Glu Mutation Research Articles

Phosphorylation of DNA-binding domains of CLOCK–BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including Period (Per). Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.

Structures of AT8 and PHF1 phosphomimetic tau: Insights into the posttranslational modification code of tau aggregation

The microtubule-associated protein tau aggregates into amyloid fibrils in Alzheimer's disease and other neurodegenerative diseases. In these tauopathies, tau is hyperphosphorylated, suggesting that this posttranslational modification (PTM) may induce tau aggregation. Tau is also phosphorylated in normal developing brains. To investigate how tau phosphorylation induces amyloid fibrils, here we report the atomic structures of two phosphomimetic full-length tau fibrils assembled without anionic cofactors. We mutated key Ser and Thr residues to Glu in two regions of the protein. One construct contains three Glu mutations at the epitope of the anti-phospho-tau antibody AT8 (AT8-3E tau), whereas the other construct contains four Glu mutations at the epitope of the antibody PHF1 (PHF1-4E tau). Solid-state NMR data show that both phosphomimetic tau mutants form homogeneous fibrils with a single set of chemical shifts. The AT8-3E tau rigid core extends from the R3 repeat to the C terminus, whereas the PHF1-4E tau rigid core spans R2, R3, and R4 repeats. Cryoelectron microscopy data show that AT8-3E tau forms a triangular multi-layered core, whereas PHF1-4E tau forms a triple-stranded core. Interestingly, a construct combining all seven Glu mutations exhibits the same conformation as PHF1-4E tau. Scalar-coupled NMR data additionally reveal the dynamics and shape of the fuzzy coat surrounding the rigid cores. These results demonstrate that specific PTMs induce structurally specific tau aggregates, and the phosphorylation code of tau contains redundancy.

The Psb27 Assembly Factor and Histidine‐252 of the D1 Protein Modify Energy Transfer to Photosystem II in Synechocystis sp. PCC 6803

AbstractPhotosystem II (PS II) is responsible for light‐driven water splitting in oxygenic photosynthesis. The Psb27 protein, an assembly factor required for biogenesis of PS II, is found associated with hydrophilic regions of the CP43 core antenna protein in the thylakoid lumen. CP43 and the D1 reaction center protein provide ligands for the Mn4CaO5 oxygen‐evolving complex (OEC). Release of Psb27 coincides with conformational changes that enable successful light‐driven assembly of the OEC. This stage in biogenesis also requires changes to allow electron transfer between plastoquinone electron acceptors on the opposite side of the membrane. We have introduced charge‐swap mutations to target the binding of Psb27 to CP43 during assembly. Here, we show that perturbation of the Psb27‐CP43 interaction results in elevated fluorescence, indicating enhanced energy transfer to PS II in fully assembled complexes. In a Psb27:Arg78 to Glu mutant, D1:His252 spontaneously mutated to Gln. D1:His252 is in the DE loop that contributes to quinone binding and protonation. Mutations targeting D1:His252 produced mutants with elevated PS II‐specific fluorescence that exceeded that observed in our Psb27 mutants and this was attenuated when the Psb27 charge‐swap mutations were introduced into H252Q cells. Perturbation of Psb27 binding to CP43 therefore modified structural changes on the opposite side of the membrane resulting from mutation of D1:His252. The peripheral phycobilisome antenna is lost during thylakoid isolation and thylakoids from our mutants did not display the increased PS II‐specific fluorescence. Hence Psb27 binding to CP43 during photoassembly of the OEC can modify phycobilisome‐dependent energy transfer into PS II.

Exploring the Sequence Space of Antimicrobial Peptide Dendrimers

AbstractThere is an urgent need to develop new antibacterial agents against multidrug resistant bacteria. Herein we report our investigation of antimicrobial peptide dendrimers (AMPDs) active against Gram‐negative bacteria, whose sequences were designed using a genetic algorithm optimizing molecular similarity to the previously reported AMPD T7 with sequence (KL)8(KKL)4(KKLL)2KKKL. Our computational approach selected analogues unlikely to emerge from a systematic study, including AMPD X66 with a non‐conservative Leu→Glu mutation at the dendrimer core which proved compatible with antibacterial effects. Circular dichroism showed that this AMPD is α‐helical. Molecular dynamics suggest that its α‐helical structure is stabilized by an intramolecular salt bridge involving the core glutamate side chain and a lysine side chain in the dendrimer branches. More substantial variations at the dendrimer core were also tolerated such as the installation of the dianionic pegylated fatty acid side chain of the drug semaglutide potentially useful for in vivo studies.

