Alternative splicing liberates a cryptic cytoplasmic isoform of mitochondrial MECR that antagonizes influenza virus.

Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners. Here we used immunocompetitive capture-mass spectrometry to identify cellular proteins that interact with human- and avian-style viral polymerases. We focused on the proviral activity of heterogenous nuclear ribonuclear protein U-like 1 (hnRNP UL1) and the antiviral activity of mitochondrial enoyl CoA-reductase (MECR). MECR is localized to mitochondria where it functions in mitochondrial fatty acid synthesis (mtFAS). While a small fraction of the polymerase subunit PB2 localizes to the mitochondria, PB2 did not interact with full-length MECR. By contrast, a minor splice variant produces cytoplasmic MECR (cMECR). Ectopic expression of cMECR shows that it binds the viral polymerase and suppresses viral replication by blocking assembly of viral ribonucleoprotein complexes (RNPs). MECR ablation through genome editing or drug treatment is detrimental for cell health, creating a generic block to virus replication. Using the yeast homolog Etr1 to supply the metabolic functions of MECR in MECR-null cells, we showed that specific antiviral activity is independent of mtFAS and is reconstituted by expressing cMECR. Thus, we propose a strategy where alternative splicing produces a cryptic antiviral protein that is embedded within a key metabolic enzyme.

Targeting intracellular WT1 in AML with a novel RMF-peptide-MHC-specific T-cell bispecific antibody.

Antibody-based immunotherapy is a promising strategy for targeting chemoresistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated by using CrossMAb and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms tumor protein (WT1) in the context of HLA-A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary acute myeloid leukemia (AML) cells was mediated in ex vivo long-term cocultures by using allogeneic (mean ± standard error of the mean [SEM] specific lysis, 67 ± 6% after 13-14 days; n = 18) or autologous, patient-derived T cells (mean ± SEM specific lysis, 54 ± 12% after 11-14 days; n = 8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean ± SEM specific lysis on days 3-4, 45.4 ± 9.0% vs 70.8 ± 8.3%; P = .015; n = 9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors exhibited a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo, and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase 1 trial in patients with relapsed/refractory AML (#NCT04580121).

A novel immuno-competitive capture mass spectrometry strategy for protein-protein interaction profiling reveals that LATS kinases regulate HCV replication through NS5A phosphorylation.

Mapping protein-protein interactions is essential to fully characterize the biological function of a protein and improve our understanding of diseases. Affinity purification coupled to mass spectrometry (AP-MS) using selective antibodies against a target protein has been commonly applied to study protein complexes. However, one major limitation is a lack of specificity as a substantial part of the proposed binders is due to nonspecific interactions. Here, we describe an innovative immuno-competitive capture mass spectrometry (ICC-MS) method to allow systematic investigation of protein-protein interactions. ICC-MS markedly increases the specificity of classical immunoprecipitation (IP) by introducing a competition step between free and capturing antibody prior to IP. Instead of comparing only one experimental sample with a control, the methodology generates a 12-concentration antibody competition profile. Label-free quantitation followed by a robust statistical analysis of the data is then used to extract the cellular interactome of a protein of interest and to filter out background proteins. We applied this new approach to specifically map the interactome of hepatitis C virus (HCV) nonstructural protein 5A (NS5A) in a cellular HCV replication system and uncovered eight new NS5A-interacting protein candidates along with two previously validated binding partners. Follow-up biological validation experiments revealed that large tumor suppressor homolog 1 and 2 (LATS1 and LATS2, respectively), two closely related human protein kinases, are novel host kinases responsible for NS5A phosphorylation at a highly conserved position required for optimal HCV genome replication. These results are the first illustration of the value of ICC-MS for the analysis of endogenous protein complexes to identify biologically relevant protein-protein interactions with high specificity.

Development of a 5-plex SILAC method tuned for the quantitation of tyrosine phosphorylation dynamics.

