Mammalian Cell Lysates Research Articles

Isolating and Analyzing Protein Containing Granules from Cells

Recent advancements in detection methods have made protein condensates, also called granules, a major area of study, but tools to characterize these assemblies need continued development to keep up with evolving paradigms. We have optimized a protocol to separate condensates from cells using chemical cross-linking followed by size-exclusion chromatography (SEC). After SEC fractionation, the samples can be characterized by a variety of approaches including enzyme-linked immunosorbent assay, dynamic light scattering, electron microscopy, and mass spectrometry. The protocol described here has been optimized for cultured mammalian cells and E. coli expressing recombinant proteins. Since the lysates are fractionated by size, this protocol can be modified to study other large protein assemblies, including the nuclear pore complex, and for other tissues or organisms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: SEC separation of cross-linked mammalian cell lysates Alternate Protocol: Preparation of non-crosslinked mammalian cells Basic Protocol 2: SEC separation of E. coli lysate Support Protocol 1: Detecting protein of interest by ELISA Support Protocol 2: TCA precipitation of SEC fractions.

Calmodulin influences MAPK signaling by binding KSR1

The mitogen-activated protein kinase (MAPK) cascade is a fundamental signaling pathway that regulates cell fate decisions in response to external stimuli. Several scaffold proteins bind directly to kinase components of this pathway and regulate their activation by growth factors. One of the best studied MAPK scaffolds is kinase suppressor of Ras1 (KSR1), which is induced by epidermal growth factor (EGF) to translocate to the plasma membrane where it activates extracellular signal-regulated kinase (ERK). While Ca2+ has been shown to modulate MAPK signaling, the molecular mechanisms by which this occurs are incompletely understood. Here we tested the hypothesis that Ca2+ alters MAPK activity at least in part via KSR1. Using several approaches, including fusion proteins, immunoprecipitation, confocal microscopy, and a cell-permeable chemical inhibitor, we investigated the functional interaction between KSR1 and calmodulin. In vitro analysis with pure proteins reveals that calmodulin binds directly to KSR1. Moreover, endogenous calmodulin and KSR1 co-immunoprecipitate from mammalian cell lysates. Importantly, Ca2+ is required for the association between calmodulin and KSR1, both in vitro and in cells. The cell-permeable calmodulin antagonist CGS9343B significantly reduced activation of ERK by EGF in mouse embryo fibroblasts that overexpress KSR1, but not in control cells. Moreover, CGS9343B impaired the ability of EGF to induce KSR1 translocation to the plasma membrane and to stimulate formation of KSR1-ERK and KSR1-pERK (phosphorylated ERK) complexes in cells. Collectively, our data identify a previously unrecognized mechanism by which the scaffold protein KSR1 couples Ca2+ and calmodulin signaling to the MAPK cascade.

Megadalton-sized Dityrosine Aggregates of α-Synuclein Retain High Degrees of Structural Disorder and Internal Dynamics

Heterogeneous aggregates of the human protein α-synuclein (αSyn) are abundantly found in Lewy body inclusions of Parkinson’s disease patients. While structural information on classical αSyn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of αSyn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-β amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict αSyn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species, rather than cytochrome c, are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining αSyn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative αSyn aggregation scavenges cytochrome c’s activity into the formation of amorphous, high molecular-weight structures that may contribute to the structural diversity of Lewy body deposits.

Generation and validation of recombinant antibodies to study human aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have noncanonical functions, and several of the aaRSs have been linked to autoimmune diseases, cancer, and neurological disorders. Deciphering these roles has been challenging because of a lack of tools to enable their study. To help solve this problem, we have generated recombinant high-affinity antibodies for a collection of thirteen cytoplasmic and one mitochondrial aaRSs. Selected domains of these proteins were produced recombinantly in Escherichia coli and used as antigens in phage display selections using a synthetic human single-chain fragment variable library. All targets yielded large sets of antibody candidates that were validated through a panel of binding assays against the purified antigen. Furthermore, the top-performing binders were tested in immunoprecipitation followed by MS for their ability to capture the endogenous protein from mammalian cell lysates. For antibodies targeting individual members of the multi-tRNA synthetase complex, we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a subset of binders for each target was also confirmed using immunofluorescence. The sequences of these proteins have been deposited in publicly available databases and repositories. We anticipate that this open source resource, in the form of high-quality recombinant proteins and antibodies, will accelerate and empower future research of the role of aaRSs in health and disease.

