Structural Insights into the BRAF Monomer-to-dimer Transition Mediated by RAS Binding

An unresolved issue in RAF kinase signaling is how binding of autoinhibited RAF monomers to activated RAS initiates the conformational changes required to form active RAF dimers. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. The RAS binding domain (RBD) of BRAF is resolved in the autoinhibited structures, and we find that neither MEK nor ATP binding is required to stabilize the autoinhibited complexes. Notably, the RBD was found to interact extensively with the 14-3-3 protomer bound to the BRAF C-terminal site. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the 14-3-3 interface have a dual function, first stabilizing RBD orientation in the autoinhibited state and then contributing to full RAS contact.

DNA damage-induced phosphorylation of histone H2A at serine 15 is linked to DNA end resection

The repair of DNA double-strand breaks (DSBs) occurs in chromatin and several histone post-translational modifications have been implicated in the process. Modifications of histone H2A N-terminal tail has also been linked to DNA damage response, through acetylation or ubiquitination of lysine residues that regulate repair pathway choice. Here, we characterize a new DNA damage-induced phosphorylation on chromatin, at serine 15 of H2A in yeast. We show that this SQ motif functions independently of the classical S129 C-terminal site (γH2A) and mutant mimicking constitutive phosphorylation increases cell sensitivity to DNA damage. H2AS129ph is induced by Tel1 ATM and Mec1 ATR , and loss of Lcd1 ATRIP or Mec1 signaling decreases γH2A spreading distal to the DSB. In contrast, H2AS15ph is completely dependent on Lcd1 ATRIP , indicating that this modification only happens when end resection is engaged. This is supported by an increase of RPA and a decrease in DNA signal near the DSB in the H2AS-15E phosphomimic mutant, indicating higher resection. This serine is replaced by a lysine in mammals (H2AK15), which undergoes an acetyl-monoubiquityl switch to regulate binding of 53BP1 and resection. This regulation seems functionally conserved with budding yeast H2AS15 and 53BP1-homolog Rad9, using different post-translational modifications between organisms but achieving the same function.

SYNCRIP facilitates porcine parvovirus viral DNA replication through the alternative splicing of NS1 mRNA to promote NS2 mRNA formation

Porcine Parvovirus (PPV), a pathogen causing porcine reproductive disorders, encodes two capsid proteins (VP1 and VP2) and three nonstructural proteins (NS1, NS2 and SAT) in infected cells. The PPV NS2 mRNA is from NS1 mRNA after alternative splicing, yet the corresponding mechanism is unclear. In this study, we identified a PPV NS1 mRNA binding protein SYNCRIP, which belongs to the hnRNP family and has been identified to be involved in host pre-mRNA splicing by RNA-pulldown and mass spectrometry approaches. SYNCRIP was found to be significantly up-regulated by PPV infection in vivo and in vitro. We confirmed that it directly interacts with PPV NS1 mRNA and is co-localized at the cytoplasm in PPV-infected cells. Overexpression of SYNCRIP significantly reduced the NS1 mRNA and protein levels, whereas deletion of SYNCRIP significantly reduced NS2 mRNA and protein levels and the ratio of NS2 to NS1, and further impaired replication of the PPV. Furthermore, we found that SYNCRIP was able to bind the 3′-terminal site of NS1 mRNA to promote the cleavage of NS1 mRNA into NS2 mRNA. Taken together, the results presented here demonstrate that SYNCRIP is a critical molecule in the alternative splicing process of PPV mRNA, while revealing a novel function for this protein and providing a potential target of antiviral intervention for the control of porcine parvovirus disease.

Detecting factorization effect in continuous-time quantum walks

We consider an continuous-time quantum walk on triple graphs with a potential well in one of the terminal sites on the main chain and study its dynamical properties in the open system by introducing a ‘spontaneous damping’ process between the terminal site and its nearest neighbour one. Calculating the stable probability of the terminal site and the time evolution of probabilities of the other sites on the main chain, we show that the system can manifest the factorization and coherence protection phenomena when the length and position of the side chain satisfy some concrete conditions. Furthermore we investigate the effect of disorder on the hopping strength and show that the existence of the disorder can destroy the coherence protection phenomena and increase the stable probability of the terminal site on the main chain.

