Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.

Protecting video games against online copyright infringement: a Russian approach to site blocking

Throughout recent years Russia has developed a strong anti-piracy and site blocking laws which claim to become effective tools against an ongoing issue of video game piracy. Thousands of pirate torrent indexing websites, illegal marketplaces with unauthorized digital goods and in-game values are shut down yearly by video game right holders in order to prevent illegal content consumption. Torrent sites loose up to 90% of traffic quickly after blocking measures are implemented. However, if the law is too good to be true, it most probably is. The strict domain-specific site blocking approach has been an ongoing issue and an obstacle for right holders to tackle site blocking circumvention tools from Russia. Most pirate sites hop to a new domain name after the right holder obtains first content-removal order making it impossible to sue the website for repeated copyright infringement. The dynamic site blocking approach developed within European case law and best practices may become a potential solution to this problem.

Allosteric control of Ubp6 and the proteasome via a bidirectional switch

The proteasome is the principal cellular protease, and recognizes target proteins that have been covalently marked by ubiquitin chains. The ubiquitin signal is subject to rapid editing at the proteasome, allowing it to reject substrates based on topological features of their attached ubiquitin chains. Editing is mediated by a key regulator of the proteasome, deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome–both by deubiquitinating proteasome-bound ubiquitin conjugates, and through a noncatalytic effect that does not involve deubiquitination. We report mutants in both Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations fall within its ILR element, defined here, which is conserved from yeast to mammals. The ILR is a component of the BL1 blocking loop, other parts of which obstruct ubiquitin access to the catalytic groove in free Ubp6. Rpt1 docking at the ILR opens the catalytic groove by rearranging not only BL1 but also a novel network of three directly interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition by Ubp6 are linked in that they were eliminated by the same Ubp6 and Rpt1 mutations. Ubp6 and ubiquitin together drive the proteasome into a unique conformational state associated with proteasome inhibition. Our results identify a multicomponent allosteric switch that exerts simultaneous control over the activity of both Ubp6 and the proteasome, and suggest that their active states are in general mutually exclusive. The findings lead to a new paradigm for allosteric control of deubiquitinating enzymes.

Thermodynamic Modelling of Hydrogen-Multicomponent Alloy Systems: Calculating Pressure-Composition-Temperature Diagrams

<p>The applicability of an alloy as a hydrogen storage media mostly relies on its pressure-composition-temperature (PCT) diagram. Since the PCT diagram is composition-dependent, the vast compositional filed of high entropy alloys, complex concentrated alloys or multicomponent alloys can be explored to design alloys with optimized properties for each application. In this work, we present a thermodynamic model to calculate PCT diagrams of body-centered (BCC) multicomponent alloys. The entropy of the phases is described using the ideal configurational entropy for interstitial solid solutions with site blocking effect. As a first approximation, it is assumed that the H partial molar enthalpy of a phase is constant, so the enthalpy of H mixing varies linearly with the H concentration. Moreover, the H partial enthalpy of a phase for a multicomponent alloy was approximated by a simple ideal mixture law of this quantity for the alloy’s components with the same structure. Experimental data and DFT calculations were used for parametrization of the enthalpy terms of eight elements (Ti, V, Cr, Ni, Zr, Nb, Hf, and Ta), which are the components of the alloys tested in this work. Experimental PCTs of six BCC multicomponent alloys of four different systems were compared against the calculated ones and the agreement was remarkable. The model and parameters presented here can be regarded as a basis for developing powerful alloy design tools for different hydrogen storage applications.</p>

Thermodynamic Modelling of Hydrogen-Multicomponent Alloy Systems: Calculating Pressure-Composition-Temperature Diagrams

<p>The applicability of an alloy as a hydrogen storage media mostly relies on its pressure-composition-temperature (PCT) diagram. Since the PCT diagram is composition-dependent, the vast compositional filed of high entropy alloys, complex concentrated alloys or multicomponent alloys can be explored to design alloys with optimized properties for each application. In this work, we present a thermodynamic model to calculate PCT diagrams of body-centered (BCC) multicomponent alloys. The entropy of the phases is described using the ideal configurational entropy for interstitial solid solutions with site blocking effect. As a first approximation, it is assumed that the H partial molar enthalpy of a phase is constant, so the enthalpy of H mixing varies linearly with the H concentration. Moreover, the H partial enthalpy of a phase for a multicomponent alloy was approximated by a simple ideal mixture law of this quantity for the alloy’s components with the same structure. Experimental data and DFT calculations were used for parametrization of the enthalpy terms of eight elements (Ti, V, Cr, Ni, Zr, Nb, Hf, and Ta), which are the components of the alloys tested in this work. Experimental PCTs of six BCC multicomponent alloys of four different systems were compared against the calculated ones and the agreement was remarkable. The model and parameters presented here can be regarded as a basis for developing powerful alloy design tools for different hydrogen storage applications.</p>

A novel antibody against the furin cleavage site of SARS-CoV-2 spike protein: effects on proteolytic cleavage and ACE2 binding

SARS-CoV-2 harbors a unique S1/S2 furin cleavage site within its spike protein, which can be cleaved by furin and other proprotein convertases. Proteolytic activation of SARS-CoV-2 spike protein at the S1/S2 boundary facilitates interaction with host ACE2 receptor for cell entry. To address this, high titer antibody was generated against the SARS-CoV-2-specific furin motif. Using a series of innovative ELISA-based assays, this furin site blocking antibody displayed high sensitivity and specificity for the S1/S2 furin cleavage site, and demonstrated effective blockage of both enzyme-mediated cleavage and spike-ACE2 interaction. The results suggest that immunological blocking of the furin cleavage site may afford a suitable approach to stem proteolytic activation of SARS-CoV-2 spike protein and curtail viral infectivity.

Web site blocking as a proxy of policy alignment

This work introduces a new metric of policy alignment between states: Web site blocking. Intuitively, we measure the degree to which states block similar content. This metric speaks to the flow of information in the digital layer of international politics, an element of international trade and cooperation that existing metrics fail to capture. In addition, our measure can be constantly scraped and updated, offering a higher temporal resolution than existing metrics. Our work suggests a link between Internet governance and other issues in international relations (e.g., trade). Since our metric can be updated in real time, future work could use our metric to detect geopolitical shifts more rapidly than would otherwise be possible.

PMO-based let-7c site blocking oligonucleotide (SBO) mediated utrophin upregulation in mdx mice, a therapeutic approach for Duchenne muscular dystrophy (DMD)

Upregulation of utrophin, a dystrophin related protein, is considered a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Utrophin expression is repressed at the post-transcriptional level by a set of miRNAs, among which let-7c is evolutionarily highly conserved. We designed PMO-based SBOs complementary to the let-7c binding site in UTRN 3′UTR, with the goal of inhibiting let-7c interaction with UTRN mRNA and thus upregulating utrophin. We used the C2C12UTRN5′luc3′ reporter cell line in which the 5′- and 3′-UTRs of human UTRN sequences flank luciferase, for reporter assays and the C2C12 cell line for utrophin western blots, to independently evaluate the site blocking efficiency of a series of let-7c PMOs in vitro. Treatment of one-month old mdx mice with the most effective let-7c PMO (i.e. S56) resulted in ca. two-fold higher utrophin protein expression in skeletal muscles and the improvement in dystrophic pathophysiology in mdx mice, in vivo. In summary, we show that PMO-based let-7c SBO has potential applicability for upregulating utrophin expression as a therapeutic approach for DMD.