Molecular basis for substrate recruitment to the PRMT5 methylosome.

PRMT5 is an essential arginine methyltransferase and a therapeutic target in MTAP-null cancers. PRMT5 uses adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (CLNS1A, RIOK1, and COPR5) and show that it is necessary and sufficient for interaction with PRMT5. We demonstrate that PRMT5 uses modular adaptor proteins containing a common binding motif for substrate recruitment, comparable with other enzyme classes such as kinases and E3 ligases. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosomal complexes. Furthermore, disruption of this site affects Sm spliceosome activity, leading to intron retention. Genetic disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells and is thus a site for development of therapeutic inhibitors of PRMT5.

Assessing optimal: inequalities in codon optimization algorithms.

BACKGROUND: Custom genes have become a common resource in recombinant biology over the last 20 years due to the plummeting cost of DNA synthesis. These genes are often "optimized" to non-native sequences for overexpression in a non-native host by substituting synonymous codons within the coding DNA sequence (CDS). A handful of studies have compared native and optimized CDSs, reporting different levels of soluble product due to the accumulation of misfolded aggregates, variable activity of enzymes, and (at least one report of) a change in substrate specificity. No study, to the best of our knowledge, has performed a practical comparison of CDSs generated from different codon optimization algorithms or reported the corresponding protein yields. RESULTS: In our efforts to understand what factors constitute an optimized CDS, we identified that there is little consensus among codon-optimization algorithms, a roughly equivalent chance that an algorithm-optimized CDS will increase or diminish recombinant yields as compared to the native DNA, a near ubiquitous use of a codon database that was last updated in 2007, and a high variability of output CDSs by some algorithms. We present a case study, using KRas4B, to demonstrate that a median codon frequency may be a better predictor of soluble yields than the more commonly utilized CAI metric. CONCLUSIONS: We present a method for visualizing, analyzing, and comparing algorithm-optimized DNA sequences for recombinant protein expression. We encourage researchers to consider if DNA optimization is right for their experiments, and work towards improving the reproducibility of published recombinant work by publishing non-native CDSs.

The Autophagy-Related Beclin-1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity.

Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3-5 mg/L in an Escherichia coli culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (KD ∼ 0.45 µM) that interacts with Bcl-2 (KD = 4.3 ± 1.2 µM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer KD 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold.

Crystal Structure of Recoverin with Calcium Ions Bound to Both Functional EF Hands.

Recoverin (Rv), a small Ca(2+)-binding protein that inhibits rhodopsin kinase (RK), has four EF hands, two of which are functional (EF2 and EF3). Activation requires Ca(2+) in both EF hands, but crystal structures have never been observed with Ca(2+) ions in both sites; all previous structures have Ca(2+) bound to only EF3. We suspected that this was due to an intermolecular crystal contact between T80 and a surface glutamate (E153) that precluded coordination of a Ca(2+) ion in EF2. We constructed the E153A mutant, determined its X-ray crystal structure to 1.2 Å resolution, and showed that two Ca(2+) ions are bound, one in EF3 and one in EF2. Additionally, several other residues are shown to adopt conformations in the 2Ca(2+) structure not seen previously and not seen in a second structure of the E153A mutant containing Na(+) instead of Ca(2+) in the EF2 site. The side-chain rearrangements in these residues form a 28 Å allosteric cascade along the surface of the protein connecting the Ca(2+)-binding site of EF2 with the active-site pocket responsible for binding RK.

A highly conserved cysteine of neuronal calcium-sensing proteins controls cooperative binding of Ca2+ to recoverin.

Recoverin, a 23-kDa Ca(2+)-binding protein of the neuronal calcium sensing (NCS) family, inhibits rhodopsin kinase, a Ser/Thr kinase responsible for termination of photoactivated rhodopsin in rod photoreceptor cells. Recoverin has two functional EF hands and a myristoylated N terminus. The myristoyl chain imparts cooperativity to the Ca(2+)-binding sites through an allosteric mechanism involving a conformational equilibrium between R and T states of the protein. Ca(2+) binds preferentially to the R state; the myristoyl chain binds preferentially to the T state. In the absence of myristoylation, the R state predominates, and consequently, binding of Ca(2+) to the non-myristoylated protein is not cooperative. We show here that a mutation, C39A, of a highly conserved Cys residue among NCS proteins, increases the apparent cooperativity for binding of Ca(2+) to non-myristoylated recoverin. The binding data can be explained by an effect on the T/R equilibrium to favor the T state without affecting the intrinsic binding constants for the two Ca(2+) sites.

meso-arylporpholactones and their reduction products.

