Kinome and phosphoproteome reprogramming underlies the aberrant immune responses in critically ill COVID-19 patients.

SARS-CoV-2 infection triggers extensive host immune reactions, leading to severe diseases in certain individuals. However, the molecular basis underlying the excessive yet non-productive immune responses in severe COVID-19 remains incompletely understood. In this study, we conducted a comprehensive analysis of the peripheral blood mononuclear cell (PBMC) proteome and phosphoproteome in sepsis patients positive or negative for SARS-CoV-2 infection, as well as healthy subjects, using quantitative mass spectrometry. Our findings demonstrate dynamic changes in the COVID-19 PBMC proteome and phosphoproteome during disease progression, with distinctive protein or phosphoprotein signatures capable of distinguishing longitudinal disease states. Furthermore, SARS-CoV-2 infection induces a global reprogramming of the kinome and phosphoproteome, resulting in defective adaptive immune response mediated by the B and T lymphocytes, compromised innate immune responses involving the SIGLEC and SLAM family of immunoreceptors, and excessive cytokine-JAK-STAT signaling. In addition to uncovering host proteome and phosphoproteome aberrations caused by SARS-CoV-2, our work recapitulates several reported therapeutic targets for COVID-19 and identified numerous new candidates, including the kinases PKG1, CK2, ROCK1/2, GRK2, SYK, JAK2/3, TYK2, DNA-PK, PKCδ, and the cytokine IL-12.

Generic Plug-and-Play Strategy for High-Throughput Analysis of PTM-Mediated Protein Complexes.

Protein complexes mediated by various post-translational modifications (PTMs) play important roles in almost every aspect of biological processes. PTM-mediated protein complexes often have weak and transient binding properties, which limit their unbiased profiling especially in complex biological samples. Here, we developed a plug-and-play chemical proteomic approach for high-throughput analyis of PTM-mediated protein complexes. Taking advantage of the glutathione-S-transferase (GST) tag, which is the gold standard for protein purification and has wide access to a variety of proteins of interest (POIs), a glutathione (GSH) group- and photo-cross-linking group-containing trifunctional chemical probe was developed to tag POIs and assembled onto a streptavidin-coated 96-well plate for affinity purification, photo-cross-linking, and proteomics sample preparation in a fully integrated manner. Compared with the previously developed photo-pTyr-scaffold strategy, by assembling the tyrosine phosphorylation (pTyr) binding domain through covalent NHS chemistry, the new plug-and-play strategy using a noncovalent GST-GSH interaction has comparable enrichment efficiency for EGF stimulation-dependent pTyr protein complexes. To further prove its feasibility, we additionally assembled four pTyr-binding domains in the 96-well plate and selectively identified their pTyr-dependent interacting proteins. Importantly, we systematically optimized and applied the plug-and-play approach for exploring protein methylation-mediated protein complexes, which are difficult to be characterized due to their weak binding affinity and the lack of efficient enrichment strategies. We explored a comprehensive protein methylation-mediated interaction network assembled by five protein methylation binding domains including the chromo domain of MPP8, tandem tudor domain of KDM4A, full-length CBX1, PHD domain of RAG2, and tandem tudor domain of TP53BP1 and validated the chromo domain- and tudor domain-mediated interaction with histone H3. Collectively, this plug-and-play approach provides a convenient and generic strategy for exploring PTM-dependent protein complexes for any POIs with the GST tag.

Dynamic interplay of two molecular switches enabled by the MEK1/2-ERK1/2 and IL-6-STAT3 signaling axes controls epithelial cell migration in response to growth factors.

