Molecular basis for modulation of the p53 target selectivity by KLF4

The tumour suppressor p53 controls transcription of various genes involved in apoptosis, cell-cycle arrest, DNA repair and metabolism. However, its DNA-recognition specificity is not nearly sufficient to explain binding to specific locations in vivo. Here, we present evidence that KLF4 increases the DNA-binding affinity of p53 through the formation of a loosely arranged ternary complex on DNA. This effect depends on the distance between the response elements of KLF4 and p53. Using nuclear magnetic resonance and fluorescence techniques, we found that the amino-terminal domain of p53 interacts with the KLF4 zinc fingers and mapped the interaction site. The strength of this interaction was increased by phosphorylation of the p53 N-terminus, particularly on residues associated with regulation of cell-cycle arrest genes. Taken together, the cooperative binding of KLF4 and p53 to DNA exemplifies a regulatory mechanism that contributes to p53 target selectivity

Prefrontal involvement in imitation learning of hand actions : effects of practice and expertise.

In this event-related fMRI study, we demonstrate the effects of a single session of practising configural hand actions (guitar chords) on cortical activations during observation, motor preparation, and imitative execution. During the observation of non-practised actions, the mirror neuron system (MNS), consisting of inferior parietal and ventral premotor areas, was more strongly activated than for the practised actions. This finding indicates a strong role of the MNS in the early stages of imitation learning. In addition, the dorsolateral prefrontal cortex (DLPFC) was selectively involved during observation and motor preparation of the non-practised chords. This finding confirms Buccino et al.’s (2004a) model of imitation learning: for actions that are not yet part of the observer’s motor repertoire, DLPFC engages in operations of selection and combination of existing, elementary representations in the MNS. The pattern of prefrontal activations further supports Shallice’s (2004) proposal of a dominant role of the left DLPFC in modulating lower-level systems, and of a dominant role of the right DLPFC in monitoring operations

OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis

Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1’ and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates

Dynamics in Fip1 regulate eukaryotic mRNA 3^′ end processing

Cleavage and polyadenylation factor (CPF/CPSF) is a multiprotein complex essential for mRNA 3′ end processing in eukaryotes. It contains an endonuclease that cleaves pre-mRNAs, and a polymerase that adds a poly(A) tail onto the cleaved 3′ end. Several CPF subunits, including Fip1, contain intrinsically disordered regions (IDRs). IDRs within multiprotein complexes can be flexible, or can become ordered upon interaction with binding partners. Here, we show that yeast Fip1 anchors the poly(A) polymerase Pap1 onto CPF via an interaction with zinc finger 4 of another CPF subunit, Yth1. We also reconstitute a fully recombinant 850-kDa CPF. By incorporating selectively labeled Fip1 into recombinant CPF, we could study the dynamics of Fip1 within the megadalton complex using nuclear magnetic resonance (NMR) spectroscopy. This reveals that a Fip1 IDR that connects the Yth1- and Pap1-binding sites remains highly dynamic within CPF. Together, our data suggest that Fip1 dynamics within the 3′ end processing machinery are required to coordinate cleavage and polyadenylation.</p

Dynamics in Fip1 regulate eukaryotic mRNA 3^′ end processing

Cleavage and polyadenylation factor (CPF/CPSF) is a multiprotein complex essential for mRNA 3′ end processing in eukaryotes. It contains an endonuclease that cleaves pre-mRNAs, and a polymerase that adds a poly(A) tail onto the cleaved 3′ end. Several CPF subunits, including Fip1, contain intrinsically disordered regions (IDRs). IDRs within multiprotein complexes can be flexible, or can become ordered upon interaction with binding partners. Here, we show that yeast Fip1 anchors the poly(A) polymerase Pap1 onto CPF via an interaction with zinc finger 4 of another CPF subunit, Yth1. We also reconstitute a fully recombinant 850-kDa CPF. By incorporating selectively labeled Fip1 into recombinant CPF, we could study the dynamics of Fip1 within the megadalton complex using nuclear magnetic resonance (NMR) spectroscopy. This reveals that a Fip1 IDR that connects the Yth1- and Pap1-binding sites remains highly dynamic within CPF. Together, our data suggest that Fip1 dynamics within the 3′ end processing machinery are required to coordinate cleavage and polyadenylation.</p

Disease-Associated Mutations in CEP120 Destabilize the Protein and Impair Ciliogenesis.

Ciliopathies are a group of genetic disorders caused by a failure to form functional cilia. Due to a lack of structural information, it is currently poorly understood how ciliopathic mutations affect protein functionality to give rise to the underlying disease. Using X-ray crystallography, we show that the ciliopathy-associated centriolar protein CEP120 contains three C2 domains. The point mutations V194A and A199P, which cause Joubert syndrome (JS) and Jeune asphyxiating thoracic dystrophy (JATD), respectively, both reduce the thermostability of the second C2 domain by targeting residues that point toward its hydrophobic core. Genome-engineered cells homozygous for these mutations have largely normal centriole numbers but show reduced CEP120 levels, compromised recruitment of distal centriole markers, and deficient cilia formation. Our results provide insight into the disease mechanism of two ciliopathic mutations in CEP120, identify putative binding partners of CEP120 C2B, and suggest a complex genotype-phenotype relation of the CEP120 ciliopathy alleles

Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties

A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life.</p

Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties

A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life.</p

An intrinsically labile α-helix abutting the BCL9-binding site of β-catenin is required for its inhibition by carnosic acid.

Wnt/β-catenin signalling controls development and tissue homeostasis. Moreover, activated β-catenin can be oncogenic and, notably, drives colorectal cancer. Inhibiting oncogenic β-catenin has proven a formidable challenge. Here we design a screen for small-molecule inhibitors of β-catenin's binding to its cofactor BCL9, and discover five related natural compounds, including carnosic acid from rosemary, which attenuates transcriptional β-catenin outputs in colorectal cancer cells. Evidence from NMR and analytical ultracentrifugation demonstrates that the carnosic acid response requires an intrinsically labile α-helix (H1) amino-terminally abutting the BCL9-binding site in β-catenin. Similarly, in colorectal cancer cells with hyperactive β-catenin signalling, carnosic acid targets predominantly the transcriptionally active ('oncogenic') form of β-catenin for proteasomal degradation in an H1-dependent manner. Hence, H1 is an 'Achilles' Heel' of β-catenin, which can be exploited for destabilization of oncogenic β-catenin by small molecules, providing proof-of-principle for a new strategy for developing direct inhibitors of oncogenic β-catenin

Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1.

Funder: Gates Cambridge Trust; Id: http://dx.doi.org/10.13039/501100005370During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD ) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly