- by Thiou, S., Martin, L., Manousaki, E., Nguyen, M., Mortelecque, J., Heidsieck, L., Cantrelle, F.-X., Blum, D., Buee-Scherrer, V., Buee, L., Landrieu, I., Dupre, E., Danis, C.Liquid-liquid phase separation (LLPS) mediates the formation of biomolecular condensates, which organize cellular processes such as synaptic plasticity and stress response. The neuronal microtubule-associated protein tau undergoes LLPS under specific conditions, regulating synaptic vesicle clustering and microtubule dynamics. In vitro, tau LLPS is induced by cofactors such as polyethylene glycol (PEG) or RNA, mainly via weak multivalent electrostatic interactions. However, the molecular mechanisms governing the formation of tau LLPS, including domain specific contribution, remain unclear. In this study, we used […]
- by Soens, R. W., Anderson, B. J., Lancaster, N. M., Kumar, M., Hansen, J. K., Galmozzi, A., Grant, T., Overmyer, K. A., Coon, J. J.Lysine acetylation has widespread ramifications from genetic regulatory effects to modulation of enzymatic function. With improved acetyl-lysine enrichment technologies and advances in mass spectrometer speed and sensitivity, we present a comprehensive atlas of the mouse acetylome comprising 17,952 unique lysine acetylation sites across 4,340 proteins and 15 tissues. This resource, which nearly doubles the known mouse acetyl-lysine catalog, shows at least 14% of the acetylome is shared across tissues. We focus our investigation on several acetylated proteins, including ribosomal acetylation […]
- by Lamme, T. D., Sanchez Arroyo, I. B., Smit, M. J., Schafer, C. T.Atypical chemokine receptors (ACKRs) indirectly mediate cell migration through chemokine scavenging, which generally requires phosphorylation by G protein-coupled receptor (GPCR) kinases (GRKs) to efficiently control chemokine levels. Despite not coupling G proteins, ACKR4 is preferentially modified by GRK3, a kinase dependent on active G protein subunits for membrane translocation and phosphorylation activity. Here we resolve the underlying mechanisms allowing ACKR4 to circumvent the G protein requirement for GRK3 function. Using live cell BRET assays, we confirm that ACKR4 is preferentially […]
- by Chuesaard, T., Chaimanee, V., Jobsri, J., Pukklay, P.Propolis, a resinous substance produced by Apis mellifera, is rich in polyphenols and flavonoids and exhibits diverse biological activities, including antioxidant and antimicrobial effects. This study aimed to characterize the chemical composition of Thai propolis and evaluate its free radical scavenging activity. Ethanolic (EEP) and aqueous (WEP) extracts were prepared and analyzed. Gas chromatography-mass spectrometry (GC-MS) identified major constituents of EEP, including phenolic acids, flavonoids, and sugars. Antioxidant activities were assessed using DPPH and hydrogen peroxide scavenging assays. EEP exhibited […]
- by Croft, J. T., Do, H. N., Leaman, D. P., Lovendahl, K. N., Ralli-Jain, P., Chase, K. J., Chen, C., Prasad, V. M., Derdeyn, C. A., Zwick, M. B., Gnanakaran, S., Lee, K. K.An effective vaccine for Human Immunodeficiency Virus type-1 (HIV-1) has yet to be developed, and detailed characterization of functional Env glycoprotein, the primary antigenic target on virions, has remained elusive. While engineered Env trimers recapitulate many aspects of functional Env, key differences in antigenicity and dynamic behavior have been reported. Here, cryo-electron tomography and subtomogram averaging of HIV-1 virus-like particles (VLPs) revealed conformational differences in critical membrane-proximal regions compared to soluble Envs. Hydrogen/Deuterium-Exchange Mass Spectrometry and Molecular Dynamics captured dynamic […]
- by Zhou, Z., Saffarian-Deemyad, I., Shi, H., Weiss, T., ur-Rehman, M. M., Vohra, K., Skopintsev, P., Yoon, P. H., Trinidad, M. I., Langeberg, C. J., Kamalu, M., Amerasekera, J., Doherty, E. E., Aris, K. D. P., Al-Sayyad, N., Thornton, B. W., Weissman, R. F., Wasko, K. M., Esain-Garcia, I., DeTurk, E. C., Savage, D. F., Jacobsen, S. E., Bryant, Z., Doudna, J. A.TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through formation of a partially unwound intermediate state to a fully unwound open state. The open state forms inefficiently and collapses readily in the absence […]
