Caskin2 is a novel talin- and Abi1-binding protein that promotes cell motility.

Talin (herein referring collectively to talin 1 and 2) couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix. Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades. Here, we demonstrate that the scaffold protein Caskin2 interacts directly with the R8 domain of talin through its C-terminal LD motif. Caskin2 also associates with the WAVE regulatory complex to promote cell migration in an Abi1-dependent manner. Furthermore, we demonstrate that the Caskin2-Abi1 interaction is regulated by growth factor-induced phosphorylation of Caskin2 on serine 878. In MCF7 and UACC893 cells, which contain an amplification of CASKIN2, Caskin2 localizes in plasma membrane-associated plaques and around focal adhesions in cortical microtubule stabilization complexes. Taken together, our results identify Caskin2 as a novel talin-binding protein that might not only connect integrin-mediated adhesion to actin polymerization but could also play a role in crosstalk between integrins and microtubules.

The functional and structural characterization of Xanthomonas campestris pv. campestris core effector XopP revealed a new kinase activity.

Exo70B1 is a protein subunit of the exocyst complex with a crucial role in a variety of cell mechanisms, including immune responses against pathogens. The calcium-dependent kinase 5 (CPK5) of Arabidopsis thaliana (hereafter Arabidopsis), phosphorylates AtExo70B1 upon functional disruption. We previously reported that, the Xanthomonas campestris pv. campestris effector XopP compromises AtExo70B1, while bypassing the host's hypersensitive response, in a way that is still unclear. Herein we designed an experimental approach, which includes biophysical, biochemical, and molecular assays and is based on structural and functional predictions, utilizing AplhaFold and DALI online servers, respectively, in order to characterize the in vivo XccXopP function. The interaction between AtExo70B1 and XccXopP was found very stable in high temperatures, while AtExo70B1 appeared to be phosphorylated at XccXopP-expressing transgenic Arabidopsis. XccXopP revealed similarities with known mammalian kinases and phosphorylated AtExo70B1 at Ser107, Ser111, Ser248, Thr309, and Thr364. Moreover, XccXopP protected AtExo70B1 from AtCPK5 phosphorylation. Together these findings show that XccXopP is an effector, which not only functions as a novel serine/threonine kinase upon its host target AtExo70B1 but also protects the latter from the innate AtCPK5 phosphorylation, in order to bypass the host's immune responses. Data are available via ProteomeXchange with the identifier PXD041405.

Distant sequence regions of JBP1 contribute to J-DNA binding.

Base-J (ß-D-glucopyranosyloxymethyluracil) is a modified DNA nucleotide that replaces 1% of thymine in kinetoplastid flagellates. The biosynthesis and maintenance of base-J depends on the base-J-binding protein 1 (JBP1) that has a thymidine hydroxylase domain and a J-DNA-binding domain (JDBD). How the thymidine hydroxylase domain synergizes with the JDBD to hydroxylate thymine in specific genomic sites, maintaining base-J during semi-conservative DNA replication, remains unclear. Here, we present a crystal structure of the JDBD including a previously disordered DNA-contacting loop and use it as starting point for molecular dynamics simulations and computational docking studies to propose recognition models for JDBD binding to J-DNA. These models guided mutagenesis experiments, providing additional data for docking, which reveals a binding mode for JDBD onto J-DNA. This model, together with the crystallographic structure of the TET2 JBP1-homologue in complex with DNA and the AlphaFold model of full-length JBP1, allowed us to hypothesize that the flexible JBP1 N-terminus contributes to DNA-binding, which we confirmed experimentally. Α high-resolution JBP1:J-DNA complex, which must involve conformational changes, would however need to be determined experimentally to further understand this unique underlying molecular mechanism that ensures replication of epigenetic information.

cFLIP suppression and DR5 activation sensitize senescent cancer cells to senolysis.