The propagation of phosphorylation downstream of receptor tyrosine kinases is a key dynamic cellular event involved in signal transduction, which is often deregulated in disease states such as cancer. Probing phosphorylation dynamics is therefore crucial for understanding receptor tyrosine kinases' function and finding ways to inhibit their effects. MS methods combined with metabolic labeling such as stable isotope labeling with amino acids in cell culture (SILAC) have already proven successful in deciphering temporal phosphotyrosine perturbations. However, they are limited in terms of multiplexing, and they also are time consuming, because several experiments need to be performed separately. Here, we introduce an innovative approach based on 5-plex SILAC that allows monitoring of phosphotyrosine signaling perturbations induced by a drug treatment in one single experiment. Using this new labeling strategy specifically tailored for phosphotyrosines, it was possible to generate the time profiles for 318 unique phosphopeptides belonging to 215 proteins from an erlotinib-treated breast cancer cell line model. Hierarchical clustering of the time profiles followed by pathway enrichment analysis highlighted epidermal growth factor receptor (EGFR or ErbB1) and ErbB2 signaling as the major pathways affected by erlotinib, thereby validating the method. Moreover, based on the similarity of its time profile to those of other proteins in the ErbB pathways, the phosphorylation at Tyr453 of protein FAM59A, a recently described adaptor of EGFR, was confirmed as tightly involved in the signaling cascade. The present investigation also demonstrates the remote effect of EGFR inhibition on ErbB3 phosphorylation sites such as Tyr1289 and Tyr1328, as well as a potential feedback effect on Tyr877 of ErbB2. Overall, the 5-plex SILAC is a straightforward approach that extends sample multiplexing and builds up the arsenal of methods for tyrosine phosphorylation dynamics.

Pharmacological Enhancement of Adult Hippocampal Neurogenesis Improves Behavioral Pattern Separation in Young and Aged Mice.

BACKGROUND: Impairments in behavioral pattern separation (BPS)-the ability to distinguish between similar contexts or experiences-contribute to memory interference and overgeneralization seen in many neuropsychiatric conditions, including depression, anxiety, PTSD, dementia, and age-related cognitive decline. While BPS relies on the dentate gyrus and is sensitive to changes in adult hippocampal neurogenesis (AHN), its significance as a pharmacological target has not been tested. METHODS: In this study, we applied a human neural stem cell high-throughput screening cascade to identify compounds that increase human neurogenesis. One compound with a favorable profile, RO6871135, was then tested in BPS in mice. RESULTS: Chronic treatment with RO6871135, 7.5 mg/kg increased AHN and improved BPS in a fear discrimination task in both young and aged mice. RO6871135 treatment also lowered innate anxiety-like behavior, which was more apparent in mice exposed to chronic corticosterone. Ablation of AHN by hippocampal irradiation supported a neurogenesis-dependent mechanism for RO6871135-induced improvements in BPS. To identify possible mechanisms of action, in vitro and in vivo kinase inhibition and chemical proteomics assays were performed. These tests indicated that RO6871135 inhibited CDK8, CDK11, CaMK2a, CaMK2b, MAP2K6, and GSK3b. An analog compound also demonstrated high affinity for CDK8, CaMK2a, and GSK3b. CONCLUSIONS: These studies demonstrate a method for empirical identification and preclinical testing of novel neurogenic compounds that can improve BPS, and points to possible novel mechanisms that can be interrogated for the development of new therapies to improve specific endophenotypes such as impaired BPS.

The physiological interactome of TCR-like antibody therapeutics in human tissues.

Selective binding of TCR-like antibodies that target a single tumour-specific peptide antigen presented by human leukocyte antigens (HLA) is the absolute prerequisite for their therapeutic suitability and patient safety. To date, selectivity assessment has been limited to peptide library screening and predictive modeling. We developed an experimental platform to de novo identify interactomes of TCR-like antibodies directly in human tissues using mass spectrometry. As proof of concept, we confirm the target epitope of a MAGE-A4-specific TCR-like antibody. We further determine cross-reactive peptide sequences for ESK1, a TCR-like antibody with known off-target activity, in human liver tissue. We confirm off-target-induced T cell activation and ESK1-mediated liver spheroid killing. Off-target sequences feature an amino acid motif that allows a structural groove-coordination mimicking that of the target peptide, therefore allowing the interaction with the engager molecule. We conclude that our strategy offers an accurate, scalable route for evaluating the non-clinical safety profile of TCR-like antibody therapeutics prior to first-in-human clinical application.

Enrichment of Glial Cells From Human Post-mortem Tissue for Transcriptome and Proteome Analysis Using Immunopanning.