A Conserved Second Sphere Residue Tunes Copper Site Reactivity in Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H2O2 splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.

The β-myosin G256E mutation impairs relaxation by altering transducer stiffness.

Associations between mutations in sarcomeric proteins and familial cardiomyopathies are myriad. However, detailed structural and functional studies are required to describe the mechanistic relationship between a mutation and its pathology. To accelerate such studies, we utilize a combined computational and experimental platform to probe how mutations to b-myosin lead to familial cardiomyopathies. Here, we report results on the HCN-associated MYH7 mutation G256E, which is located in the myosin transducer. Experimentally, the specific force generation of mutant G256E myofibrils was ∼2x greater than isogenic control myofibrils. The activation rate was similarly increased. The initial slow phase relaxation kinetics of G256E myosin were significantly reduced relative to isogenic controls, indicating a slower cross-bridge detachment rate. Using stopped flow kinetics, the ATP binding rate was reduced in G256E relative to WT. Molecular dynamics simulations of the rigor and myosin.ATP states showed that G256E led to reorganization of hydrogen bonding networks within the transducer region. Enhanced sampling MD simulations modeled the rigor (nucleotide free) to post-rigor (ATP-bound) structural transition along multiple reaction coordinates. The transition required that G256E myosins surmount a greater energetic barrier that describes the binding of switch 1 to ATP, rotation of the transducer backbone, and opening of the actin binding cleft. This increased barrier is attributed to salt bridges formed by the mutant Glu residue. Based on our combined computational and experimental results, we hypothesize that G256E leads to a hypercontractile and delayed relaxation phenotype via modulation of the transducer structure. This alters communication between the nucleotide binding pocket and the actin binding surface during steps in the chemo-mechanical cycle that involve transducer motion during nucleotide release-induced strong actin binding and ATP binding-induced release from actin. Other effects on myosin structure and its activation during contraction are being investigated.

MYH7 G256E induced changes in myosin biomechanics influence function of myofibril contractile organelles.

We studied the contractile properties of the MYH7 G256E mutation using engineered human stem cell derived cardiomyocytes (hSC-CMs). When hSC-CMs were cultured to 40-45 days post-differentiation and Western blot analysis demonstrated near 100% β-myosin (MYH7). Single sub-cellular contractile organelles (myofibrils) were mounted in a custom built apparatus between a force transducer and linear motor. Rapid solutions switching to maximal activation solution (pCa 4.0) resulted in a doubling of steady-state force and rate of force development for G256E myofibrils compared with wild type (WT) controls. Upon switching to relaxation solution (pCa 9.0), the slope of the initial linear, slow phase relaxation was significantly slower (∼4-fold) for G256E vs WT myofibrils, suggesting the mutation slows the crossbridge detachment rate in loaded contractions. Crossbridge detachment rate can be reduced by slower ADP release or slower ATP binding to myosin. Analysis using stopped flow kinetics showed the ATP binding rate was slightly, but significantly reduced for G256E vs WT myofibrils. Preliminary data from myofibril mechanical analysis suggests that ADP release is also slower with the G256E mutation. Molecular dynamics (MD) simulations of the rigor and myosin.ATP states showed that G256E led to reorganization of hydrogen bonding networks within the transducer region. Enhanced sampling MD simulations of the rigor to post-rigor (ATP-bound) structural transition indicated G256E myosin surmounts a greater energetic barrier describing the binding of switch 1 to ATP, rotation of the transducer backbone, and opening of the actin binding cleft. This increased barrier is attributed to salt bridges formed by the mutant Glu residue. Combined, our results suggest that G256E leads to hypercontraction and delayed relaxation phenotype via modulation of the transducer structure that alters communication between the nucleotide binding pocket and the actin binding surface during the chemo-mechanical cycle.