- by Reynolds, A. J., Sue, A., MacRenaris, K., O'Halloran, T. V., Qiu, T.Metal homeostasis is a complex process wherein essential metals serving structural, catalytic and regulatory roles are acquired, trafficked, and exported once they are present in excess. Understanding changes in metal content and localization in heterogenous tissue types is critical to understanding fundamental physiology as well as a growing number of disease states. Laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) imaging is a powerful technique for untargeted quantitation and mapping of metals in biological systems. While the nematode Caenorhabditis […]
- by Nguyen, L., Hicks, L., Jodoin, L., Carby, S., Sanford, D. S., Mishra, A., Yadav, R.Human serum albumin (HSA) is the most abundant plasma protein and serves as a major transporter and reservoir for a wide range of endogenous ligands, including fatty acids, heme, bilirubin, prostaglandins, and metal ions, as well as numerous exogenous compounds, such as pharmaceuticals. HSA is a 66 kDa monomeric, multidomain protein containing two principal ligand-binding sites, Sudlow sites I and II. Sudlow site I contains Trp214, the sole tryptophan residue in HSA, whose intrinsic fluorescence provides a sensitive probe for […]
- by Walter, J., Sidl, L., Gutenbrunner, K., Skibinski, D., Kolberg, T., Hofacker, I. L., Yao, H.-T., Mörl, M., Wolfinger, M. T.Mechanically active RNAs represent an emerging class of biomolecules whose function derives from resisting molecular forces. Among them, exoribonuclease-resistant RNAs (xr-RNAs) achieve this by folding into a ring-like topology that physically blocks 5 [->] 3 degra-dation. However, despite years of structural insight, the rational design of such mechanically functional RNA devices has remained elusive. Here, we describe a mechanics-aware RNA design approach that enables de novo engineering of functional xrRNAs. We first identify structural determinants of force resistance by perturbing […]
- by Martinez-Costa, O. H., Ben-Salah, A., Valerio, G. N., Cordas, C. M., Samhan-Arias, A. K.In enzymology, hysteresis is manifested as a time-dependent shift in the kinetic behavior of an enzyme. Through hysteresis, the activation or inhibition of a biological pathway can be regulated by a molecule or metabolite that acts as a hysteretic modulator of the enzyme within that metabolic route. This mechanism of regulation contrasts with those that act on gene expression leading to modulation of enzyme protein levels. Through hysteresis, the amplitude of natural oscillations in metabolic pathways can be adjusted according […]
- by Prucker, I., Milanov, M., Moser, P., Popp, C., Haas, T. M., Qiu, D., Koch, H.-G., Jessen, H. J.To survive stressful conditions, bacteria enter the stringent response mediated by the magic spot nucleotides (MSN) guanosine 35-bispyrophosphate (ppGpp) and guanosine 3-diphosphate 5-triphosphate (pppGpp). Another group of so-called alarmones elevated under stress are dinucleoside polyphosphates (NpnNs), such as diadenosine triphosphate (Ap3A). How these two groups of alarmones intersect with one another is poorly understood. Here, we present a sensitive method using capillary electrophoresis coupled to mass spectrometry (CE-MS) with heavy isotope labeled internal references to separate and quantify magic spot […]
- by Wu, C., Lin, Y., Wang, X., Gong, W., Liu, B., Lu, Y., Tang, F., Yang, M., Lin, G., Zhang, J., Zhang, S.Protein phosphorylation is a critical mechanism in cellular signal transduction, playing essential roles in various cellular processes. However, significant challenges of their low abundance, high dynamic range, and complexity are especially prominent for accurate phosphoproteome analysis in nanoscale samples, such as oocytes, where misregulation of phosphorylation can lead to infertility. In this study, we developed a novel phosphopeptide enrichment tip (Ti-PAN) using highly hydrophilic textile PAN fibers, enabling rapid in-situ one-tip IMAC enrichment in just 8 minutes. This approach not […]
- by Abanti, R. R., Wu, D., Kielkowski, P.Chemical proteomics is a powerful method to track proteins labelled by reactive small molecules in living cells on proteome-wide scale. The strategy relies on reactivity and specificity of bioorthogonal click reactions. Although a variety of bioorthogonal reactions have been developed to facilitate chemical proteomics, their reactivity and specificity might not be comparable with enzymatic reactions. Here we describe an iodoacetamide chloroalkane cysteine reactive probe that is, upon the reaction with nucleophilic cysteine of thioredoxin (TrxA), efficiently and specifically conjugated with […]