The rational syntheses of meso-tetraaryl-3-oxo-2-oxaporphyrins 5, known as porpholactones, via MnO(4)(-)-mediated oxidations of the corresponding meso-tetraaryl-2,3-dihydroxychlorins (7) is detailed. Since chlorin 7 is prepared from the parent porphyrin 1, this amounts to a 2-step replacement of a pyrrole moiety in 1 by an oxazolone moiety. The stepwise reduction of the porpholactone 5 results in the formation of chlorin analogues, meso-tetraaryl-3-hydroxy-2-oxachlorin (11) and meso-tetraaryl-2-oxachlorins (12). The reactivity of 11 with respect to nucleophilic substitution by O-, N-, and S-nucleophiles is described. The profound photophysical consequences of the formal replacement of a pyrrole with an oxazolone (porphyrin-like chromophore) or (substituted) oxazole moiety (chlorin-like chromophore with, for the parent oxazolochlorin 12, red-shifted Q(x) band with enhanced oscillator strengths) are detailed and rationalized on the basis of SAC-CI and MNDO-PSDCI molecular orbital theory calculations. The single crystal X-ray structures of the porpholactones point at a minor steric interaction between the carbonyl oxygen and the flanking phenyl group. The essentially planar structures of all chromophores in all oxidation states prove that the observed optical properties originate from the intrinsic electronic properties of the chromophores and are not subject to conformational modulation.

Green proteorhodopsin reconstituted into nanoscale phospholipid bilayers (nanodiscs) as photoactive monomers.

Over 4000 putative proteorhodopsins (PRs) have been identified throughout the oceans and seas of the Earth. The first of these eubacterial rhodopsins was discovered in 2000 and has expanded the family of microbial proton pumps to all three domains of life. With photophysical properties similar to those of bacteriorhodopsin, an archaeal proton pump, PRs are also generating interest for their potential use in various photonic applications. We perform here the first reconstitution of the minimal photoactive PR structure into nanoscale phospholipid bilayers (nanodiscs) to better understand how protein-protein and protein-lipid interactions influence the photophysical properties of PR. Spectral (steady-state and time-resolved UV-visible spectroscopy) and physical (size-exclusion chromatography and electron microscopy) characterization of these complexes confirms the preparation of a photoactive PR monomer within nanodiscs. Specifically, when embedded within a nanodisc, monomeric PR exhibits a titratable pK(a) (6.5-7.1) and photocycle lifetime (∼100-200 ms) that are comparable to the detergent-solubilized protein. These ndPRs also produce a photoactive blue-shifted absorbance, centered at 377 or 416 nm, that indicates that protein-protein interactions from a PR oligomer are required for a fast photocycle. Moreover, we demonstrate how these model membrane systems allow modulation of the PR photocycle by variation of the discoidal diameter (i.e., 10 or 12 nm), bilayer thickness (i.e., 23 or 26.5 Å), and degree of saturation of the lipid acyl chain. Nanodiscs also offer a highly stable environment of relevance to potential device applications.

Activation of retinal ganglion cells using a biomimetic artificial retina.

Objective.Biomimetic protein-based artificial retinas offer a new paradigm for restoring vision for patients blinded by retinal degeneration. Artificial retinas, comprised of an ion-permeable membrane and alternating layers of bacteriorhodopsin (BR) and a polycation binder, are assembled using layer-by-layer electrostatic adsorption. Upon light absorption, the oriented BR layers generate a unidirectional proton gradient. The main objective of this investigation is to demonstrate the ability of the ion-mediated subretinal artificial retina to activate retinal ganglion cells (RGCs) of degenerated retinal tissue.Approach. Ex vivoextracellular recording experiments with P23H line 1 rats are used to measure the response of RGCs following selective stimulation of our artificial retina using a pulsed light source. Single-unit recording is used to evaluate the efficiency and latency of activation, while a multielectrode array (MEA) is used to assess the spatial sensitivity of the artificial retina films.Main results.The activation efficiency of the artificial retina increases with increased incident light intensity and demonstrates an activation latency of ∼150 ms. The results suggest that the implant is most efficient with 200 BR layers and can stimulate the retina using light intensities comparable to indoor ambient light. Results from using an MEA show that activation is limited to the targeted receptive field.Significance.The results of this study establish potential effectiveness of using an ion-mediated artificial retina to restore vision for those with degenerative retinal diseases, including retinitis pigmentosa.

Discovery of a First-in-Class Inhibitor of the PRMT5-Substrate Adaptor Interaction.

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action studies revealed the formation of a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts PRMT5-RIOK1 complexes, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive inhibitor that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.

Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase.

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.

High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl-2 Binding.

Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy induction-specifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ∼300 K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC50 in the micromolar range and increase autophagic flux. These compounds do not demonstrate significant cytotoxicity, and they exert selectivity for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared with the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have identified candidate molecules that serve as lead templates for developing potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as autophagy-inducing agents.

