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Autophosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is a de facto c-Src autophosphorylation site with slow time-resolution kinetics and a strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes faster kinetics and a cis-to-trans phosphorylation switch that controls c-terminal Tyr 530 autophosphorylation, enzyme specificity, and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular autophosphorylation. In an asymmetric arrangement of both catalytic domains, a c-terminal palindromic phospho-motif flanking Tyr 530 on the substrate molecule engages the G-loop of the active kinase adopting a position ready for entry into the catalytic cleft. Perturbation of the phospho-motif accounts for c-Src dysfunction as indicated by viral and colorectal cancer (CRC)-associated c-terminal deleted variants. We show that c-terminal residues 531 to 536 are required for c-Src Tyr 530 autophosphorylation, and such a detrimental effect is caused by the substrate molecule inhibiting allosterically the active kinase. Our work reveals a crosstalk between the activation and c-terminal segments that control the allosteric interplay between substrate- and enzyme-acting kinases during autophosphorylation. Protein kinase transition between different conformational states is controlled by autophosphorylation. Here, the authors demonstrate that the c-terminal Tyr530 is a de facto c-Src autophosphorylation site  and identify a critical c-terminal palindromic phospho-motif that controls the interplay between substrate and enzyme-acting kinases during autophosphorylation.

Background Germline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases. Methods A training set of 23 variants considered pathogenic or benign was used to define the usability and strength of the ACMG/AMP criteria. Population frequencies, computational predictions, co-segregation data, phenotypic and tumor data, and functional results, among other features, were considered. Results Gene-specific variant classification recommendations for non-disruptive variants located in the exonuclease domain of POLE and POLD1 were defined. The resulting recommendations were applied to 128 exonuclease domain variants reported in the literature and/or public databases. A total of 17 variants were classified as pathogenic or likely pathogenic, and 17 as benign or likely benign. Conclusions Our recommendations, with room for improvement in the coming years as more information become available on carrier families, tumor molecular characteristics and functional assays, are intended to serve the clinical and scientific communities and help improve diagnostic performance, avoiding variant misclassifications.

MDM2 and MDM4 are major negative regulators of tumor suppressor p53. Beyond regulating p53, MDM2 possesses p53-independent activity in promoting cell cycle progression and tumorigenesis via its RING domain ubiquitin E3 ligase activity. MDM2 and MDM4 form heterodimer polyubiquitin E3 ligases via their RING domain interaction. Inhibitors disrupting p53 interaction with MDM2/MDM4 are in clinical trials in patients bearing wild-type p53 cancers. However, these inhibitors are not designed to work for p53-null/mutant cancer cells. Owing to the importance of the E3 ligase of MDM2 in its p53-independent oncogenic activity, inhibitors targeting the E3 ligase activity of MDM2-MDM4 are desirable for p53-mutant cancer cells. Here, we report the development of such inhibitors with pro-apoptotic activity in p53-null leukemic cells. Among analogues of MDM2-MDM4 E3 ligase inhibitors, we initially identified MMRi36 as a potent pro-apoptotic compound in p53-null leukemic cells with acquired drug resistance. MMRi36 acts as an activator of MDM2-MDM4 E3 ligase by stabilizing MDM2-MDM4 heterodimers and promotes MDM2/MDM4 degradation in cells. Interestingly, replacement of the sulfur in 1,3,4-thiadiazole MMRi36 with a carbon led to identification of pyrazole MMRi36C that dissociates the MDM2-MDM4 RING heterodimers, inhibits the E3 ligase activity of the complex, and induces p53 protein accumulation, but retains the p53-independent pro-apoptotic activity. A brief SAR study identified a fluorine derivative of MMRi36C with improved pro-apoptotic activity. This study discovered a novel class of compound that targets MDM2-MDM4 ubiquitin E3 ligase activity for apoptosis induction in p53-mutant cancer cells.

Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhibitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs. The Tousled-like kinase (TLKs) family belongs to a distinct branch of Ser/Thr kinases that exhibit the highest levels of activity during DNA replication. Here the authors present the crystal structure of the kinase domain from human TLK2 and propose an activation model for TLK2 based on biochemical and phosphoproteomics experiments.

Homing endonucleases (HE) are double-stranded DNAses that target large recognition sites (12-40 bp). HE-encoding sequences are usually embedded in either introns or inteins. Their recognition sites are extremely rare, with none or only a few of these sites present in a mammalian-sized genome. However, these enzymes, unlike standard restriction endonucleases, tolerate some sequence degeneracy within their recognition sequence. Several members of this enzyme family have been used as templates to engineer tools to cleave DNA sequences that differ from their original wild-type targets. These custom HEs can be used to stimulate double-strand break homologous recombination in cells, to induce the repair of defective genes with very low toxicity levels. The use of tailored HEs opens up new possibilities for gene therapy in patients with monogenic diseases that can be treated ex vivo. This review provides an overview of recent advances in this field.