Senolytics, drugs that kill senescent cells, have been proposed to improve the response to pro-senescence cancer therapies; however, this remains challenging due to a lack of broadly acting senolytic drugs. Using CRISPR/Cas9-based genetic screens in different senescent cancer cell models, we identify loss of the death receptor inhibitor cFLIP as a common vulnerability of senescent cancer cells. Senescent cells are primed for apoptotic death by NF-κB-mediated upregulation of death receptor 5 (DR5) and its ligand TRAIL, but are protected from death by increased cFLIP expression. Activation of DR5 signaling by agonistic antibody, which can be enhanced further by suppression of cFLIP by BRD2 inhibition, leads to efficient killing of a variety of senescent cancer cells. Moreover, senescent cells sensitize adjacent non-senescent cells to killing by DR5 agonist through a bystander effect mediated by secretion of cytokines. We validate this 'one-two punch' cancer therapy by combining pro-senescence therapy with DR5 activation in different animal models.

The host exocyst complex is targeted by a conserved bacterial type-III effector that promotes virulence.

For most Gram-negative bacteria, pathogenicity largely depends on the type-III secretion system that delivers virulence effectors into eukaryotic host cells. The subcellular targets for the majority of these effectors remain unknown. Xanthomonas campestris, the causal agent of black rot disease of crucifers such as Brassica spp., radish, and turnip, delivers XopP, a highly conserved core-effector protein produced by X. campestris, which is essential for virulence. Here, we show that XopP inhibits the function of the host-plant exocyst complex by direct targeting of Exo70B, a subunit of the exocyst complex, which plays a significant role in plant immunity. XopP interferes with exocyst-dependent exocytosis and can do this without activating a plant NOD-like receptor that guards Exo70B in Arabidopsis. In this way, Xanthomonas efficiently inhibits the host's pathogen-associated molecular pattern (PAMP)-triggered immunity by blocking exocytosis of pathogenesis-related protein-1A, callose deposition, and localization of the FLAGELLIN SENSITIVE2 (FLS2) immune receptor to the plasma membrane, thus promoting successful infection. Inhibition of exocyst function without activating the related defenses represents an effective virulence strategy, indicating the ability of pathogens to adapt to host defenses by avoiding host immunity responses.

Cell cycle dynamics of lamina-associated DNA.

In mammalian interphase nuclei, more than one thousand large genomic regions are positioned at the nuclear lamina (NL). These lamina-associated domains (LADs) are involved in gene regulation and may provide a backbone for the folding of interphase chromosomes. Little is known about the dynamics of LADs during interphase, in particular at the onset of G1 phase and during DNA replication. We developed an antibody-based variant of the DamID technology (named pA-DamID) that allows us to map and visualize genome-NL interactions with high temporal resolution. Application of pA-DamID combined with synchronization and cell sorting experiments reveals that LAD-NL contacts are generally rapidly established early in G1 phase. However, LADs on the distal ~25 Mb of most chromosomes tend to contact the NL first and then gradually detach, while centromere-proximal LADs accumulate gradually at the NL. Furthermore, our data indicate that S-phase chromatin shows transiently increased lamin interactions. These findings highlight a dynamic choreography of LAD-NL contacts during interphase progression and illustrate the usefulness of pA-DamID to study the dynamics of genome compartmentalization.

A fragment-like approach to PYCR1 inhibition.

Pyrroline-5-carboxylate reductase 1 (PYCR1) is the final enzyme involved in the biosynthesis of proline and has been found to be upregulated in various forms of cancer. Due to the role of proline in maintaining the redox balance of cells and preventing apoptosis, PYCR1 is emerging as an attractive oncology target. Previous PYCR1 knockout studies led to a reduction in tumor growth. Accordingly, a small molecule inhibitor of PYCR1 could lead to new treatments for cancer, and a focused screening effort identified pargyline as a fragment-like hit. We report the design and synthesis of the first tool compounds as PYCR1 inhibitors, derived from pargyline, which were assayed to assess their ability to attenuate the production of proline. Structural activity studies have revealed the key determinants of activity, with the most potent compound (4) showing improved activity in vitro in enzyme (IC50 = 8.8 µM) and pathway relevant effects in cell-based assays.

Crystal structure of the tubulin tyrosine carboxypeptidase complex VASH1-SVBP.