129Xe NMR-Protein Sensor Reveals Cellular Ribose Concentration.

Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable via a sensitive NMR technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST). We demonstrate that RBP, with a tunable ribose-binding site and further engineered to bind xenon, enables the quantitation of ribose over a wide concentration range (nM to mM). Ribose binding induces the RBP "closed" conformation, which slows Xe exchange to a rate detectable by hyper-CEST. Such detection is remarkably specific for ribose, with the minimal background signal from endogenous sugars of similar size and structure, for example, glucose or ribose-6-phosphate. Ribose concentration was measured for mammalian cell lysate and serum, which led to estimates of low-mM ribose in a HeLa cell line. This highlights the potential for using genetically encoded periplasmic binding proteins such as RBP to measure metabolites in different biological fluids, tissues, and physiologic states.

Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-Protein Interactions.

Throughout their cellular lifetime, RNA transcripts are bound to proteins, playing crucial roles in RNA metabolism, trafficking, and function. Despite the importance of these interactions, identifying the proteins that interact with an RNA of interest in mammalian cells represents a major challenge in RNA biology. Leveraging the ability to site-specifically and covalently label an RNA of interest using E. coli tRNA guanine transglycosylase and an unnatural nucleobase substrate, we establish the identification of RNA-protein interactions and the selective enrichment of cellular RNA in mammalian systems. We demonstrate the utility of this approach through the identification of known binding partners of 7SK snRNA via mass spectrometry. Through a minimal 4-nucleotide mutation of the long noncoding RNA HOTAIR, enzymatic biotinylation enables identification of putative HOTAIR binding partners in MCF7 breast cancer cells that suggest new potential pathways for oncogenic function. Furthermore, using RNA sequencing and qPCR, we establish that an engineered enzyme variant achieves high levels of labeling selectivity against the human transcriptome allowing for 145-fold enrichment of cellular RNA directly from mammalian cell lysates. The flexibility and breadth of this approach suggests that this system could be routinely applied to the functional characterization of RNA, greatly expanding the toolbox available for studying mammalian RNA biology.

Development of a cell-free split-luciferase biochemical assay as a tool for screening for inhibitors of challenging protein-protein interaction targets

Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc ® Binary Technology (NanoBiT ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.

Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context

Hepatitis B virus (HBV) core protein (HBc) is essential to the formation of the HBV capsid. HBc contains two domains: the N-terminal domain corresponding to residues 1–140 essential to form the icosahedral shell and the C-terminal domain corresponding to a basic and phosphorylated peptide, and required for DNA replication. The role of these two domains for HBV capsid assembly was essentially studied in vitro with HBc purified from mammalian or non-mammalian cell lysates, but their respective role in living cells remains to be clarified. We therefore investigated the assembly of the HBV capsid in Huh7 cells by combining fluorescence lifetime imaging microscopy/Förster's resonance energy transfer, fluorescence correlation spectroscopy and transmission electron microscopy approaches. We found that wild-type HBc forms oligomers early after transfection and at a sub-micromolar concentration. These oligomers are homogeneously diffused throughout the cell. We quantified a stoichiometry ranging from ~170 to ~230 HBc proteins per oligomer, consistent with the visualization of eGFP-containingHBV capsid shaped as native capsid particles by transmission electron microscopy. In contrast, no assembly was observed when HBc–N-terminal domain was expressed. This highlights the essential role of the C-terminal domain to form capsid in mammalian cells. Deletion of either the third helix or of the 124–135 residues of HBc had a dramatic impact on the assembly of the HBV capsid, inducing the formation of mis-assembled oligomers and monomers, respectively. This study shows that our approach using fluorescent derivatives of HBc is an innovative method to investigate HBV capsid formation.

A Chemical Probe for Dehydrobutyrine.

Bacterial phosphothreonine lyases, or phospholyases, catalyze a unique post-translational modification that introduces dehydrobutyrine (Dhb) or dehydroalanine (Dha) in place of phosphothreonine or phosphoserine residues, respectively. We report the use of a phospha-Michael reaction to label proteins and peptides modified with Dha or Dhb. We demonstrate that a nucleophilic phosphine probe is able to modify Dhb-containing proteins and peptides that were recalcitrant to reaction with thiol or amine nucleophiles under mild aqueous conditions. Furthermore, we used this reaction to detect multiple Dhb-modified proteins in mammalian cell lysates, including histone H3, a previously unknown target of phospholyases. This method should prove useful for identifying new phospholyase targets, profiling the biomarkers of bacterial infection, and developing enzyme-mediated strategies for bioorthogonal labeling in living cells.