Cell migration is an essential physiological process, and aberrant migration of epithelial cells underlies many pathological conditions. However, the molecular mechanisms governing cell migration are not fully understood. We report here that growth factor-induced epithelial cell migration is critically dependent on the crosstalk of two molecular switches, namely phosphorylation switch (P-switch) and transcriptional switch (T-switch). P-switch refers to dynamic interactions of deleted in liver cancer 1 (DLC1) and PI3K with tensin-3 (TNS3), phosphatase and tensin homolog (PTEN), C-terminal tension, and vav guanine nucleotide exchange factor 2 (VAV2) that are dictated by mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated protein kinase 1/2-dependent phosphorylation of TNS3, PTEN, and VAV2. Phosphorylation of TNS3 and PTEN on specific Thr residues led to the switch of DLC1-TNS3 and PI3K-PTEN complexes to DLC1-PTEN and PI3K-TNS3 complexes, whereas Ser phosphorylation of VAV2 promotes the transition of the PI3K-TNS3/PTEN complexes to PI3K-VAV2 complex. T-switch denotes an increase in C-terminal tension transcription/expression regulated by both extracellular signal-regulated protein kinase 1/2 and signal transducer and activator of transcription 3 (STAT3) via interleukin-6-Janus kinase-STAT3 signaling pathway. We have found that, the P-switch is indispensable for both the initiation and continuation of cell migration induced by growth factors, whereas the T-switch is only required to sustain cell migration. The interplay of the two switches facilitated by the interleukin-6-Janus kinase-STAT3 pathway governs a sequence of dynamic protein-protein interactions for sustained cell migration. That a similar mechanism is employed by both normal and tumorigenic epithelial cells to drive their respective migration suggests that the P-switch and T-switch are general regulators of epithelial cell migration and potential therapeutic targets.

Rapid and accurate agglutination-based testing for SARS-CoV-2 antibodies.

We have developed a rapid, accurate, and cost-effective serologic test for SARS-CoV-2 virus, which caused the COVID-19 pandemic, on the basis of antibody-dependent agglutination of antigen-coated latex particles. When validated using plasma samples that are positive or negative for SARS-CoV-2, the agglutination assay detected antibodies against the receptor-binding domain of the spike (S-RBD) or the nucleocapsid protein of SARS-CoV-2 with 100% specificity and ∼98% sensitivity. Furthermore, we found that the strength of the S-RBD antibody response measured by the agglutination assay correlated with the efficiency of the plasma in blocking RBD binding to the angiotensin-converting enzyme 2 in a surrogate neutralization assay, suggesting that the agglutination assay might be used to identify individuals with virus-neutralizing antibodies. Intriguingly, we found that >92% of patients had detectable antibodies on the day of a positive viral RNA test, suggesting that the agglutination antibody test might complement RNA testing for the diagnosis of SARS-CoV-2 infection.

Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern.

BACKGROUNDThe role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome.METHODSUsing SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients' plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution.RESULTSWe identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811-825, S-881-895, and N-156-170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes.CONCLUSIONEpitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats.FUNDINGOntario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund.

DLC1 SAM domain-binding peptides inhibit cancer cell growth and migration by inactivating RhoA.

Deleted-in-liver cancer 1 (DLC1) exerts its tumor suppressive function mainly through the Rho-GTPase-activating protein (RhoGAP) domain. When activated, the domain promotes the hydrolysis of RhoA-GTP, leading to reduced cell migration. DLC1 is kept in an inactive state by an intramolecular interaction between its RhoGAP domain and the DLC1 sterile α motif (SAM) domain. We have shown previously that this autoinhibited state of DLC1 may be alleviated by tensin-3 (TNS3) or PTEN. We show here that the TNS3/PTEN-DLC1 interactions are mediated by the C2 domains of the former and the SAM domain of the latter. Intriguingly, the DLC1 SAM domain was capable of binding to specific peptide motifs within the C2 domains. Indeed, peptides containing the binding motifs were highly effective in blocking the C2-SAM domain-domain interaction. Importantly, when fused to the tat protein-transduction sequence and subsequently introduced into cells, the C2 peptides potently promoted the RhoGAP function in DLC1, leading to decreased RhoA activation and reduced tumor cell growth in soft agar and migration in response to growth factor stimulation. To facilitate the development of the C2 peptides as potential therapeutic agents, we created a cyclic version of the TNS3 C2 domain-derived peptide and showed that this peptide readily entered the MDA-MB-231 breast cancer cells and effectively inhibited their migration. Our work shows, for the first time, that the SAM domain is a peptide-binding module and establishes the framework on which to explore DLC1 SAM domain-binding peptides as potential therapeutic agents for cancer treatment.