Glia cells have a crucial role in the central nervous system and are involved in the majority of neurological diseases. While glia isolation techniques are well established for rodent brain, only recent advances in isolating glial cells from human brain enabled analyses of human-specific glial-cell profiles. Immunopanning that is the prospective purification of cells using cell type-specific antibodies, has been successfully established for isolating glial cells from human fetal brain or from tissue obtained during brain surgeries. Here, we describe an immunopanning protocol to acutely isolate glial cells from post-mortem human brain tissue for e.g. transcriptome and proteome analyses. We enriched for microglia, oligodendrocytes and astrocytes from cortical gray matter tissue from three donors. For each enrichment, we assessed the presence of known glia-specific markers at the RNA and protein levels. In this study we show that immunopanning can be employed for acute isolation of glial cells from human post-mortem brain, which allows characterization of glial phenotypes depending on age, disease and brain regions.

Symmetric signal transduction and negative allosteric modulation of heterodimeric mGlu1/5 receptors.

For a long time metabotropic glutamate receptors (mGluRs) were thought to regulate neuronal functions as obligatory homodimers. Recent reports, however, indicate the existence of heterodimers between group-II and -III mGluRs in the brain, which differ from the homodimers in their signal transduction and sensitivity to negative allosteric modulators (NAMs). Whether the group-I mGluRs, mGlu1 and mGlu5, form functional heterodimers in the brain is still a matter of debate. We now show that mGlu1 and mGlu5 co-purify from brain membranes and hippocampal tissue and co-localize in cultured hippocampal neurons. Complementation assays with mutants deficient in agonist-binding or G protein-coupling reveal that mGlu1/5 heterodimers are functional in heterologous cells and transfected cultured hippocampal neurons. In contrast to heterodimers between group-II and -III mGluRs, mGlu1/5 receptors exhibit a symmetric signal transduction, with both protomers activating G proteins to a similar extent. NAMs of either protomer in mGlu1/5 receptors partially inhibit signaling, showing that both protomers need to be able to reach an active conformation for full receptor activity. Complete heterodimer inhibition is observed when both protomers are locked in their inactive state by a NAM. In summary, our data show that mGlu1/5 heterodimers exhibit a symmetric signal transduction and thus intermediate signaling efficacy and kinetic properties. Our data support the existence of mGlu1/5 heterodimers in neurons and highlight differences in the signaling transduction of heterodimeric mGluRs that influence allosteric modulation.

Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers.

Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.

Quantitative chemical proteomics profiling differentiates erlotinib from gefitinib in EGFR wild-type non-small cell lung carcinoma cell lines.

Although both erlotinib and gefitinib target the EGF receptor (EGFR), erlotinib is effective in patients with EGFR wild-type or mutated tumors, whereas gefitinib is only beneficial for patients with activating mutations. To determine whether these differences in clinical outcomes can be attributed to their respective protein interaction profiles, a label-free, quantitative chemical proteomics study was conducted. Using this method, 24 proteins were highlighted in the binding profiles of erlotinib and gefitinib. Unlike gefinitib, erlotinib displaced the ternary complex formed by integrin-linked kinase (ILK), α-parvin, and PINCH (IPP). The docking of erlotinib in the three-dimensional structure of ILK showed that erlotinib has the ability to bind to the ATP-binding site, whereas gefitinib is unlikely to bind with high affinity. As the IPP complex has been shown to be involved in epithelial-to-mesenchymal transition (EMT) and erlotinib sensitivity has been correlated with EMT status, we used a cellular model of inducible transition and observed that erlotinib prevented EMT in a more efficient way than gefitinib by acting on E-cadherin expression as well as on IPP levels. A retrospective analysis of the MERIT trial indicated that, besides a high level of E-cadherin, a low level of ILK could be linked to clinical benefit with erlotinib. In conclusion, we propose that, in an EGFR wild-type context, erlotinib may have a complementary mode of action by inhibiting IPP complex activities, resulting in the slowing down of the metastatic process of epithelial tumors.

Cholesterol independent effect of LXR agonist TO-901317 on gamma-secretase.

