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Background: Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. Seeding into the complex cytoplasmic milieu happens within hours, implying the existence of unknown factors that regulate this process. Methods: We used proximity labeling to identify proteins that control seed amplification within 5 h of seed exposure. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity 5 h after seeded intracellular tau aggregation. Valosin containing protein (VCP/p97) was the top hit. VCP harbors dominant mutations that underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We used immortalized cells and human neurons to study the effects of VCP on tau seeding. We exposed cells to fibrils or brain homogenates in cell culture media and measured effects on uptake and induction of intracellular tau aggregation following various genetic and pharmacological manipulations of VCP. Results: VCP knockdown reduced tau seeding. Chemical inhibitors had opposing effects on seeding in HEK293T tau biosensor cells and human neurons: ML-240 increased seeding efficiency, whereas NMS-873 decreased it. The inhibitors only functioned when administered within 8 h of seed exposure, indicating a role for VCP early in seed processing. We screened 30 VCP co-factors in HEK293T biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. By contrast, reduction of FAF2 increased tau seeding. Conclusions: Divergent effects on tau seeding of chemical inhibitors and cofactor reduction indicate that VCP regulates this process. This is consistent with a cytoplasmic processing complex centered on VCP that directs seeds acutely towards degradation vs. amplification. [ABSTRACT FROM AUTHOR]

ATTR amyloidosis results from the conversion of transthyretin into amyloid fibrils that deposit in tissues causing organ failure and death. This conversion is facilitated by mutations in ATTRv amyloidosis, or aging in ATTRwt amyloidosis. ATTRv amyloidosis exhibits extreme phenotypic variability, whereas ATTRwt amyloidosis presentation is consistent and predictable. Previously, we found unique structural variabilities in cardiac amyloid fibrils from polyneuropathic ATTRv-I84S patients. In contrast, cardiac fibrils from five genotypically different patients with cardiomyopathy or mixed phenotypes are structurally homogeneous. To understand fibril structure's impact on phenotype, it is necessary to study the fibrils from multiple patients sharing genotype and phenotype. Here we show the cryo-electron microscopy structures of fibrils extracted from four cardiomyopathic ATTRwt amyloidosis patients. Our study confirms that they share identical conformations with minimal structural variability, consistent with their homogenous clinical presentation. Our study contributes to the understanding of ATTR amyloidosis biopathology and calls for further studies. Cryo-EM analysis of fibrils from four cardiomyopathic ATTRwt amyloidosis patients reveals identical structures with minimal variability. This finding contributes to the understanding of ATTR amyloidosis biopathology. [ABSTRACT FROM AUTHOR]

Wnt16 is expressed in bone and arteries, and maintains bone mass in mice and humans, but its role in cardiovascular physiology is unknown. We show that Wnt16 protein accumulates in murine and human vascular smooth muscle (VSM). WNT16 genotypes that convey risk for bone frailty also convey risk for cardiovascular events in the Dallas Heart Study. Murine Wnt16 deficiency, which causes postnatal bone loss, also reduced systolic blood pressure. Electron microscopy demonstrated abnormal VSM mitochondrial morphology in Wnt16-null mice, with reductions in mitochondrial respiration. Following angiotensin-II (AngII) infusion, thoracic ascending aorta (TAA) dilatation was greater in Wnt16 −/− vs Wnt16+/+ mice (LDLR −/− background). Acta2 (vascular smooth muscle alpha actin) deficiency has been shown to impair contractile phenotype and worsen TAA aneurysm with concomitant reductions in blood pressure. Wnt16 deficiency reduced expression of Acta2 , SM22 (transgelin), and other contractile genes, and reduced VSM contraction induced by TGFβ. Acta2 and SM22 proteins were reduced in Wnt16 −/− VSM as was Ankrd1, a prototypic contractile target of Yap1 and Taz activation via TEA domain (TEAD)-directed transcription. Wnt16 −/− VSM exhibited reduced nuclear Taz and Yap1 protein accumulation. SiRNA targeting Wnt16 or Taz, but not Yap1, phenocopied Wnt16 deficiency, and Taz siRNA inhibited contractile gene upregulation by Wnt16. Wnt16 incubation stimulated mitochondrial respiration and contraction (reversed by verteporfin, a Yap/Taz inhibitor). SiRNA targeting Taz inhibitors Ccm2 and Lats1/2 mimicked Wnt16 treatment. Wnt16 stimulated Taz binding to Acta2 chromatin and H3K4me3 methylation. TEAD cognates in the Acta2 promoter conveyed transcriptional responses to Wnt16 and Taz. Wnt16 regulates cardiovascular physiology and VSM contractile phenotype, mediated via Taz signaling. [ABSTRACT FROM AUTHOR]

Density functional theory and blackbody infrared radiative dissociation experiments are used to investigate the structures of lithiated lysine, ornithine and related molecules, both with and without a water molecule. The results have indicated that loss of a water molecule from the lysine complexes is both energetically and entropically favored compared to interconversion between the nonzwitterionic and zwitterionic structures and the comparisons to binding energies of water to the various structures have shown that experimental results are most consistent with the nonzwitterionic forms.