Reversible Assembly of an Artificial Protein Nanocage Using Alkaline Earth Metal Ions

Protein nanocages are of increasing interest for use as drug capsules, but the encapsulation and release of drug molecules at appropriate times require the reversible association and dissociation of the nanocages. One promising approach to addressing this challenge is the design of metal-dependent associating proteins. Such designed proteins typically have Cys or His residues at the protein surface for connecting the associating proteins through metal-ion coordination. However, Cys and His residues favor interactions with soft and borderline metal ions, such as Au+ and Zn2+, classified by the hard and soft acids and bases concept, restricting the types of metal ions available to drive association. Here, we show the alkaline earth (AE) metal-dependent association of the recently designed artificial protein nanocage TIP60, which is composed of 60-mer fusion proteins. The introduction of a Glu (hard base) mutation to the fusion protein (K67E mutant) prevented the formation of the 60-mer but formed the expected cage structure in the presence of Ca, Sr, or Ba ions (hard acids). Cryogenic electron microscopy (cryo-EM) analysis indicated a Ba ion at the interface of the subunits. Furthermore, we demonstrated the encapsulation and release of single-stranded DNA molecules using this system. Our results provide insights into the design of AE metal-dependent association and dissociation mechanisms for proteins.

PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the CORB GTPase domain.

Leucine-rich-repeat-kinase 1 (LRRK1) and its homolog LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause Parkinson's disease. Previous work suggested that LRRK1 but not LRRK2, is activated via a Protein Kinase C (PKC)-dependent mechanism. Here we demonstrate that phosphorylation and activation of LRRK1 in HEK293 cells is blocked by PKC inhibitors including LXS-196 (Darovasertib), a compound that has entered clinical trials. We show multiple PKC isoforms phosphorylate and activate recombinant LRRK1 in a manner reversed by phosphatase treatment. PKCα unexpectedly does not activate LRRK1 by phosphorylating the kinase domain, but instead phosphorylates a cluster of conserved residues (Ser1064, Ser1074 and Thr1075) located within a region of the CORB domain of the GTPase domain. These residues are positioned at the equivalent region of the LRRK2 DK helix reported to stabilize the kinase domain αC-helix in the active conformation. Thr1075 represents an optimal PKC site phosphorylation motif and its mutation to Ala, blocked PKC-mediated activation of LRRK1. A triple Glu mutation of Ser1064/Ser1074/Thr1075 to mimic phosphorylation, enhanced LRRK1 kinase activity ∼3-fold. From analysis of available structures, we postulate that phosphorylation of Ser1064, Ser1074 and Thr1075 activates LRRK1 by promoting interaction and stabilization of the αC-helix on the kinase domain. This study provides new fundamental insights into the mechanism controlling LRRK1 activity and reveals a novel unexpected activation mechanism.

Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c.

A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial membrane can be induced by the peroxidase activity of cytochrome c in a complex with cardiolipin. Using a number of mutant variants of cytochrome c, we showed that both substitutions of Lys residues from the universal binding site for oppositely charged Glu residues and mutations leading to a decrease in the conformational mobility of the red Ω-loop in almost all cases did not affect the ability of cytochrome c to bind to cardiolipin. At the same time, the peroxidase activity of all mutant variants in a complex with cardiolipin was three to five times higher than that of the wild type. A pronounced increase in the ability to permeabilize the lipid membrane in the presence of hydrogen peroxide, as measured by calcein leakage from liposomes, was observed only in the case of four substitutions in the red Ω-loop (M4 mutant). According to resonance and surface-enhanced Raman spectroscopy, the mutations caused significant changes in the heme of oxidized cytochrome c molecules resulting in an increased probability of the plane heme conformation and the enhancement of the rigidity of the protein surrounding the heme. The binding of wild-type and mutant forms of oxidized cytochrome c to cardiolipin-containing liposomes caused the disordering of the acyl lipid chains that was more pronounced for the M4 mutant. Our findings indicate that the Ω-loop is important for the pore formation in cardiolipin-containing membranes.

Elongation of Fibrils Formed by a Tau Fragment is Inhibited by a Transient Dimeric Intermediate.