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8 N/C EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8 N/C EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8 N/C EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy. Cancer therapy using systemically administrated 4-1BB-targeting antibodies is often associated with severe toxicity due to the nonspecific activation of autoreactive T cells. Here, the authors have developed a trimeric antibody targeting both 4-1BB and EGFR, which activates T cells effectively and shows negligible cytotoxicity.

Iron-sulfur clusters are essential cofactors for the accurate cellular function of many proteins. In eukaryotic cells, the biogenesis of most iron-sulfur clusters occurs in the mitochondria and involves the action of the Cys desulfurase supercomplex, which is activated by the protein frataxin (FXN). The decrease of FXN expression and/or function results in Friedreich's ataxia (FRDA).In this work, several nanobodies specific to human FXN were selected via phage display, demonstrating a wide range of effects on Cys desulfurase activity and a strong interaction with FXN. Nanobody interaction stabilized wild-type and FRDA-related FXN variants in vitro. FXN-nanobody complexes were characterized by NMR, SAXS, and X-ray crystallography. Additionally, Nanobody expression was studied in human cells. The subcellular localization, direct interaction with FXN by in situ proximity ligation assay, effect on cell viability, Fe-S-dependent enzymatic activities, and oxygen consumption rates were analyzed. Significantly, nanobody expression did not alter these key metabolic variables, suggesting that the interaction with FXN did not disrupt the pathway.As a whole, our results suggest that nanobodies can serve as binding partners for mitochondrial FXN. However, the specific effect of the nanobodies on the conformational stability of FRDA-related FXN variants in cells should be investigated.

For the gene therapy toolkit Homing endonucleases or 'meganucleases' are a type of very rare-cutting endonuclease. They recognize much larger DNA sequences than do 'classical' restriction enzymes, so they produce a low frequency of cleavage in the human genome. This makes them of interest for gene therapy, as highly specific molecular scalpels to target specific genes. In a proof-of-principle experiment, Redondo et al . have engineered homing endonucleases with the potential to aid the repair of the gene mutated in xeroderma pigmentosum, a disorder that affects the nucleotide excision repair machinery, compromising the body's ability to remove damage caused by ultraviolet light. Xeroderma pigmentosum patients have high frequencies of mutations and hence a high predisposition to develop skin cancer. The newly designed enzymes, called Amel3–4 and Ini3–4, are derivatives of the homing endonuclease I-CreI that cleave the human XPC gene both in vitro and in vivo . Structural analysis suggests that their catalytic mechanism of cleavage is similar to that of the wild type homodimeric I-CreI, and both Amel3–4 and Ini3–4 induce high levels of gene targeting in mammalian cells. This work demonstrates a gene repair technology with the potential to repair genes in xeroderma pigmentosum that produce other monogenetic diseases. Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers 1 . The use of rare cutting DNA endonucleases—such as homing endonucleases, also known as meganucleases—constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules—Amel3–Amel4 and Ini3–Ini4—cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo . Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo .

CCT/TRiC is a eukaryotic multi-subunit chaperonin that promotes the correct folding of many cytosolic proteins, including tubulin, within its cavity. Now the crystal structure of CCT in its open state is solved to 5.5-Å resolution and, together with EM and biochemical analysis, allows the observation of a bound tubulin molecule interacting with CCT loops in the apical and equatorial domains. Protein folding is assisted by molecular chaperones. CCT (chaperonin containing TCP-1, or TRiC) is a 1-MDa oligomer that is built by two rings comprising eight different 60-kDa subunits. This chaperonin regulates the folding of important proteins including actin, α-tubulin and β-tubulin. We used an electron density map at 5.5 Å resolution to reconstruct CCT, which showed a substrate in the inner cavities of both rings. Here we present the crystal structure of the open conformation of this nanomachine in complex with tubulin, providing information about the mechanism by which it aids tubulin folding. The structure showed that the substrate interacts with loops in the apical and equatorial domains of CCT. The organization of the ATP-binding pockets suggests that the substrate is stretched inside the cavity. Our data provide the basis for understanding the function of this chaperonin.

Separation of daughter cells during bacterial cell division requires splitting of the septal cross wall by peptidoglycan hydrolases. In Streptococcus pneumoniae, PcsB is predicted to perform this operation. Recent evidence shows that PcsB is recruited to the septum by the transmembrane FtsEX complex, and that this complex is required for cell division. However, PcsB lacks detectable catalytic activity in vitro , and while it has been proposed that FtsEX activates PcsB, evidence for this is lacking. Here we demonstrate that PcsB has muralytic activity, and report the crystal structure of full-length PcsB. The protein adopts a dimeric structure in which the V-shaped coiled–coil (CC) domain of each monomer acts as a pair of molecular tweezers locking the catalytic domain of each dimeric partner in an inactive configuration. This suggests that the release of the catalytic domains likely requires an ATP-driven conformational change in the FtsEX complex, conveyed towards the catalytic domains through coordinated movements of the CC domain. The peptidoglycan hydrolase PcsB is required for cell wall splitting during cell division in Streptococci. Bartual et al. show that PcsB adopts an autoinhibited dimeric structure, and demonstrate the muralytic activity of the uninhibited catalytic domain.