The modes of metal ion and water binding in doubly hydrated complexes of lithiated and sodiated glutamine (Gln) are probed using blackbody infrared radiative dissociation (BIRD) experiments and density functional theory (DFT) calculations. The results show that the doubly hydrated complexes containing sodium are more stable than those containing lithium.

The structures of lithiated and sodiated glutamine, both with and without a water molecule, are investigated using experiment and theory. Addition of water molecule does not affect the lowest-energy structure of lithiated glutamine, and for sodiated glutamine, the lowest-energy zwitterionic and nonzwitterionic structures are essentially isoenergetic.

Theory and experiments are used for examining the structures of valine (Val) and methylaminoisobutyric acid (Maiba) bond to a sodium ion, either with or without a water molecule. The results indicate that the threshold dissociation energy for loss of a water molecule from these salt-bridges is less than that as compared to the loss of water from the charge-solvated complexes.

The mode of metal ion and water binding to the amino acid valine is investigated. The results indicate that metal ion in the lithiated and sodiated clusters is NO-coordinated to nonzwitterionic valine and in potassiated cluster has OO coordination.

The intrinsic effect of divalent alkaline earth metal ions (Be, Mg, Ca, Sr, Ba) on the structure of glycine in the absence of solvent is examined. In the presence of larger multivalent ions, stable zwitterions and salt-bridge structures play an important role in the gas-phase structure and chemistry of these ions.

Reactive oxygen species (ROS) are by-products of cellular respiration that can promote oxidative stress and damage cellular proteins and lipids. One canonical role of ROS is to defend the cell against invading bacterial and viral pathogens. Curiously, some viruses, including herpesviruses, thrive despite the induction of ROS, suggesting that ROS are beneficial for the virus. However, the underlying mechanisms remain unclear. Here, we found that ROS impaired interferon response during murine herpesvirus infection and that the inhibition occurred downstream of cytoplasmic DNA sensing. We further demonstrated that ROS suppressed the type I interferon response by oxidizing Cysteine 147 on murine stimulator of interferon genes (STING), an ER-associated protein that mediates interferon response after cytoplasmic DNA sensing. This inhibited STING polymerization and activation of downstream signaling events. These data indicate that redox regulation of Cysteine 147 of mouse STING, which is equivalent to Cysteine 148 of human STING, controls interferon production. Together, our findings reveal that ROS orchestrates anti-viral immune responses, which can be exploited by viruses to evade cellular defenses. [ABSTRACT FROM AUTHOR]

To explore the role of molecular structure in collisions that relax highly excited polyatomic molecules, we have studied collisional deactivation of a series of highly vibrationally excited methylated pyridines (E[sub vib] ∼ 38 500 cm[sup -1]) in a CO[sub 2] bath. Complementary studies that investigated quenching by H[sub 2]O have been presented in Part I of this series [M. S. Elioff, M. Fang, and A. S. Mullin, J. Chem. Phys. 115, 6990 (2001)]. We have used high-resolution transient infrared absorption probing to measure rotational and translational energy gain in individual quantum levels of CO[sub 2] (00[sup 0]0) that are populated via collisions with vibrationally excited picoline (2-methylpyridine) and lutidine (2,6-dimethylpyridine). Vibrationally excited picoline and lutidine were prepared by absorption of pulsed λ = 266 nm light and fast internal decay to the ground electronic state. The nascent distribution of CO[sub 2] (00[sub 0]0) rotational states was measured for Or =60-78. Translational energy gain distributions were determined for the J=60-80 states of CO[sub 2] (00[sup 0]0) using Doppler-broadened linewidth measurements. Energy transfer probabilities were determined by measuring absolute energy transfer rate constants for energy gain into specific CO[sub 2] quantum states. These results are compared to previous single-collision energy transfer studies on hot pyridine [M. C. Wall, B. Stewart, and A. S. Mullin, J. Chem. Phys. 108, 9658 (1998)] and hot pyrazine [M. C. Wall and A. S. Mullin, J. Chem. Phys. 108, 9658 (1998)] initially excited with 266 nm light and quenched via collisions with CO[sub 2]. We find that donor methylation reduces the amount of translational and rotational energy imparted to the CO[sub 2] (00[sup 0]0) high-or states, but that the cross section for exciting the high-or states of CO[sub 2] (00[sup 0]0) increases upon donor methylation. Fermi's golden rule is used to describe the relaxation process, and the energy... [ABSTRACT FROM AUTHOR]