The formation and propagation of aggregates of the tau protein in the brain are associated with the tauopathy group of neurodegenerative diseases. Different tauopathies have been shown to be associated with structurally distinct aggregates of tau. However, the mechanism by which different structural folds arise remains poorly understood. In this study of fibril formation by the fragment tau-K18 of tau, it is shown that the Lys 280 → Glu mutation in the variant tau-K18 K280E forms fibrils that are morphologically distinct from those formed by wild-type (wt) tau-K18. The mutant fibrils appear to have two protofilaments twisted around each other, whereas the wt fibrils are straight and appear to have a single protofilament. Modeling the kinetics of seeded aggregation, using a simple Michaelis-Menten-like mechanism, reveals that the two morphologically distinct fibrils are elongated with different catalytic efficiencies. Surprisingly, when the elongation of monomeric tau-K18 is seeded with tau-K18 K280E fibrils, it is seen to be inhibited at high monomer concentrations. Such inhibition is not seen when elongation is seeded with tau-K18 fibrils. The mechanism of inhibition is shown to be describable as uncompetitive inhibition, in which a transient dimeric form of tau-K18 acts as an uncompetitive inhibitor. Importantly, a dimeric form of tau-K18 is seen to be populated to a detectable extent early during aggregation. A covalently linked tau dimer, with an inter-molecular disulphide linkage, is shown to be capable of acting as an inhibitor. In summary, a quantitative kinetic approach has provided an understanding of how the formation of distinct structural folds of tau fibrils can be modulated by mutation and how the elongation of one fibril type, but not the other, is inhibited by a transiently formed dimer.

Oncohistone Mutations Occur at Functional Sites of Regulatory ADP-Ribosylation.

This study identifies cancer-driving mutations in histones as sites of PARP1-mediated ADP-ribosylation in breast and ovarian cancers, providing a molecular pathway by which cancers may subvert the growth-regulating effects of PARP1.

Dermal Fibroblast Migration and Proliferation Upon Wounding or Lipopolysaccharide Exposure is Mediated by Stathmin.

The dermal fibroblast is a crucial executor involved in wound healing, and lipopolysaccharide is a key factor in initiating the migration and proliferation of the dermal fibroblasts, followed by wound healing. However, the underlying molecular mechanism is still unknown. In this study, we demonstrated that stathmin increased concomitantly with p38/MAPK pathway activation by lipopolysaccharide stimulation of the human dermal fibroblast (HDF), which induced microtubule (MT) depolymerization followed by increased HDF migration and proliferation. In contrast, the application of taxol, the small interfering RNA transfection of stathmin, or the application of the p38/MAPK inhibitor SB203580 suppressed MT depolymerization and HDF migration and proliferation. Additionally, the overexpression of a MKK6(Glu) mutant, which constitutively activated p38/MAPK, resulted in MT depolymerization and, subsequently, promoted HDF migration and proliferation. Our data reveal a crucial role of stathmin in HDF migration and proliferation. These findings will provide new targets and strategies for clinical interventions in wound healing.

Akt Phosphorylation of Hepatitis C Virus NS5B Regulates Polymerase Activity and Hepatitis C Virus Infection.

Hepatitis C virus (HCV) is a single-stranded RNA virus of positive polarity [ssRNA(+)] that replicates its genome through the activity of one of its proteins, called NS5B. This viral protein is responsible for copying the positive-polarity RNA genome into a negative-polarity RNA strand, which will be the template for new positive-polarity RNA genomes. The NS5B protein is phosphorylated by cellular kinases, including Akt. In this work, we have identified several amino acids of NS5B that are phosphorylated by Akt, with positions S27, T53, T267, and S282 giving the most robust results. Site-directed mutagenesis of these residues to mimic (Glu mutants) or prevent (Ala mutants) their phosphorylation resulted in a reduced NS5B in vitro RNA polymerase activity, except for the T267E mutant, the only non-conserved position of all those that are phosphorylated. In addition, in vitro transcribed RNAs derived from HCV complete infectious clones carrying mutations T53E/A and S282E/A were transfected in Huh-7.5 permissive cells, and supernatant viral titers were measured at 6 and 15 days post-transfection. No virus was rescued from the mutants except for T53A at 15 days post-transfection whose viral titer was statistically lower as compared to the wild type. Therefore, phosphorylation of NS5B by cellular kinases is a mechanism of viral polymerase inactivation. Whether this inactivation is a consequence of interaction with cellular kinases or a way to generate inactive NS5B that may have other functions are questions that need further experimental work.

Amino Acids 785, 787 of the Na+/H+ Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser785 and Ser787, that were similar in location and context to two amino acids of the Arabidopsis Na+/H+ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783–790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.