A Chemical Probe for Dehydrobutyrine

AbstractBacterial phosphothreonine lyases, or phospholyases, catalyze a unique post‐translational modification that introduces dehydrobutyrine (Dhb) or dehydroalanine (Dha) in place of phosphothreonine or phosphoserine residues, respectively. We report the use of a phospha‐Michael reaction to label proteins and peptides modified with Dha or Dhb. We demonstrate that a nucleophilic phosphine probe is able to modify Dhb‐containing proteins and peptides that were recalcitrant to reaction with thiol or amine nucleophiles under mild aqueous conditions. Furthermore, we used this reaction to detect multiple Dhb‐modified proteins in mammalian cell lysates, including histone H3, a previously unknown target of phospholyases. This method should prove useful for identifying new phospholyase targets, profiling the biomarkers of bacterial infection, and developing enzyme‐mediated strategies for bioorthogonal labeling in living cells.

Development of a cell-free split-luciferase biochemical assay as a tool for screening for inhibitors of challenging protein-protein interaction targets.

Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc â® Binary Technology (NanoBiT â®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.

Target Identification of a 1,3,4‐Oxadiazin‐5(6H)‐One Anticancer Agent via Photoaffinity Labelling

AbstractRational design and synthetic feasibility are critical factors for the photoaffinity labelling (PAL) approach, which can identify protein targets of bioactive small molecules under native cellular conditions. In this study, we developed photoaffinity labelling probes derived from 1,3,4‐oxadiazin‐5(6H)‐ones (OPALs) for LL‐2003, a previously reported potential anticancer agent against IGF‐1R and Src. Our photoaffinity labelling strategy enabled successful photo‐crosslinking of the probes (OPAL‐6 and OPAL‐8) with the target proteins in both mammalian cell lysates and live MCF7, A549, HepG2 and HeLa cells in situ. In vitro and in situ labelling demonstrated different patterns and expression levels of the proteome, and the strongest band for Src appeared in the A549 cell line. An in‐gel fluorescence scan combined with MS/MS analysis of the IGF‐1R overexpressed insect proteome labelled by OPAL‐6 and OPAL‐8 identified the binding location of the synthesized probes.

Facile single-molecule pull-down assay for analysis of endogenous proteins

The single-molecule pull-down (SiMPull) assay analyzes molecular complexes in physiological conditions from cell or tissue lysates. Currently the approach requires a lengthy sample preparation process, which has largely prevented the widespread adoption of this technique in bioanalysis. Here, we present a simplified SiMPull assay based upon dichlorodimethylsilane—Tween-20 passivation and F(ab) fragment labeling. Our passivation is a much shorter process than the standard polyethylene glycol passivation used in most single-molecule studies. The use of F(ab) fragments for indirect fluorescent labeling rather than divalent F(ab′)2 or whole IgG antibodies allows for the pre-incubation of the detection antibodies, reducing the sample preparation time for single-molecule immunoprecipitation samples. We examine the applicability of our approach to recombinant proteins and endogenous proteins from mammalian cell lysates.

Chemically reprogramming the phospho-transfer reaction to crosslink protein kinases to their substrates.

The proteomic mapping of enzyme-substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide-based substrate analog capable of reprogramming the wild-type phosphoryl-transfer reaction to produce a kinase-acrylamide-based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP-based methacrylate crosslinker (ATP-MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP-MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP-based methacrylate (ADP-MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP-MA over ATP-MA is due to reduced inhibition of the second step of the kinase-substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP-MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site-specific manner. The kinase-substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.

A Novel Single Molecule Assay Reveals the Deafness‐Associated Protein Otoferlin as a Multivalent Calcium‐Sensitive Scaffold Linking SNAREs and Calcium Channels