Sequential binding of zinc triggers tau aggregation

Tau protein has been extensively studied due to its key roles in microtubular cytoskeleton regulation and in the formation of aggregates found in some neurodegenerative diseases. Recently it has been shown that zinc is able to induce tau aggregation by interacting with several binding sites. However, precise location of these sites and the molecular mechanism of zinc-induced aggregation remain unknown. Here we used Isothermal Titration Calorimetry (ITC) and Nuclear Magnetic Resonance (NMR) to identify zinc binding sites on hTau40 isoform. These experiments revealed three distinct zinc binding sites on tau, located in the N-terminal part (H14, H32, H94, and H121), the repeat region (H299, C322, H329 and H330) and the C-terminal part (H362, H374, H388 and H407). We then demonstrated that one zinc ion binds first to the repeat region, thus allowing the binding of a second zinc ion to the C-terminal part, while the N-terminal site is independant. Using molecular simulations, we modeled the structure of each site in complex with zinc. Finally, using turbidity and Dynamic Light Scattering (DLS) assays, we showed that the C-terminal site (in particular H388 and H407) is critical for zinc-induced aggregation of tau. Our study highlights key residues involved in zinc induced aggregation of tau. Given the clinical importance of tau aggregation, our findings pave the way for designing potential therapies for tauopathies. Based on our results, we propose a model of zinc-induced aggregation of tau, allowing a better understanding of both the physiological and pathological processes associated with tau-zinc interaction.

DNA damage-induced phosphorylation of histone H2A at serine 15 is linked to DNA end resection

ABSTRACTThe repair of DNA double-strand breaks (DSBs) occurs in chromatin and several histone post-translational modifications have been implicated in the process. Modifications of histone H2A N-terminal tail has also been linked to DNA damage response, through acetylation or ubiquitination of lysine residues that regulate repair pathway choice. Here, we characterize a new DNA damage-induced phosphorylation event on chromatin, at serine 15 of H2A in yeast. We show that this SQ motif functions independently of the classical S129 C-terminal site (γH2A) and mutant mimicking constitutive phosphorylation increases cell sensitivity to DNA damage. H2AS129ph is induced by both Tel1ATM and Mec1ATR, and loss of Lcd1ATRIP or Mec1 signaling decreases γH2A spreading distal to DSB. In contrast, H2AS15ph is completely dependent on Lcd1ATRIP, indicating that this modification only happens when end resection is engaged. This link is supported by an increase of RPA binding in the H2AS15E phosphomimic mutant, reflecting higher resection. This serine on H2A is replaced by a lysine in higher eukaryotes (H2AK15), which undergoes an acetyl-monoubiquityl switch to regulate binding of 53BP1 and therefore resection. This regulation seems functionally conserved with budding yeast H2AS15 and the 53BP1-homolog Rad9, utilizing different post-translational modifications between organisms but achieving the same function.

Selective profiling of N- and C-terminal nucleotide-binding sites in a TRPM2 channel