NUMB regulates the endocytosis and activity of the anaplastic lymphoma kinase in an isoform-specific manner.

NUMB is an evolutionarily conserved protein that plays an important role in cell adhesion, migration, polarity, and cell fate determination. It has also been shown to play a role in the pathogenesis of certain cancers, although it remains controversial whether NUMB functions as an oncoprotein or tumor suppressor. Here, we show that NUMB binds to anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase aberrantly activated in several forms of cancer, and this interaction regulates the endocytosis and activity of ALK. Intriguingly, the function of the NUMB-ALK interaction is isoform-dependent. While both p66-NUMB and p72-NUMB isoforms are capable of mediating the endocytosis of ALK, the former directs ALK to the lysosomal degradation pathway, thus decreasing the overall ALK level and the downstream MAP kinase signal. In contrast, the p72-NUMB isoform promotes ALK recycling back to the plasma membrane, thereby maintaining the kinase in its active state. Our work sheds light on the controversial role of different isoforms of NUMB in tumorigenesis and provides mechanistic insight into ALK regulation.

Surface Loops in a Single SH2 Domain Are Capable of Encoding the Spectrum of Specificity of the SH2 Family.

Src homology 2 (SH2) domains play an essential role in cellular signal transduction by binding to proteins phosphorylated on Tyr residue. Although Tyr phosphorylation (pY) is a prerequisite for binding for essentially all SH2 domains characterized to date, different SH2 domains prefer specific sequence motifs C-terminal to the pY residue. Because all SH2 domains adopt the same structural fold, it is not well understood how different SH2 domains have acquired the ability to recognize distinct sequence motifs. We have shown previously that the EF and BG loops that connect the secondary structure elements on an SH2 domain dictate its specificity. In this study, we investigated if these surface loops could be engineered to encode diverse specificities. By characterizing a group of SH2 variants selected by different pY peptides from phage-displayed libraries, we show that the EF and BG loops of the Fyn SH2 domain can encode a wide spectrum of specificities, including all three major specificity classes (p + 2, p + 3 and p + 4) of the SH2 domain family. Furthermore, we found that the specificity of a given variant correlates with the sequence feature of the bait peptide used for its isolation, suggesting that an SH2 domain may acquire specificity by co-evolving with its ligand. Intriguingly, we found that the SH2 variants can employ a variety of different mechanisms to confer the same specificity, suggesting the EF and BG loops are highly flexible and adaptable. Our work provides a plausible mechanism for the SH2 domain to acquire the wide spectrum of specificity observed in nature through loop variation with minimal disturbance to the SH2 fold. It is likely that similar mechanisms may have been employed by other modular interaction domains to generate diversity in specificity.

Identification and characterization of a large family of superbinding bacterial SH2 domains.

Src homology 2 (SH2) domains play a critical role in signal transduction in mammalian cells by binding to phosphorylated Tyr (pTyr). Apart from a few isolated cases in viruses, no functional SH2 domain has been identified to date in prokaryotes. Here we identify 93 SH2 domains from Legionella that are distinct in sequence and specificity from mammalian SH2 domains. The bacterial SH2 domains are not only capable of binding proteins or peptides in a Tyr phosphorylation-dependent manner, some bind pTyr itself with micromolar affinities, a property not observed for mammalian SH2 domains. The Legionella SH2 domains feature the SH2 fold and a pTyr-binding pocket, but lack a specificity pocket found in a typical mammalian SH2 domain for recognition of sequences flanking the pTyr residue. Our work expands the boundary of phosphotyrosine signalling to prokaryotes, suggesting that some bacterial effector proteins have acquired pTyr-superbinding characteristics to facilitate bacterium-host interactions.