Photochromic bacteriorhodopsin mutant with high holographic efficiency and enhanced stability via a putative self-repair mechanism.

The Q photoproduct of bacteriorhodopsin (BR) is the basis of several biophotonic technologies that employ BR as the photoactive element. Several blue BR (bBR) mutants, generated by using directed evolution, were investigated with respect to the photochemical formation of the Q state. We report here a new bBR mutant, D85E/D96Q, which is capable of efficiently converting the entire sample to and from the Q photoproduct. At pH 8.5, where Q formation is optimal, the Q photoproduct requires 65 kJ mol(-1) of amber light irradiation (590 nm) for formation and 5 kJ mol(-1) of blue light (450 nm) for reversion, respectively. The melting temperature of the resting state and Q photoproduct, measured via differential scanning calorimetry, is observed at 100 °C and 89 °C at pH 8.5 or 91 °C and 82 °C at pH 9.5, respectively. We hypothesize that the protein stability of D85E/D96Q compared to other blue mutants is associated with a rapid equilibrium between the blue form E85(H) and the purple form E85(-) of the protein, the latter providing enhanced structural stability. Additionally, the protein is shown to be stable and functional when suspended in an acrylamide matrix at alkaline pH. Real-time photoconversion to and from the Q state is also demonstrated with the immobilized protein. Finally, the holographic efficiency of an ideal thin film using the Q state of D85E/D96Q is calculated to be 16.7%, which is significantly better than that provided by native BR (6-8%) and presents the highest efficiency of any BR mutant to date.

Directed evolution of bacteriorhodopsin for applications in bioelectronics.

In nature, biological systems gradually evolve through complex, algorithmic processes involving mutation and differential selection. Evolution has optimized biological macromolecules for a variety of functions to provide a comparative advantage. However, nature does not optimize molecules for use in human-made devices, as it would gain no survival advantage in such cooperation. Recent advancements in genetic engineering, most notably directed evolution, have allowed for the stepwise manipulation of the properties of living organisms, promoting the expansion of protein-based devices in nanotechnology. In this review, we highlight the use of directed evolution to optimize photoactive proteins, with an emphasis on bacteriorhodopsin (BR), for device applications. BR, a highly stable light-activated proton pump, has shown great promise in three-dimensional optical memories, real-time holographic processors and artificial retinas.

Photochemical and thermal stability of green and blue proteorhodopsins: implications for protein-based bioelectronic devices.

The photochemical and thermal stability of the detergent-solubilized blue- and green-absorbing proteorhodpsins, BPR and GPR, respectively, are investigated to determine the viability of these proteins for photonic device applications. Photochemical stability is studied by using pulsed laser excitation and differential UV-vis spectroscopy to assign the photocyclicity. GPR, with a cyclicity of 7 × 10(4) photocycles protein(-1), is 4-5 times more stable than BPR (9 × 10(3) photocycles protein(-1)), but is less stable than native bacteriorhodopsin (9 × 10(5) photocycles protein(-1)) or the 4-keto-bacteriorhodopsin analogue (1 × 10(5) photocycles protein(-1)). The thermal stabilities are assigned by using differential scanning calorimetry and thermal bleaching experiments. Both proteorhodopsins display excellent thermal stability, with melting temperatures above 85 °C, and remain photochemically stable up to 75 °C. The biological relevance of our results is also discussed. The lower cyclicity of BPR is found to be adequate for the long-term biological function of the host organism at ocean depths of 50 m or more.

A second-site suppressor of a folding defect functions via interactions with a chaperone network to improve folding and assembly in vivo.

Single amino acid substitutions in a protein can cause misfolding and aggregation to occur. Protein misfolding can be rescued by second-site amino acid substitutions called suppressor substitutions (su), commonly through stabilizing the native state of the protein or by increasing the rate of folding. Here we report evidence that su substitutions that rescue bacteriophage P22 temperature-sensitive-folding (tsf) coat protein variants function in a novel way. The ability of tsf:su coat proteins to fold and assemble under a variety of cellular conditions was determined by monitoring levels of phage production. The tsf:su coat proteins were found to more effectively utilize P22 scaffolding protein, an assembly chaperone, as compared with their tsf parents. Phage-infected cells were radioactively labelled to quantify the associations between coat protein variants and folding and assembly chaperones. Phage carrying the tsf:su coat proteins induced more GroEL and GroES, and increased formation of protein:chaperone complexes as compared with their tsf parents. We propose that the su substitutions result in coat proteins that are more assembly competent in vivo because of a chaperone-driven kinetic partitioning between aggregation-prone intermediates and the final assembled state. Through more proficient use of this chaperone network, the su substitutions exhibit a novel means of suppression of a folding defect.