- by Earp, J. C., Lichti, N. P., Garaeva, A., Meikle, V., Niederweis, M., Seeger, M. A.Mycobacterium tuberculosis contains thirteen Mycobacterial membrane protein Large (MmpL) transporters, which belong to the family of secondary active RND transporters. MmpL4 and MmpL5, together with their operon partners MmpS4 and MmpS5, export the mycobacterial siderophore mycobactin and the last resort TB drug bedaquiline. Recently, we determined a structure of the MmpL4 monomer in complex with desferrated mycobactin, which lacked a functionally essential coiled-coil domain predicted to extend far into the periplasm. Here, we present a cryo-EM structure of the hexameric […]
- by Karalkeviciute, V., Rapalyte, S., Baronaite, I., Veiveris, D., Smirnovas, V., Ziaunys, M., Sulskis, D.The 14-3-3 protein family, with over 1300 binding partners, is one of the largest regulators of protein-protein interactions (PPIs) in eukaryotic cells. They recognise and bind to phosphorylation-related motifs in their partner proteins, creating scaffolds to stabilise proteins and allowing or inhibiting kinases access to their targets. 14-3-3 consists of seven isoforms ({beta}, {gamma}, {varepsilon}, {zeta}, , {theta} and {sigma}), which are expressed across all tissues. Given their important role in PPIs, their dysregulation can contribute to a variety of […]
- by Collingridge, J., Cole, B. A., Liddle, J., Gatfield, K. M., Biggin, P. C., Becker, E. B. E.The transient receptor channel canonical family member 3 (TRPC3) is a non-selective, calcium-permeable cation channel within the larger TRP superfamily. TRPC3 is expressed in both excitable and non-excitable cells, where it integrates multiple signaling pathways and is directly activated by binding of diacylglycerol following activation of G-protein coupled receptors. Increased TRPC3 signaling is implicated in several diseases, highlighting TRPC3 as a potential therapeutic target. However, the development of TRPC3 inhibitors with suitable potency and selectivity has been challenging due to […]
- by Prins, S., Sternbach, J., Mendelson, J. B., Markowski, T., Higgins, L., Carney, J., Tollison, W., Choudhary, G., Prins, K.Right heart failure is due to both right atrial and right ventricular dysfunction. While the two chambers are distinct, the proteomic response to pressure overload is undefined. Here, we used quantitative proteomics to evaluate changes in both chambers in pulmonary artery banded pigs. We found common alterations in oxidative phosphorylation, ribosome regulation, integrin-mediated adhesion, and glycolysis enzymes. However, the right atrium had more pronounced deficits in the TCA cycle and branched chain amino acid metabolism. Thus, these data highlight metabolic […]
- by Quail, S., Bickers, S., Tymczak, A., Kanelis, V.Many ATP binding cassette (ABC) proteins function in active transport of solutes across biological membranes. At minimum, ABC proteins are composed of two repeats, each consisting of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). In many ABC proteins, the TMD-NBD halves are connected by an intrinsically disordered linker that regulates the activity of the ABC protein through phosphorylation. These regulatory (R) regions are often invisible or at low-resolution in electron cryo-microscopy maps, and thus information about how […]
- by Hay, I. M., Bourguet, M., Ahsan, B., Perisic, O., Anandapadamanaban, M., Williams, R. L.The mammalian target of rapamycin complex 2 (mTORC2) regulates metabolism, growth, survival and cytoskeletal organization, yet its activation mechanism is poorly understood. We show that mTORC2 is directly activated by membranes and our cryo-electron tomography structure of membrane-bound mTORC2 reveals the N-HEAT region of mTOR is at the major membrane interface. mTORC2 is further potently activated by a positive feedback loop involving reciprocal phosphorylation of mTORC2 and its substrate kinase AKT. Cryo-EM structures of dephosphorylated, autophosphorylated and AKT-phosphorylated mTORC2 reveal […]
- by Kotsarenko, K., Hajdusek, O., Rievajova, M., Bezdekova, K., Vechtova, P., Malinovska, L., Paulenova, E., Houser, J., Sterba, J., Grubhoffer, L., Wimmerova, M.Ixodes ricinus ticks are widely distributed throughout Europe and represent major vectors of tick-borne encephalitis virus and the Lyme borreliosis agent Borrelia burgdorferi sensu lato. In invertebrates, C-type lectins are commonly associated with innate immune functions, and several such lectins have been predicted in I. ricinus. Given the limited knowledge of lectin function in ticks, we characterized three carbohydrate-recognition domains (CRDs) of a novel C-type lectin identified in the I. ricinus transcriptome (IrCLec). The tertiary structures of CRD1, CRD2, and […]
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