Affinity Purification of Methyllysine Proteome by Site-Specific Covalent Conjugation.

A basic but critical step in targeted proteomics by mass spectrometry is the separation of the targeted proteins from the complex mixture of the whole proteome by affinity purification. The bait protein is usually immobilized on the surface of a solid support to enable affinity-based purification of the targeted proteome. Here, we developed a site-specific covalent immobilization of the bait protein through affinity-guided covalent coupling (AGCC) of a single cysteine residue of an SH2 domain (utilized as an affinity tag for the protein target) with an engineered ligand peptide. Site-specific covalent immobilization of a methyllysine-binding protein HP1ß chromodomain on the agarose resin was used to purify the methyllysine proteome from the whole-protein mixture. This new bait immobilization led to a notably low background in the affinity purification step, markedly outperforming the conventional (His)6 tag-nickel nitrilotriacetic acid (Ni-NTA) immobilization method. Subsequent analysis of the purified proteome identified 275 lysine methylated sites and 184 methylated proteins from 332 HP1ß CD-binding proteins, including 30 novel methylated proteins. This work demonstrates that a robust site-specific covalent protein immobilization method is well-suited for proteomic analysis of low-abundance proteins. This method also enables the identification of new methylated proteins and methylation sites in the methyllysine proteome.

(Arg)9-SH2 superbinder: a novel promising anticancer therapy to melanoma by blocking phosphotyrosine signaling.

BACKGROUND: Melanoma is a malignant tumor with high misdiagnosis rate and poor prognosis. The bio-targeted therapy is a prevailing method in the treatment of melanoma; however, the accompanying drug resistance is inevitable. SH2 superbinder, a triple-mutant of the Src Homology 2 (SH2) domain, shows potent antitumor ability by replacing natural SH2-containing proteins and blocking multiple pY-based signaling pathways. Polyarginine (Arg)9, a powerful vector for intracellular delivery of large molecules, could transport therapeutic agents across cell membrane. The purpose of this study is to construct (Arg)9-SH2 superbinder and investigate its effects on melanoma cells, expecting to provide potential new approaches for anti-cancer therapy and overcoming the unavoidable drug resistance of single-targeted antitumor agents. METHODS: (Arg)9 and SH2 superbinder were fused to form (Arg)9-SH2 superbinder via genetic engineering. Pull down assay was performed to identify that (Arg)9-SH2 superbinder could capture a wide variety of pY proteins. Immunofluorescence was used to detect the efficiency of (Arg)9-SH2 superbinder entering cells. The proliferation ability was assessed by MTT and colony formation assay. In addition, wound healing and transwell assay were performed to evaluate migration of B16F10, A375 and A375/DDP cells. Moreover, apoptosis caused by (Arg)9-SH2 superbinder was analyzed by flow cytometry-based Annexin V/PI. Furthermore, western blot revealed that (Arg)9-SH2 superbinder influenced some pY-related signaling pathways. Finally, B16F10 xenograft model was established to confirm whether (Arg)9-SH2 superbinder could restrain the growth of tumor. RESULTS: Our data showed that (Arg)9-SH2 superbinder had the ability to enter melanoma cells effectively and displayed strong affinities for various pY proteins. Furthermore, (Arg)9-SH2 superbinder could repress proliferation, migration and induce apoptosis of melanoma cells by regulating PI3K/AKT, MAPK/ERK and JAK/STAT signaling pathways. Importantly, (Arg)9-SH2 superbinder could significantly inhibit the growth of tumor in mice. CONCLUSIONS: (Arg)9-SH2 superbinder exhibited high affinities for pY proteins, which showed effective anticancer ability by replacing SH2-containing proteins and blocking diverse pY-based pathways. The remarkable ability of (Arg)9-SH2 superbinder to inhibit cancer cell proliferation and tumor growth might open the door to explore the SH2 superbinder as a therapeutic agent for cancer treatment.

