSH
• Protein Conformation MeSH
• Membrane Proteins MeSH
• Trypanosoma * MeSH
• Deuterium Exchange Measurement * methods   MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH

A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.

• Keywords
• CD20, comparative medicine, hydrogen deuterium exchange mass spectrometry, lymphoma, monoclonal antibody,
• MeSH
• Antigens, CD20 immunology   MeSH
• Chromatography, Liquid MeSH
• Immunoglobulin G chemistry   MeSH
• Kinetics MeSH
• Immunoglobulin Light Chains genetics   metabolism   MeSH
• Humans MeSH
• Antibodies, Monoclonal chemistry   genetics   MeSH
• Cell Line, Tumor MeSH
• Peptide Library MeSH
• Dogs MeSH
• Recombinant Fusion Proteins MeSH
• Amino Acid Sequence MeSH
• Tandem Mass Spectrometry MeSH
• Immunoglobulin Heavy Chains genetics   metabolism   MeSH
• Binding Sites, Antibody MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, CD20 MeSH
• Immunoglobulin G MeSH
• Immunoglobulin Light Chains MeSH
• Antibodies, Monoclonal MeSH
• Peptide Library MeSH
• Recombinant Fusion Proteins MeSH
• Immunoglobulin Heavy Chains MeSH

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

• Keywords
• Autophagosome, LC3, cancer, flux, lysosome, macroautophagy, neurodegeneration, phagophore, stress, vacuole,
• MeSH
• Autophagy * physiology   MeSH
• Autophagosomes MeSH
• Biomarkers MeSH
• Biological Assay standards   MeSH
• Humans MeSH
• Lysosomes MeSH
• Autophagy-Related Proteins metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Guideline MeSH
• Names of Substances
• Biomarkers MeSH
• Autophagy-Related Proteins MeSH