Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites.

Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.

Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.

Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.

Conformational landscape of the type V-K CRISPR-associated transposon integration assembly.

CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.

Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages.

Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.

DAXX adds a de novo H3.3K9me3 deposition pathway to the histone chaperone network.

A multitude of histone chaperones are required to support histones from their biosynthesis until DNA deposition. They cooperate through the formation of histone co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using exploratory interactomics, we define the interplay between human histone H3-H4 chaperones in the histone chaperone network. We identify previously uncharacterized histone-dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX provides a unique functionality to the histone chaperone network, recruiting histone methyltransferases to promote H3K9me3 catalysis on new histone H3.3-H4 prior to deposition onto DNA. Hereby, DAXX provides a molecular mechanism for de novo H3K9me3 deposition and heterochromatin assembly. Collectively, our findings provide a framework for understanding how cells orchestrate histone supply and employ targeted deposition of modified histones to underpin specialized chromatin states.

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.

RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.

TnpB structure reveals minimal functional core of Cas12 nuclease family.

The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells1,2. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases3-5, which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally6, the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB-reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools.

Structures of the Cmr-ß Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas.

Cmr-ß is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-ß, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-ß and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.

A Consensus Binding Motif for the PP4 Protein Phosphatase.

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.

Chemogenetic profiling reveals PP2A-independent cytotoxicity of proposed PP2A activators iHAP1 and DT-061.

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.

A Conserved Motif Provides Binding Specificity to the PP2A-B56 Phosphatase.

Dynamic protein phosphorylation is a fundamental mechanism regulating biological processes in all organisms. Protein phosphatase 2A (PP2A) is the main source of phosphatase activity in the cell, but the molecular details of substrate recognition are unknown. Here, we report that a conserved surface-exposed pocket on PP2A regulatory B56 subunits binds to a consensus sequence on interacting proteins, which we term the LxxIxE motif. The composition of the motif modulates the affinity for B56, which in turn determines the phosphorylation status of associated substrates. Phosphorylation of amino acid residues within the motif increases B56 binding, allowing integration of kinase and phosphatase activity. We identify conserved LxxIxE motifs in essential proteins throughout the eukaryotic domain of life and in human viruses, suggesting that the motifs are required for basic cellular function. Our study provides a molecular description of PP2A binding specificity with broad implications for understanding signaling in eukaryotes.

Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA.

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here, we selected a target site on bacteriophage λ-DNA and used optical tweezers combined with fluorescence to provide mechanistic insight into wild type Cas12a and three engineered variants, where the specific dsDNA and the unspecific ssDNA cleavage are dissociated (M1 and M2) and a third one which nicks the target DNA (M3). At low forces wtCas12a and the variants display two main off-target binding sites, while on stretched dsDNA at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. The multiple binding events onto dsDNA at high tension do not lead to cleavage, which is observed on the target site at low forces when the DNA is flexible. In addition, activity assays also show that the preferential off-target sites for this crRNA are not cleaved by wtCas12a, indicating that λ-DNA is only severed at the target site. Our single molecule data indicate that the Cas12a scaffold presents singular mechanical properties, which could be used to generate new endonucleases with biomedical and biotechnological applications.

Molecular basis of cyclic tetra-oligoadenylate processing by small standalone CRISPR-Cas ring nucleases.

Standalone ring nucleases are CRISPR ancillary proteins, which downregulate the immune response of Type III CRISPR-Cas systems by cleaving cyclic oligoadenylates (cA) second messengers. Two genes with this function have been found within the Sulfolobus islandicus (Sis) genome. They code for a long polypeptide composed by a CARF domain fused to an HTH domain and a short polypeptide constituted by a CARF domain with a 40 residue C-terminal insertion. Here, we determine the structure of the apo and substrate bound states of the Sis0455 enzyme, revealing an insertion at the C-terminal region of the CARF domain, which plays a key role closing the catalytic site upon substrate binding. Our analysis reveals the key residues of Sis0455 during cleavage and the coupling of the active site closing with their positioning to proceed with cA4 phosphodiester hydrolysis. A time course comparison of cA4 cleavage between the short, Sis0455, and long ring nucleases, Sis0811, shows the slower cleavage kinetics of the former, suggesting that the combination of these two types of enzymes with the same function in a genome could be an evolutionary strategy to regulate the levels of the second messenger in different infection scenarios.