Interactome Mapping Uncovers a General Role for Numb in Protein Kinase Regulation.

Cellular functions are frequently regulated by protein-protein interactions involving the binding of a modular domain in one protein to a specific peptide sequence in another. This mechanism may be explored to identify binding partners for proteins harboring a peptide-recognition domain. Here we report a proteomic strategy combining peptide and protein microarray screening with biochemical and cellular assays to identify modular domain-mediated protein-protein interactions in a systematic manner. We applied this strategy to Numb, a multi-functional protein containing a phosphotyrosine-binding (PTB) domain. Through the screening of a protein microarray, we identified >100 protein kinases, including both Tyr and Ser/Thr kinases, that could potentially interact with the Numb PTB domain, suggesting a general role for Numb in regulating kinase function. The putative interactions between Numb and several tyrosine kinases were subsequently validated by GST pull-down and/or co-immunoprecipitation assays. Furthermore, using the Oriented Peptide Array Library approach, we defined the specificity of the Numb PTB domain which, in turn, allowed us to predict binding partners for Numb at the genome level. The combination of the protein microarray screening with computer-aided prediction produced the most expansive interactome for Numb to date, implicating Numb in regulating phosphorylation signaling through protein kinases and phosphatases. Not only does the data generated from this study provide an important resource for hypothesis-driven research to further define the function of Numb, the proteomic strategy described herein may be employed to uncover the interactome for other peptide-recognition domains whose consensus motifs are known or can be determined.

Using Reciprocal Protein-Peptide Array Screening to Unravel Protein Interaction Networks.

Protein-protein interactions (PPIs) play a central role in almost all cellular processes. Recent technological advances have enabled the elucidation of an incredibly complex PPI network within the cell. However, protein interactions driven by posttranslational modifications (PTMs) such as phosphorylation, which comprises a significant part of the PPI network, have proven difficult to decipher systematically. Herein, we describe a reciprocal protein-peptide array strategy to uncover PPIs mediated by tyrosine phosphorylation and the Src homology 2 (SH2) domain. This strategy, namely combining peptide and protein domain arrays for PPI mapping, may be applicable for other peptide-binding modules.

Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder.

We present a new strategy for systematic identification of phosphotyrosine (pTyr) by affinity purification mass spectrometry (AP-MS) using a Src homology 2 (SH2)-domain-derived pTyr superbinder as the affinity reagent. The superbinder allows for markedly deeper coverage of the Tyr phosphoproteome than anti-pTyr antibodies when an optimal amount is used. We identified ∼20,000 distinct phosphotyrosyl peptides and >10,000 pTyr sites, of which 36% were 'novel', from nine human cell lines using the superbinder approach. Tyrosine kinases, SH2 domains and phosphotyrosine phosphatases were preferably phosphorylated, suggesting that the toolkit of kinase signaling is subject to intensive regulation by phosphorylation. Cell-type-specific global kinase activation patterns inferred from label-free quantitation of Tyr phosphorylation guided the design of experiments to inhibit cancer cell proliferation by blocking the highly activated tyrosine kinases. Therefore, the superbinder is a highly efficient and cost-effective alternative to conventional antibodies for systematic and quantitative characterization of the tyrosine phosphoproteome under normal or pathological conditions.

A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration.

Although cell migration plays a central role in development and disease, the underlying molecular mechanism is not fully understood. Here we report that a phosphorylation-mediated molecular switch comprising deleted in liver cancer 1 (DLC1), tensin-3 (TNS3), phosphatase and tensin homologue (PTEN) and phosphoinositide-3-kinase (PI3K) controls the spatiotemporal activation of the small GTPases, Rac1 and RhoA, thereby initiating directional cell migration induced by growth factors. On epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) stimulation, TNS3 and PTEN are phosphorylated at specific Thr residues, which trigger the rearrangement of the TNS3-DLC1 and PTEN-PI3K complexes into the TNS3-PI3K and PTEN-DLC1 complexes. Subsequently, the TNS3-PI3K complex translocates to the leading edge of a migrating cell to promote Rac1 activation, whereas PTEN-DLC1 translocates to the posterior for localized RhoA activation. Our work identifies a core signalling mechanism by which an external motility stimulus is coupled to the spatiotemporal activation of Rac1 and RhoA to drive directional cell migration.