Sensory hair cells rely on otoferlin as the calcium sensor for exocytosis and encoding of sound preferentially over the neuronal calcium sensor synaptotagmin. Although it is established that synaptotagmin cannot rescue the otoferlin KO phenotype, the large size and low solubility of otoferlin have prohibited direct biochemical comparisons that could establish functional differences between these two proteins. To address this challenge, we have developed a singlemolecule colocalization binding titration assay (smCoBRA) that can quantitatively characterize full-length otoferlin from mammalian cell lysate. Using smCoBRA, we found that, although both otoferlin and synaptotagmin bind membrane fusion SNARE proteins, only otoferlin interacts with the L-type calcium channel Cav1.3, showing a significant difference between the synaptic proteins. Furthermore, otoferlin was found capable of interacting with multiple SNARE and Cav1.3 proteins simultaneously, forming a heterooligomer complex. We also found that a deafness-causing missense mutation in otoferlin attenuates binding between otoferlin and Cav1.3, suggesting that deficiencies in this interaction may form the basis for otoferlin-related hearing loss. Based on our results, we propose a model in which otoferlin acts as a calcium-sensitive scaffolding protein, localizing SNARE proteins proximal to the calcium channel so as to synchronize calcium influx with membrane fusion. Our findings also provide a molecular-level explanation for the observation that synaptotagmin and otoferlin are not functionally redundant. This study also validates Q:9 a generally applicable methodology for quantitatively characterizing large, multivalent membrane proteins. This presentation is based on our recent publication (Hams et al. Proc Natl Acad Sci 2017 114:8023-8028). Support or Funding Information NIH NIDCD Grant 1R01DC014588 Schematic of sensory hair cell and neuronal presynapses. (A) Synapticribbons within the sensory hair cells of the cochlea position synaptic vesiclesproximal to the presynaptic membrane. Otoferlin resides on synaptic vesicles, whereas Cav1.3 localizes to the presynapse. (B) The synaptic vesicles of neurons harbor synaptotagmin I/II and Cav2.2). (C) Diagram of otoferlin depicting six C2 domains, labeled C2A–C2F, and the transmembrane domain (TMD). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IQGAP1 Binds The Axl Receptor Tyrosine Kinase And Inhibits Its Signaling

IQGAP1 is a scaffold protein that integrates cell signaling pathways by binding several growth factor receptors and intracellular signaling molecules (Trends Cell Biol 2015; 25:171–184). The Axl tyrosine kinase receptor is important in hematopoiesis, the innate immune response, platelet aggregation, engulfment of apoptotic cells and cell survival (Cold Spring Harb Perspect Biol 2013; 5:a009076). Binding of growth arrest‐specific protein 6 (Gas6) to immunoglobulin‐like (Ig‐like) domains of Axl activates intracellular signaling. However, the mechanism of inactivation of the Axl receptor is poorly understood. Here, we investigate the interaction between IQGAP1 and Axl. Using GST pulldown assays and immunoprecipitation from mammalian cell lysates, we found that IQGAP1 associates with Axl in cells. Analysis with multiple discrete fragments of each protein revealed that the binding is direct and mediated by the IQ domain of IQGAP1 and the Ig‐like domains of Axl. We observed by proximity ligation assays that Axl:IQGAP1 complexes are located in the cytoplasm, and this interaction is reduced by 64% when cells are incubated with Gas6. Consistent with these findings, reducing IQGAP1 levels in cells by CRISPR/Cas9 enhanced the ability of Gas6 to stimulate both Axl phosphorylation and activation of Akt. Moreover, reduction of IQGAP1 in cells promoted Gas6‐stimulated transcription of matrix metalloproteinase (MMP)‐2, MMP‐3, MMP‐9 and urokinase‐type plasminogen activator genes. Finally, we observed by confocal microscopy that IQGAP1 downregulates the heterodimerization of Axl with the epidermal growth factor receptor. In summary, we identify IQGAP1 as a previously undescribed suppressor of Axl. These findings differ from the previously reported functions for IQGAP1 as a scaffold that promotes signaling by growth factor receptors (J Biol Chem 2007; 292:3273–3289; J Biol Chem 2011; 286:29734–29747; J Biol Chem 2011; 286:15010–15021). Our data expand the repertoire of binding partners for both Axl and IQGAP1, and provide additional insight into the regulation of Axl function.Support or Funding InformationThis work was supported by the National Institutes of Health Intramural Research Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enzymatic Site‐Specific Labeling of RNA for Affinity Isolation of RNA‐Protein Complexes

The role of RNA in regulating cellular processes has generated tremendous interest in methods to image RNAs and profile their interactions within cells. Building on our recently developed enzymatic technique, RNA‐TAG (Transglycosylation At Guanosine), we demonstrate the site‐specific labeling and affinity purification of RNA transcripts. This technology takes advantage of a bacterial tRNA guanine transglycosylase (TGT) for the incorporation of nucleobase derivatives bearing functional probes, such as biotin, into an encodable RNA stem loop recognition motif. Affinity purification enables high levels of enrichment of target RNA complexes and analysis of bound proteins. Using this methodology, we have demonstrated that an RNA of interest can be selectively biotinylated in live mammalian cells or cell lysate. Through the development of RNA‐TAG, we aim to create a robust, convenient methodology for the elucidation of RNA function via affinity purification of cellular RNA‐protein complexes.Support or Funding InformationThis work is supported by the NIH under grant R01 GM123285. K.N.B. is supported by the Chemical Biology Interfaces Training Grant, NIH Grant T32 GM112584.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows.

