Uncovering the mode of action of engineered T cells in patient cancer organoids.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF.

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B. Here, we show that the acidic domain of DNA repair factor APLF (APLFAD) can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes. The crystal structure of the APLFAD-histone octamer complex shows that APLFAD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLFAD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted.

Engineering caspase 7 as an affinity reagent to capture proteolytic products.

Many proteases recognize their substrates with high specificities, with this in mind, it should theoretically be possible to utilize the substrate binding cleft of a protease as a scaffold to engineer an affinity reagent. In this study, we sought to develop reagents that would differentiate between substrates and products of proteolysis, based on a caspase 7 scaffold. Firstly, we engineered a form of caspase 7 that can undergo conversion to a substrate binding conformation without catalysis. Seeking to generate a product-only trap, we further engineered this construct by incorporating mutations that compensate for the generation of a negative charge in the neo C terminus of a newly generated product. This was accomplished with only three substitutions within the substrate binding cleft. Moreover, the affinity of the product trap for peptides was comparable to the affinity of caspase 7 to parental substrates. Finally, generation of a hybrid fluorescent protein with the product trap provided a reagent that specifically recognized apoptotic cells and highlights the versatility of such an approach in developing affinity and imaging agents for a variety of cysteine and serine proteases.

γ9δ2T cell diversity and the receptor interface with tumor cells.

γ9δ2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded γ9δ2T cells, we explored the clonal diversity of γ9δ2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded γ9δ2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected γ9δ2 T cell receptors (γ9δ2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the γ9δ2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR γ chain and modulated by the affinity of the CDR3 region of the TCRδ chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of γ9δ2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the γ9δ2TCR and required membrane flexibility of the γ9δ2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.

Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome.

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.

Diarylcarbonates are a new class of deubiquitinating enzyme inhibitor.

Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be similarly valuable. The currently available promiscuous DUB inhibitors are highly reactive electrophilic compounds that can crosslink proteins. Herein we introduce diarylcarbonate esters as a novel class of promiscuous DUB inhibitors that do not have the liabilities associated with the previously reported compounds. Diarylcarbonates stabilize the high molecular weight ubiquitin pools in cells and lysates. They also elicit cellular phenotypes associated with DUB inhibition, demonstrating their utility in ubiquitin discovery. Diarylcarbonates may also be a useful scaffold for the development of specific DUB inhibitors.

Efficient and robust proteome-wide approaches for cross-linking mass spectrometry.

Cross-linking mass spectrometry (XL-MS) has received considerable interest, owing to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison with other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by the use of bifunctional cross-linking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by MS. XL-MS has received the required boost to tackle more-complex samples with recent advances in cross-linking chemistry with MS-cleavable or reporter-based cross-linkers and faster, more sensitive and more versatile MS platforms. This protocol provides a detailed description of our optimized conditions for a full-proteome native protein preparation followed by cross-linking using the gas-phase cleavable cross-linking reagent disuccinimidyl sulfoxide (DSSO). Following cross-linking, we demonstrate extensive sample fractionation and substantially simplified data analysis with XlinkX in Proteome Discoverer, as well as subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within ~10 d, including structural investigations.

Histone Interaction Landscapes Visualized by Crosslinking Mass Spectrometry in Intact Cell Nuclei.

Cells organize their actions partly through tightly controlled protein-protein interactions-collectively termed the interactome. Here we use crosslinking mass spectrometry (XL-MS) to chart the protein-protein interactions in intact human nuclei. Overall, we identified ∼8,700 crosslinks, of which 2/3 represent links connecting distinct proteins. From these data, we gain insights on interactions involving histone proteins. We observed that core histones on the nucleosomes expose well-defined interaction hot spots. For several nucleosome-interacting proteins, such as USF3 and Ran GTPase, the data allowed us to build low-resolution models of their binding mode to the nucleosome. For HMGN2, the data guided the construction of a refined model of the interaction with the nucleosome, based on complementary NMR, XL-MS, and modeling. Excitingly, the analysis of crosslinks carrying posttranslational modifications allowed us to extract how specific modifications influence nucleosome interactions. Overall, our data depository will support future structural and functional analysis of cell nuclei, including the nucleoprotein assemblies they harbor.

Mix and match of the tumor metastasis suppressor Nm23 protein isoforms in vitro and in vivo.

Nm23/NME was identified 30 years ago as the first metastatic gene suppressor family. Despite extensive studies, the mechanism of action behind the observed antimetastatic potential of Nm23 has remained largely unresolved. Human Nm23 is present in various isoforms, of which Nm23-H1 and Nm23-H2 are by far the most dominant. Both isoforms are multifunctional enzymes involved in important cellular processes, through their nucleic acid binding ability, their protein-protein interactions and/or their histidine kinase activity. Although Nm23-H1 and Nm23-H2 exhibit 88% sequence homology, they often are considered to have distinct biological functions. Here, we developed an efficient and robust purification protocol to pull-down Nm23 isoforms in their native state. We applied this protocol to purify both overexpressed isoform pure as well as endogenous Nm23 proteins from several human cell lines and mouse brain tissue. Subsequent native mass spectrometry (MS) analysis revealed that all purified Nm23 samples form hexamers, whereby the endogenous protein assembly is primarily present as heterohexamers formed by statistical association of the Nm23-H1 and Nm23-H2 isoforms. Therefore, we conclude that isoform-pure hexameric Nm23 complexes scarcely exist in vivo. We also used native and top-down MS to investigate the histidine autophosphorylation activity of purified Nm23 assemblies. Our data in fine challenge the biological relevance of studying the genes/proteins Nm23-H1 and Nm23-H2 individually.