A Comprehensive Immunoreceptor Phosphotyrosine-based Signaling Network Revealed by Reciprocal Protein-Peptide Array Screening.

Cells of the immune system communicate with their environment through immunoreceptors. These receptors often harbor intracellular tyrosine residues, which, when phosphorylated upon receptor activation, serve as docking sites to recruit downstream signaling proteins containing the Src Homology 2 (SH2) domain. A systematic investigation of interactions between the SH2 domain and the immunoreceptor tyrosine-based regulatory motifs (ITRM), including inhibitory (ITIM), activating (ITAM), or switching (ITSM) motifs, is critical for understanding cellular signal transduction and immune function. Using the B cell inhibitory receptor CD22 as an example, we developed an approach that combines reciprocal or bidirectional phosphopeptide and SH2 domain array screens with in-solution binding assays to identify a comprehensive SH2-CD22 interaction network. Extending this approach to 194 human ITRM sequences and 78 SH2 domains led to the identification of a high-confidence immunoreceptor interactome containing 1137 binary interactions. Besides recapitulating many previously reported interactions, our study uncovered numerous novel interactions. The resulting ITRM-SH2 interactome not only helped to fill many gaps in the immune signaling network, it also allowed us to associate different SH2 domains to distinct immune functions. Detailed analysis of the NK cell ITRM-mediated interactions led to the identification of a network nucleated by the Vav3 and Fyn SH2 domains. We showed further that these SH2 domains have distinct functions in cytotoxicity. The bidirectional protein-peptide array approach described herein may be applied to the numerous other peptide-binding modules to identify potential protein-protein interactions in a systematic and reliable manner.

A method for systematic mapping of protein lysine methylation identifies functions for HP1ß in DNA damage response.

Lysine methylation occurs on both histone and nonhistone proteins. However, our knowledge on the prevalence and function of nonhistone protein methylation is poor. We describe an approach that combines peptide array, bioinformatics, and mass spectrometry to systematically identify lysine methylation sites and map methyllysine-driven protein-protein interactions. Using this approach, we identified a high-confidence and high-resolution interactome of the heterochromatin protein 1ß (HP1ß) and uncovered, simultaneously, numerous methyllysine sites on nonhistone proteins. We found that HP1ß binds to DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and regulates its localization to double-strand breaks (DSBs) during DNA damage response (DDR). Mutation of the methylation sites in DNA-PKcs or depletion of HP1ß in cells caused defects in DDR. Furthermore, we showed that the methylation of DNA-PKcs and many other proteins in the HP1ß interactome undergoes large changes in response to DNA damage, indicating that Lys methylation is a highly dynamic posttranslational modification.

Dynamic methylation of Numb by Set8 regulates its binding to p53 and apoptosis.

Although Numb exhibits its tumor-suppressive function in breast cancer in part by binding to and stabilizing p53, it is unknown how the Numb-p53 interaction is regulated in cells. We found that Numb is methylated in its phosphotyrosine-binding (PTB) domain by the lysine methyltransferase Set8. Moreover, methylation uncouples Numb from p53, resulting in increased p53 ubiquitination and degradation. While Numb promotes apoptosis in a p53-dependent manner, the apoptotic function is abolished when Numb is methylated by Set8 or the Lys methylation sites in Numb are mutated. Conversely, the Numb-p53 interaction and Numb-mediated apoptosis are significantly enhanced by depletion of Set8 from cancer cells or by treating the cells with doxorubicin, a chemotherapeutic drug that causes a reduction in the mRNA and protein levels of Set8. Our work identifies the Set8-Numb-p53 signaling axis as an important regulatory pathway for apoptosis and suggests a therapeutic strategy by targeting Numb methylation.