Transient receptor potential melastatin 2 (TRPM2) is a homotetrameric Ca2+-permeable cation channel important for the immune response, body temperature regulation, and insulin secretion, and is activated by cytosolic Ca2+ and ADP ribose (ADPR). ADPR binds to two distinct locations, formed by large N- and C-terminal cytosolic domains, respectively, of the channel protein. In invertebrate TRPM2 channels, the C-terminal site is not required for channel activity but acts as an active ADPR phosphohydrolase that cleaves the activating ligand. In vertebrate TRPM2 channels, the C-terminal site is catalytically inactive but cooperates with the N-terminal site in channel activation. The precise functional contributions to channel gating and the nucleotide selectivities of the two sites in various species have not yet been deciphered. For TRPM2 of the sea anemone Nematostella vectensis (nvTRPM2), catalytic activity is solely attributable to the C-terminal site. Here, we show that nvTRPM2 channel gating properties remain unaltered upon deletion of the C-terminal domain, indicating that the N-terminal site is single-handedly responsible for channel gating. Exploiting such functional independence of the N- and C-terminal sites, we selectively measure their affinity profiles for a series of ADPR analogues, as reflected by apparent affinities for channel activation and catalysis, respectively. Using site-directed mutagenesis, we confirm that the same N-terminal site observed in vertebrate TRPM2 channels was already present in ancient cnidarians. Finally, by characterizing the functional effects of six amino acid side chain truncations in the N-terminal site, we provide first insights into the mechanistic contributions of those side chains to TRPM2 channel gating.

Insights into the molecular mechanisms underlying the inhibition of acid-sensing ion channel 3 gating by stomatin

Stomatin (STOM) is a monotopic integral membrane protein found in all classes of life that has been shown to regulate members of the acid-sensing ion channel (ASIC) family. However, the mechanism by which STOM alters ASIC function is not known. Using chimeric channels, we combined patch-clamp electrophysiology and FRET to search for regions of ASIC3 critical for binding to and regulation by STOM. With this approach, we found that regulation requires two distinct sites on ASIC3: the distal C-terminus and the first transmembrane domain (TM1). The C-terminal site is critical for formation of the STOM–ASIC3 complex, while TM1 is required only for the regulatory effect. We then looked at the mechanism of STOM-dependent regulation of ASIC3 and found that STOM does not alter surface expression of ASIC3 or shift the pH dependence of channel activation. However, a point mutation (Q269G) that prevents channel desensitization also prevents STOM regulation, suggesting that STOM may alter ASIC3 currents by stabilizing the desensitized state of the channel. Based on these findings, we propose a model whereby STOM is anchored to the channel via a site on the distal C-terminus and stabilizes the desensitized state of the channel via an interaction with TM1.

Effect of 20-Hydroxyecdysone and S-adenosylmethionine on the ecdysone receptor gene EcR transcriptional activity in housefly Musca domestica L.

Steroid hormone 20-hydroxyecdysone (20E) initiates larval molting start and metamorphosis and regulates reproduction. Its basic receptor is heterodimer including proteins EcR and USP. Ecdysone receptor gene EcR coding protein EcR is a key regulatory element of gene circuits cover considerable part of genes, implicated in growth and development as well as in reproduction of progeny and reactions of organisms to unfavorable factors of environment. The source of methyl groups S-adenosylmethionine (SAM) is in use for biosynthesis of juvenile hormone (JH), methylation of histone proteins and DNA. The main aim of our investigation was evaluation of transcriptional activity of housefly Musca domestica ecdysone receptor gene EcR under adding into ration of 20E and SAM in non-lethal concentrations. Experiments were carried out with larvae and adults of housefly from laboratory strains Shgen and Lgen differ in life span of adults. Change of gene EcR transcripts content in common pool of mRNA in the cells of muscles and gonads, as well as DNA methylation level in 5’-terminal site registered by quantitative real time PCR (RT-PCR). The results of our investigations allow us to suggest existence of mechanism for regulating expression of the EcR gene in M. domestica which is sensitive to exogenic20E and heat stress action as well as to presence of SAM in food. Variations in the mRNA quantitative ratios of EcR gene 5’-and 3’-terminal regions depending on tissue type, gender and age support the hypothesis that this gene can encode several isoforms of the protein EcR. The detected changes in the status of DNA methylation in the 5'-terminal region of the gene and fluctuations in the representation of different mRNA sites after SAM processing suggest the involvement of DNA methylation/demethylation processes in the regulation of EcR gene expression in M. domestica.

Intracellular delivery of therapeutic proteins through N-terminal site-specific modification

Adopting orthogonal dual-labeling strategies, a cell-permeable RNase A prodrug was designed for ROS-responsive targeted cancer therapy.