Response to Comment on "SUMO deconjugation is required for arsenic-triggered ubiquitylation of PML".

Arsenic trioxide chemotherapy cures acute promyelocytic leukemia by inducing the ubiquitylation of an oncogenic fusion protein containing promyelocytic leukemia protein (PML) subsequent to modification of PML by SUMO1 and SUMO2. We proposed that the SUMO switch at Lys(65) of PML enhanced subsequent SUMO2 conjugation to Lys(160) and consequent RNF4-dependent ubiquitylation of PML. Ferhi et al note differences between their experimental system and ours regarding the outcome and mechanisms of SUMO-dependent PML signaling. When confronted by apparently contradictory data, it is appropriate to drill down to where the differences could lie.

SUMO deconjugation is required for arsenic-triggered ubiquitylation of PML.

Acute promyelocytic leukemia is characterized by a chromosomal translocation that produces an oncogenic fusion protein of the retinoic acid receptor α (RARα) and promyelocytic leukemia protein (PML). Arsenic trioxide chemotherapy of this cancer induces the PML moiety to organize nuclear bodies, where the oncoprotein is degraded. This process requires the participation of two SUMO paralogs (SUMO1 and SUMO2) to promote PML ubiquitylation mediated by the ubiquitin E3 ligase RNF4 and reorganization of PML nuclear bodies. We demonstrated that the ubiquitylation of PML required the SUMO deconjugation machinery, primarily the deconjugating enzyme SENP1, and was suppressed by expression of non-deconjugatable SUMO2. We hypothesized that constitutive SUMO2 conjugation and deconjugation occurred basally and that arsenic trioxide treatment caused the exchange of SUMO2 for SUMO1 on a fraction of Lys(65) in PML. On the basis of data obtained with mutational analysis and quantitative proteomics, we propose that the SUMO switch at Lys(65) of PML enhanced nuclear body formation, subsequent SUMO2 conjugation to Lys(160), and consequent RNF4-dependent ubiquitylation of PML. Our work provides insights into how the SUMO system achieves selective SUMO paralog modification and highlights the crucial role of SENPs in defining the specificity of SUMO signaling.

Glycine fluoromethylketones as SENP-specific activity based probes.

We report here the synthesis and biochemical properties of a new peptidyl activity-based probe 1 for SUMO proteases, SENPs. The activity-based probe has at its C terminus a glycine-derived fluoromethylketone moiety as a reactive group designed to target the active-site cysteine of SENPs. Based on a study of the interactions between SENPs and SUMOs, we introduced further design elements that allow the activity-based probe to selectively target SENPs at low micromolar to high nanomolar concentrations. Moreover, 1 out-competes SUMO1 from the reversible SUMO1-SENP1 complex, thus suggesting that 1 and SUMO1 share a common binding site on SENP1.

Development of small molecule inhibitors and probes of human SUMO deconjugating proteases.

Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compound therefore represents promising new reagents to study the process of SUMO deconjugation.

Structural determinants of integrin binding to the talin rod.

The adaptor protein talin serves both to activate the integrin family of cell adhesion molecules and to couple integrins to the actin cytoskeleton. Integrin activation has been shown to involve binding of the talin FERM domain to membrane proximal sequences in the cytoplasmic domain of the integrin beta-subunit. However, a second integrin-binding site (IBS2) has been identified near the C-terminal end of the talin rod. Here we report the crystal structure of IBS2 (residues 1974-2293), which comprises two five-helix bundles, "IBS2-A" (1974-2139) and "IBS2-B" (2140-2293), connected by a continuous helix with a distinct kink at its center that is stabilized by side-chain H-bonding. Solution studies using small angle x-ray scattering and NMR point to a fairly flexible quaternary organization. Using pull-down and enzyme-linked immunosorbent assays, we demonstrate that integrin binding requires both IBS2 domains, as does binding to acidic phospholipids and robust targeting to focal adhesions. We have defined the membrane proximal region of the integrin cytoplasmic domain as the major binding region, although more membrane distal regions are also required for strong binding. Alanine-scanning mutagenesis points to an important electrostatic component to binding. Thermal unfolding experiments show that integrin binding induces conformational changes in the IBS2 module, which we speculate are linked to vinculin and membrane binding.

Contribution of alpha4beta1 integrin to the antiallergic effect of levocabastine.

Levocabastine is an antiallergic drug acting as a histamine H1-receptor antagonist. In allergic conjunctivitis (AC), it may also antagonize up-regulation of the intercellular adhesion molecule-1 (ICAM-1) expressed on epithelial conjunctival cells. However, little is known about its effects on eosinophils, important effector cells in AC. The adhesion molecule integrin alpha(4)beta(1) is expressed in eosinophils; it interacts with the vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) in vascular endothelial cells and contributes to eosinophil activation and infiltration in AC. This study provides evidence that in a scintillation proximity assay levocabastine (IC(50) 406 microM), but not the first-generation antihistamine chlorpheniramine, displaced (125)I-FN binding to human integrin alpha(4)beta(1) and, in flow cytometry analysis, levocabastine antagonized the binding of a primary antibody to integrin alpha(4) expressed on the Jurkat cell surface. Levocabastine, but not chlorpheniramine, binds the alpha(4)beta(1) integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vascular endothelial cells (HUVEC) in vitro. Similarly, levocabastine affects alpha(L)beta(2)/ICAM-1-mediated adhesion of Jurkat cells. In a model of AC levocabastine eye drops reduced the clinical aspects of the late-phase reaction and the conjunctival expression of alpha(4)beta(1) integrin by reducing infiltrated eosinophils. We propose that blockade of integrin-mediated cell adhesion might be a target of the antiallergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in AC.