The cyclic enzymatic removal and ligation of the C-terminal tyrosine of α-tubulin generates heterogeneous microtubules and affects their functions. Here we describe the crystal and solution structure of the tubulin carboxypeptidase complex between vasohibin (VASH1) and small vasohibin-binding protein (SVBP), which folds in a long helix, which stabilizes the VASH1 catalytic domain. This structure, combined with molecular docking and mutagenesis experiments, reveals which residues are responsible for recognition and cleavage of the tubulin C-terminal tyrosine.

Baculovirus-driven protein expression in insect cells: A benchmarking study.

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.

Vasohibins encode tubulin detyrosinating activity.

Tubulin is subjected to a number of posttranslational modifications to generate heterogeneous microtubules. The modifications include removal and ligation of the C-terminal tyrosine of ⍺-tubulin. The enzymes responsible for detyrosination, an activity first observed 40 years ago, have remained elusive. We applied a genetic screen in haploid human cells to find regulators of tubulin detyrosination. We identified SVBP, a peptide that regulates the abundance of vasohibins (VASH1 and VASH2). Vasohibins, but not SVBP alone, increased detyrosination of ⍺-tubulin, and purified vasohibins removed the C-terminal tyrosine of ⍺-tubulin. We found that vasohibins play a cell type-dependent role in detyrosination, although cells also contain an additional detyrosinating activity. Thus, vasohibins, hitherto studied as secreted angiogenesis regulators, constitute a long-sought missing link in the tubulin tyrosination cycle.

Recombinant cloning strategies for protein expression.

A variety of methods to create specific constructs for protein expression, in a broad range of organisms, are available nowadays. Restriction enzyme-free, ligation-independent and recombinase-based cloning methods have enabled high-throughput protein expression for structural and functional studies. These methods are also instrumental for modification of target genes including gene truncations, site-specific mutagenesis and domain swapping. Here, we describe the most common cloning techniques that are currently at hand for recombinant protein expression studies, including a brief overview of techniques associated with co-expression experiments. We also provide an inventory of many of the available reagents for the various cloning methods, and an overview for some computational tools that can help with the design of expression constructs.

Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening.

Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin's highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin's two ATPase sites.

REV7 counteracts DNA double-strand break resection and affects PARP inhibition.

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

The first step of peptide selection in antigen presentation by MHC class I molecules.

MHC class I molecules present a variable but limited repertoire of antigenic peptides for T-cell recognition. Understanding how peptide selection is achieved requires mechanistic insights into the interactions between the MHC I and candidate peptides. We find that, at first encounter, MHC I H-2K(b) considers a wide range of peptides, including those with expanded N termini and unfitting anchor residues. Discrimination occurs in the second step, when noncanonical peptides dissociate with faster exchange rates. This second step exhibits remarkable temperature sensitivity, as illustrated by numerous noncanonical peptides presented by H-2K(b) in cells cultured at 26 °C relative to 37 °C. Crystallographic analyses of H-2K(b)-peptide complexes suggest that a conformational adaptation of H-2K(b) drives the decisive step in peptide selection. We propose that MHC class I molecules consider initially a large peptide pool, subsequently refined by a temperature-sensitive induced-fit mechanism to retain the canonical peptide repertoire.

Altered peptide ligands revisited: vaccine design through chemically modified HLA-A2-restricted T cell epitopes.

Virus or tumor Ag-derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.

Integrating chemical and genetic silencing strategies to identify host kinase-phosphatase inhibitor networks that control bacterial infection.

Every year three million people die as a result of bacterial infections, and this number may further increase due to resistance to current antibiotics. These antibiotics target almost all essential bacterial processes, leaving only a few new targets for manipulation. The host proteome has many more potential targets for manipulation in order to control bacterial infection, as exemplified by the observation that inhibiting the host kinase Akt supports the elimination of different intracellular bacteria including Salmonella and M. tuberculosis. If host kinases are involved in the control of bacterial infections, phosphatases could be as well. Here we present an integrated small interference RNA and small molecule screen to identify host phosphatase-inhibitor combinations that control bacterial infection. We define host phosphatases inhibiting intracellular growth of Salmonella and identify corresponding inhibitors for the dual specificity phosphatases DUSP11 and 27. Pathway analysis places many kinases and phosphatases controlling bacterial infection in an integrated pathway centered around Akt. This network controls host cell metabolism, survival, and growth and bacterial survival and reflect a natural host cell response to bacterial infection. Inhibiting two enzyme classes with opposite activities-kinases and phosphatases-may be a new strategy to overcome infections by antibiotic-resistant bacteria.