An investigation of the structures of lithiated and sodiated alpha-methyl-proline (alpha-Me-Pro) and structural isomers, both with and without a water molecule is conducted using blackbody infrared radiative dissociation (BIRD) and density functional theory. The proton affinity of proline is 8kJ/mol higher than that of alpha-Me-Pro, indicating that lithiated and sodiated singly hydrated proline should also be zwitterionic.

Investigates the energy dependence of collisional relaxation of highly vibrationally excited pyrazine through collisions with carbon dioxide for pyrazine vibrational energies. Energy-dependent transient absorption measurements; Energy-dependent translational energy gain CO[sub 2]; Energy-dependent distributions of CO[sub 2] high rotational states.

Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy. [ABSTRACT FROM AUTHOR]

Polyamines are essential for cell growth of eukaryotes including the etiologic agent of human African trypanosomiasis (HAT), Trypanosoma brucei. In trypanosomatids, a key enzyme in the polyamine biosynthetic pathway, S-adenosylmethionine decarboxylase (TbAdoMetDC) heterodimerizes with a unique catalytically-dead paralog called prozyme to form the active enzyme complex. In higher eukaryotes, polyamine metabolism is subject to tight feedback regulation by spermidine-dependent mechanisms that are absent in trypanosomatids. Instead, in T. brucei an alternative regulatory strategy based on TbAdoMetDC prozyme has evolved. We previously demonstrated that prozyme protein levels increase in response to loss of TbAdoMetDC activity. Herein, we show that prozyme levels are under translational control by monitoring incorporation of deuterated leucine into nascent prozyme protein. We furthermore identify pathway factors that regulate prozyme mRNA translation. We find evidence for a regulatory feedback mechanism in which TbAdoMetDC protein and decarboxylated AdoMet (dcAdoMet) act as suppressors of prozyme translation. In TbAdoMetDC null cells expressing the human AdoMetDC enzyme, prozyme levels are constitutively upregulated. Wild-type prozyme levels are restored by complementation with either TbAdoMetDC or an active site mutant, suggesting that TbAdoMetDC possesses an enzyme activity-independent function that inhibits prozyme translation. Depletion of dcAdoMet pools by three independent strategies: inhibition/knockdown of TbAdoMetDC, knockdown of AdoMet synthase, or methionine starvation, each cause prozyme upregulation, providing independent evidence that dcAdoMet functions as a metabolic signal for regulation of the polyamine pathway in T. brucei. These findings highlight a potential regulatory paradigm employing enzymes and pseudoenzymes that may have broad implications in biology. [ABSTRACT FROM AUTHOR]

Background: Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. In vitro seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu happens within hours, implicating a machinery with unknown players that controls this process in the acute phase., Methods: We used proximity labeling to identify factors that control seed amplification within 5h of seed exposure. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity 5h after seeded intracellular tau aggregation. Valosin containing protein (VCP/p97) was the top hit. VCP harbors dominant mutations that underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We used immortalized cells and human neurons to study the effects of VCP on tau seeding. We exposed cells to fibrils or brain homogenates in cell culture media and measured effects on uptake and induction of intracellular tau aggregation following various genetic and chemical manipulations of VCP., Results: VCP knockdown reduced tau seeding. Chemical inhibitors had opposing effects on aggregation in HEK293T tau biosensor cells and human neurons alike: ML-240 increased seeding efficiency, whereas NMS-873 decreased it. The inhibitors were effective only when administered within 8h of seed exposure, indicating a role for VCP early in seed processing. We screened 30 VCP co-factors in HEK293T biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. By contrast, reduction of FAF2 increased tau seeding., Conclusions: Divergent effects on tau seeding of chemical inhibitors and cofactor reduction indicate that VCP regulates this process. This is consistent with a dedicated cytoplasmic processing complex based on VCP that directs seeds acutely towards degradation vs. amplification., Competing Interests: Competing interests The authors declare no competing interests.