Probing the molecular basis for signal transduction through the Zinc-Activated Channel (ZAC)

The molecular basis for the signal transduction through the classical Cys-loop receptors (CLRs) has been delineated in great detail. The Zinc-Activated Channel (ZAC) constitutes a so far poorly elucidated fifth branch of the CLR superfamily, and in this study we explore the molecular mechanisms underlying ZAC signaling in Xenopus oocytes by two-electrode voltage clamp electrophysiology. In studies of chimeric receptors fusing either the extracellular domain (ECD) or the transmembrane/intracellular domain (TMD-ICD) of ZAC with the complementary domains of 5-HT3A serotonin or α1 glycine receptors, serotonin and Zn2+/H+ evoked robust concentration-dependent currents in 5-HT3A/ZAC- and ZAC/α1-Gly-expressing oocytes, respectively, suggesting that Zn2+ and protons activate ZAC predominantly through its ECD. The molecular basis for Zn2+-mediated ZAC signaling was probed further by introduction of mutations of His, Cys, Glu and Asp residues in this domain, but as none of the mutants tested displayed substantially impaired Zn2+ functionality compared to wild-type ZAC, the location of the putative Zn2+ binding site(s) in the ECD was not identified. Finally, the functional importance of Leu246 (Leu9′) in the transmembrane M2 α-helix of ZAC was investigated by Ala, Val, Ile and Thr substitutions. In concordance with findings for this highly conserved residue in classical CLRs, the ZACL9′X mutants exhibited left-shifted agonist concentration-response relationships, markedly higher degrees of spontaneous activity and slower desensitization kinetics compared to wild-type ZAC. In conclusion, while ZAC is an atypical CLR in terms of its (identified) agonists and channel characteristics, its signal transduction seems to undergo similar conformational transitions as those in the classical CLR.

Discovery of SARS-CoV-2-E channel inhibitors as antiviral candidates

Lack of efficiency has been a major problem shared by all currently developed anti-SARS-CoV-2 therapies. Our previous study shows that SARS-CoV-2 structural envelope (2-E) protein forms a type of cation channel, and heterogeneously expression of 2-E channels causes host cell death. In this study we developed a cell-based high throughput screening (HTS) assay and used it to discover inhibitors against 2-E channels. Among 4376 compounds tested, 34 hits with cell protection activity were found. Followed by an anti-viral analysis, 15 compounds which could inhibit SARS-CoV-2 replication were identified. In electrophysiological experiments, three representatives showing inhibitory effect on 2-E channels were chosen for further characterization. Among them, proanthocyanidins directly bound to 2-E channel with binding affinity (KD) of 22.14 μM in surface plasmon resonance assay. Molecular modeling and docking analysis revealed that proanthocyanidins inserted into the pore of 2-E N-terminal vestibule acting as a channel blocker. Consistently, mutations of Glu 8 and Asn 15, two residues lining the proposed binding pocket, abolished the inhibitory effects of proanthocyanidins. The natural product proanthocyanidins are widely used as cosmetic, suggesting a potential of proanthocyanidins as disinfectant for external use. This study further demonstrates that 2-E channel is an effective antiviral drug target and provides a potential antiviral candidate against SARS-CoV-2.

Small Molecule Activators of Protein Phosphatase 2A (SMAPs) Cause c-Myc Degradation and Tumor Growth Suppression in Preclinical Models of Lymphoid Malignancies