The balance of intracellular cholesterol has proven to be critical to the production of beta-amyloid (A beta). Reducing cholesterol in vitro leads to decreased production of A beta, whereas an increase in cellular cholesterol induces A beta production. Liver X Receptor (LXR) agonists are known to increase cholesterol efflux from cells, but there are conflicting reports as to the effects of these agonists on A beta production. We therefore examined the effects of efflux-inducing agents on A beta production in vitro. We used methyl-beta-cyclodextrin and an LXR agonist (TO-901317) to induce cholesterol efflux and studied the resulting A beta production in a stable amyloid precursor protein (APP) -transfected cell line. When cholesterol efflux was induced with methyl-beta-cyclodextrin there was a >60% decrease in A beta(40) and A beta(42) production. However, while activation of LXR using TO-901317-induced cholesterol efflux in the presence of a cholesterol acceptor, no changes in A beta levels were recorded. When cells were incubated with TO-901317 above the concentration required for maximal cholesterol efflux, there was a 150% increase in A beta(42) levels. The absence of a cholesterol acceptor from the culture media (preventing cholesterol efflux) did not blunt this increase in A beta(42), suggesting that the effects of TO-901317 on A beta(42) are efflux independent. These results were confirmed in APP stably transfected human H4 cells, which revealed in addition to a 200% increase in A beta(42) levels, a concomitant 80% reduction in A beta(38). A cell-free gamma-secretase assay confirmed that TO-901317 can directly alter gamma-secretase activity. These data demonstrate that TO-901317 can directly modulate the site of cleavage of APP by gamma-secretase in vitro.

Biomarker discovery by imaging mass spectrometry: transthyretin is a biomarker for gentamicin-induced nephrotoxicity in rat.

Adverse drug effects are often associated with pathological changes in tissue. An accurate depiction of the undesired affected area, possibly supported by mechanistic data, is important to classify the effects with regard to relevance for human patients. MALDI imaging MS represents a new analytical tool to directly provide the spatial distribution and the relative abundance of proteins in tissue. Here we evaluate this technique to investigate potential toxicity biomarkers in kidneys of rats that were administered gentamicin, a well known nephrotoxicant. Differential analysis of the mass spectrum profiles revealed a spectral feature at 12,959 Da that strongly correlates with histopathology alterations of the kidney. We unambiguously identified this spectral feature as transthyretin (Ser(28)-Gln(146)) using an innovative combination of tissue microextraction and fractionation by reverse-phase liquid chromatography followed by a top-down tandem mass spectrometric approach. Our findings clearly demonstrate the emerging role of imaging MS in the discovery of toxicity biomarkers and in obtaining mechanistic insights concerning toxicity mechanisms.

Evaluation of biomarker discovery approaches to detect protein biomarkers of acute renal allograft rejection.

Management of host responses to allografts by immunosuppressive therapy is the cornerstone of transplantation medicine, but it is still deficient in one important element: biomarkers that are readily accessible and predict the fate of the transplant early, specifically, and reliably. Using a Brown Norway (BN)-to-Lewis rat renal allograft model of kidney transplantation, this study aims at evaluating two proteomic approaches to discover biomarkers for acute rejection: SELDI-MS technology and 2D gel electrophoresis combined with mass spectrometry. Several novel potential serum biomarkers have been identified for follow up. Overall, the conclusion is that apparently at the serum protein level, dramatic changes only occur at a stage where kidney function is already severely affected. Multivariate analysis of serum profiles suggests that there is an ensemble of subtle changes, comprising a proteomic signature of acute rejection at an early stage, a more detailed evaluation of which might provide novel opportunities for the diagnosis of acute rejection. Profiling of the excreted proteins indicates that urine might even present the earliest signs of the rejection process.

PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression.

A novel member of the poly(ADP-ribose) polymerase (PARP) family, hPARP-3, is identified here as a core component of the centrosome. hPARP-3 is preferentially localized to the daughter centriole throughout the cell cycle. The N-terminal domain (54 amino acids) of hPARP-3 is responsible for its centrosomal localization. Full-length hPAPR-3 (540 amino acids, with an apparent mass of 67 kDa) synthesizes ADP-ribose polymers during its automodification. Overexpression of hPARP-3 or its N-terminal domain does not influence centrosomal duplication or amplification but interferes with the G1/S cell cycle progression. PARP-1 also resides for part of the cell cycle in the centrosome and interacts with hPARP-3. The presence of both PARP-1 and PARP-3 at the centrosome may link the DNA damage surveillance network to the mitotic fidelity checkpoint.