A PTIP-PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex.

Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.

A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis.

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A-B56 phosphatase through a coiled-coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A-B56-hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A-B56 substrates containing a canonical B56 binding motif. We find that PP2A-B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.

Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage.

Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1.

Erratum: Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage.

This corrects the article DOI: 10.1038/nature22398.

Structural basis of cyclic oligoadenylate degradation by ancillary Type III CRISPR-Cas ring nucleases.

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.

Pharmacokinetic assessment and phytochemical triterpene control from Cecropia angustifolia using plant biotechnology.

INTRODUCTION: Cecropia angustifolia Trécul. is a native Andean plant containing high levels of pentacyclic triterpenes (PTs), including several isobaric molecules that serve as chemical markers. Preclinical studies suggest that PTs positively modulate metabolic and vascular diseases. However, their low oral absorption reduces their bioactive effects. OBJECTIVE: The objective of this study was (1) to improve the absorption of PTs from C. angustifolia and (2) to establish a platform to produce biomass or botanical reference material using a strategy for their accumulation. METHODS: MALDI-TOF and UPLC-MS were used to characterize and quantify PTs in different matrices. An in vitro platform for PT production was established. Chemical profiles of triterpenes were also evaluated from wild and in vitro herbal material using TLC coupled with mass spectrometry. RESULTS: To overcome the low absorption of PTs, a premier raw material was used, which increased their bioavailability to 9.2%. Active ingredients in herbal material can vary, and there is an urgent need for standardized extracts using pharmacokinetics as an effective tool to reveal the dynamics of active ingredients in vivo. A temporary immersion system was produced as a promising platform with a total PT accumulation exceeding 50% of the content in the dry fraction, indicating it is a feasible mechanism to produce biomass or botanical reference material. CONCLUSIONS: Plant tissue culture is a promising eco-friendly technology for phytochemical production and a modern strategy to protect biodiversity in natural assets. Alternative and modern, yet environmentally friendly production methods are needed to meet the large demand for herbal products.

Transposons and CRISPR: Rewiring Gene Editing.

CRISPR-Cas is driving a gene editing revolution because of its simple reprogramming. However, off-target effects and dependence on the double-strand break repair pathways impose important limitations. Because homology-directed repair acts primarily in actively dividing cells, many of the current gene correction/replacement approaches are restricted to a minority of cell types. Furthermore, current approaches display low efficiency upon insertion of large DNA cargos (e.g., sequences containing multiple gene circuits with tunable functionalities). Recent research has revealed new links between CRISPR-Cas systems and transposons providing new scaffolds that might overcome some of these limitations. Here, we comment on two new transposon-associated RNA-guided mechanisms considering their potential as new gene editing solutions. Initially, we focus on a group of small RNA-guided endonucleases of the IS200/IS605 family of transposons, which likely evolved into class 2 CRISPR effector nucleases (Cas9s and Cas12s). We explore the diversity of these nucleases (named OMEGA, obligate mobile element-guided activity) and analyze their similarities with class 2 gene editors. OMEGA nucleases can perform gene editing in human cells and constitute promising candidates for the design of new compact RNA-guided platforms. Then, we address the co-option of the RNA-guided activity of different CRISPR effector nucleases by a specialized group of Tn7-like transposons to target transposon integration. We describe the various mechanisms used by these RNA-guided transposons for target site selection and integration. Finally, we assess the potential of these new systems to circumvent some of the current gene editing challenges.