High-throughput proteome profiling requires thorough optimization to achieve comprehensive analysis. We developed a filter aided sample preparation (FASP)-like, detergent-free method, termed Filter-Based Protein Digestion (FPD). We compared FPD to protein extraction methods commonly used in isobaric tag-based proteome profiling, namely trichloroacetic acid (TCA) and chloroform-methanol (C-M) precipitation. We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma cell line for parallel protein processing with TCA (n = 3), C-M (n = 2), and FPD using either 10 kDa (n = 3) or 30 kDa (n = 3) molecular weight cutoff membranes. We labeled each sample with tandem mass tag (TMT) reagents to construct a TMT11-plex experiment. In total, 8654 proteins were quantified across all samples. Pairwise comparisons showed very little deviation for individual protein abundance measurements between the two FPD methods, whereas TCA and FPD showed the most difference. Specifically, membrane proteins were more readily quantified when samples were processed using TCA precipitation than other methods tested. However, globally, only 4% of proteins differed greater than 4-fold in the most divergent pair of protein extraction methods (i.e., FPD10 and TCA). We conclude that the detergent-free FPD strategy, particularly using the faster-flowing 30 kDa filter, is a seamless alteration to high-throughput TMT workflows.

Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

Sensory hair cells rely on otoferlin as the calcium sensor for exocytosis and encoding of sound preferentially over the neuronal calcium sensor synaptotagmin. Although it is established that synaptotagmin cannot rescue the otoferlin KO phenotype, the large size and low solubility of otoferlin have prohibited direct biochemical comparisons that could establish functional differences between these two proteins. To address this challenge, we have developed a single-molecule colocalization binding titration assay (smCoBRA) that can quantitatively characterize full-length otoferlin from mammalian cell lysate. Using smCoBRA, we found that, although both otoferlin and synaptotagmin bind membrane fusion SNARE proteins, only otoferlin interacts with the L-type calcium channel Cav1.3, showing a significant difference between the synaptic proteins. Furthermore, otoferlin was found capable of interacting with multiple SNARE and Cav1.3 proteins simultaneously, forming a heterooligomer complex. We also found that a deafness-causing missense mutation in otoferlin attenuates binding between otoferlin and Cav1.3, suggesting that deficiencies in this interaction may form the basis for otoferlin-related hearing loss. Based on our results, we propose a model in which otoferlin acts as a calcium-sensitive scaffolding protein, localizing SNARE proteins proximal to the calcium channel so as to synchronize calcium influx with membrane fusion. Our findings also provide a molecular-level explanation for the observation that synaptotagmin and otoferlin are not functionally redundant. This study also validates a generally applicable methodology for quantitatively characterizing large, multivalent membrane proteins.

Chemoproteomic profiling of targets of lipid-derived electrophiles by bioorthogonal aminooxy probe

Redox imbalance in cells induces lipid peroxidation and generates a class of highly reactive metabolites known as lipid-derived electrophiles (LDEs) that can modify proteins and affects their functions. Identifying targets of LDEs is critical to understand how such modifications are functionally implicated in oxidative-stress associated diseases. Here we report a quantitative chemoproteomic method to globally profile protein targets and sites modified by LDEs. In this strategy, we designed and synthesized an alkyne-functionalized aminooxy probe to react with LDE-modified proteins for imaging and proteomic profiling. Using this probe, we successfully quantified >4000 proteins modified by 4-hydroxy-2-nonenal (HNE) of high confidence in mammalian cell lysate and combined with a tandem-orthogonal proteolysis activity-based protein profiling (TOP-ABPP) strategy, we identified ~400 residue sites targeted by HNE including reactive cysteines in peroxiredoxins, an important family of enzymes with anti-oxidant roles. Our method expands the toolbox to quantitatively profile protein targets of endogenous electrophiles and the enlarged inventory of LDE-modified proteins and sites will contribute to functional elucidation of cellular pathways affected by oxidative stress.