Transient transfection coupled to baculovirus infection for rapid protein expression screening in insect cells.

Baculovirus infected insect cells are widely used for heterologous protein expression. Despite the power of this system, the use of baculovirus techniques for protein expression screening is hampered by the time and resources needed to generate each recombinant baculovirus. Here, we show that a transfection/infection based expression system is suitable for screening of expression constructs in insect cells and represents a valid alternative to other traditional screening methodologies using recombinant baculovirus. The described method is based on gene delivery by transfection coupled to the induction of protein expression by non-recombinant baculovirus infection. Vectors that control expression by a combination of the baculovirus promoters ie1 and p10 and the enhancer element hr5 are among the ones suitable for this method. Infection with non-recombinant baculovirus drastically increases the basal activity of these elements, leading to protein over-expression. Multiple vectors can be simultaneously co-transfected/infected, making transfection/infection amenable for screening of multiple co-expressed proteins and protein complexes. Taken together, our results prove that the transfection/infection protocol is a valid and innovative approach for increasing speed and reducing costs of protein expression screening for structural and functional studies.

Crystallization and preliminary X-ray analysis of crinumin, a chymotrypsin-like glycosylated serine protease with thrombolytic and antiplatelet activity.

Crinumin, a novel glycosylated serine protease with chymotrypsin-like catalytic specificity, was purified from the medicinally important plant Crinum asiaticum. Crinumin is a 67.7 kDa protease with an extraordinary stability and activity over a wide range of pH and temperature and is functional in aqueous, organic and chaotropic solutions. The purified protease has thrombolytic and antiplatelet activity. The use of C. asiaticum extracts has also been reported for the treatment of a variety of disorders such as injury, joint inflammation and arthritis. In order to understand its structure-function relationship, the enzyme was purified from the plant latex and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from a single crystal and processed to 2.8 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 121.61, b = 95.00, c = 72.10 Å, α = γ = 90, ß = 114.19°. The Matthews coefficient was 2.81 Å(3) Da(-1), corresponding to a solvent content of 56%, assuming one molecule in the asymmetric unit. Structure determination of the enzyme is in progress.

Crystal structure of the EphA4 protein tyrosine kinase domain in the apo- and dasatinib-bound state.

The Eph family of receptor tyrosine kinases regulates diverse cellular processes while the over-expression of a member of this family, EphA4, has been reported in a variety of malignant carcinomas. To gain insight into molecular mechanisms and to facilitate structure-based inhibitor design, we solved the crystal structure of the native EphA4 kinase domain in both the apo and dasatinib bound forms. Analysis of the two structures provides insight into structural features of inhibitor binding and revealed a hydrophobic back-pocket in the ATP- binding site of EphA4 which was previously unidentified. The structures suggest a route towards development of novel and specific inhibitors.

Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases.

High-throughput methods to produce a large number of soluble recombinant protein variants are particularly important in the process of determining the three-dimensional structure of proteins and their complexes. Here, we describe a collection of protein expression vectors for ligation-independent cloning, which allow co-expression strategies by implementing different affinity tags and antibiotic resistances. Since the same PCR product can be inserted in all but one of the vectors, this allows efficiency in versatility while screening for optimal expression strategies. We first demonstrate the use of these vectors for protein expression in Escherichia coli, on a set of proteins belonging to the ubiquitin specific protease (USP) Family. We have selected 35 USPs, created 145 different expression constructs into the pETNKI-His-3C-LIC-kan vector, and obtained 38 soluble recombinant proteins for 21 different USPs. Finally, we exemplify the use of our vectors for bacterial co-expression and for expression in insect cells, with USP4 and USP7 respectively. We conclude that our ligation-independent cloning strategy allows for high-throughput screening for the expression of soluble proteins in a variety of vectors in E. coli and in insect cells. In addition, the same vectors can be used for co-expression studies, at least for simple binary complexes. Application in the family of ubiquitin specific proteases led to a number of soluble USPs that are used for functional and crystallization studies.