Introduction: c-myc is an oncogene implicated in the initiation and progression of multiple hematologic malignancies. Characteristic translocations of the c-myc gene with the immunoglobulin heavy chain gene (in Burkitt lymphoma and “double-hit” non-Hodgkin lymphoma) and with the T cell receptor gene (in T cell acute lymphoblastic leukemia, T-ALL) lead to constitutive expression of the c-myc transcription factor, ultimately driving dysregulated proliferation (Boxer & Dang, Oncogene 2001; 20(40):5595-5610). Strategies for inhibition of c-myc remain an area of active research; however, to date there are no approved myc-targeted agents within the cancer armamentarium, and cytotoxic chemotherapy remains the standard of care for c-myc-driven lymphoid malignancies. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase tumor suppressor protein that contributes to c-myc degradation through both direct dephosphorylation of an identified phosphodegron as well as by indirect mechanisms (Farrell & Sears, Cold Spring Harbor Perspect Med 2014; 4(3):1-15). Our laboratory has developed a series of first in class, orally bioavailable small molecule activators of PP2A (SMAPs) by derivatizing tricyclic antipsychotics to maximize antiproliferative activity and minimize psychotropic properties (Sangodkar et al., J Clin Invest 2017;127(6):2081-2090). We hypothesized that activation of PP2A with SMAPs would lead to c-myc degradation and tumor growth inhibition in models of c-myc-driven lymphoid malignancies.Methods: We investigated the effects of SMAP treatment on c-myc content, c-myc half-life (t1/2), cell viability, and xenograft tumor growth in Burkitt lymphoma (BL) and T-ALL cell lines with wild type and mutant c-myc phosphodegrons.Results: SMAP treatment induced apoptosis in Daudi (BL) cells, as evidenced by decreased viability and increased PARP and caspase cleavage. In Daudi (BL) and CCRF-CEM (T-ALL) cell lines, SMAP treatment led to rapid loss of phosphorylated and total c-myc protein. Cycloheximide (CHX)-chase experiments performed in CCRF-CEM cells confirmed that SMAP treatment affects c-myc degradation without affecting protein synthesis. This effect was abrogated by Ser to Glu mutation of residue 62 within the c-myc phosphodegron, suggesting that SMAP-mediated c-myc degradation is PP2A-dependent. Treatment of NOD/SCID mice bearing Daudi xenografts with twice daily oral SMAP decreased tumor c-myc content and inhibited tumor growth without obvious toxicity. Xenografts overexpressing c-myc with phosphodegron mutations (S62D, T58I) were resistant to SMAP treatment.Conclusions: These in vitro and in vivo observations establish PP2A activation as a potential pharmacologic strategy to target c-myc in cancers such as BL, T-ALL, and “double hit” non-Hodgkin lymphoma. SMAPs are well-tolerated, orally bioavailable activators of PP2A poised for further exploration in the treatment of c-myc-driven malignancies, either alone or in combination with other agents. Sequencing of the c-myc phosphodegron prior to initiation of treatment may facilitate patient selection in future clinical trials of PP2A activators. [Display omitted] DisclosuresNarla:Mount Sinai School of Medicine: Patents & Royalties: patents on the small molecule PP2A activators (GN).

Akt Kinase Intervenes in Flavivirus Replication by Interacting with Viral Protein NS5.

Arthropod-borne flaviviruses, such as Zika virus (ZIKV), Usutu virus (USUV), and West Nile virus (WNV), are a growing cause of human illness and death around the world. Presently, no licensed antivirals to control them are available and, therefore, search for broad-spectrum antivirals, including host-directed compounds, is essential. The PI3K/Akt pathway controls essential cellular functions involved in cell metabolism and proliferation. Moreover, Akt has been found to participate in modulating replication in different viruses including the flaviviruses. In this work we studied the interaction of flavivirus NS5 polymerases with the cellular kinase Akt. In vitro NS5 phosphorylation experiments with Akt showed that flavivirus NS5 polymerases are phosphorylated and co-immunoprecipitate by Akt. Polymerase activity assays of Ala- and Glu-generated mutants for the Akt-phosphorylated residues also indicate that Glu mutants of ZIKV and USUV NS5s present a reduced primer-extension activity that was not observed in WNV mutants. Furthermore, treatment with Akt inhibitors (MK-2206, honokiol and ipatasertib) reduced USUV and ZIKV titers in cell culture but, except for honokiol, not WNV. All these findings suggest an important role for Akt in flavivirus replication although with specific differences among viruses and encourage further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral potential target.

The mechanism of activation of monomeric B-Raf V600E

Oncogenic mutations in the serine/threonine kinase B-Raf, particularly the V600E mutation, are frequent in cancer, making it a major drug target. Although much is known about B-Raf’s active and inactive states, questions remain about the mechanism by which the protein changes between these two states. Here, we utilize molecular dynamics to investigate both wild-type and V600E B-Raf to gain mechanistic insights into the impact of the Val to Glu mutation. The results show that the wild-type and mutant follow similar activation pathways involving an extension of the activation loop and an inward motion of the αC-helix. The V600E mutation, however, destabilizes the inactive state by disrupting hydrophobic interactions present in the wild-type structure while the active state is stabilized through the formation of a salt bridge between Glu600 and Lys507. Additionally, when the activation loop is extended, the αC-helix is able to move between an inward and outward orientation as long as the DFG motif adopts a specific orientation. In that orientation Phe595 rotates away from the αC-helix, allowing the formation of a salt bridge between Lys483 and Glu501. These mechanistic insights have implications for the development of new Raf inhibitors.