- by Kwarteng, E. O., Li, Y., Nguyen, D. L., Agathocleous, M.Carbohydrates are classically catabolized by fermentation or oxidation, a choice that impacts many cellular functions including proliferation. Proliferating cells including somatic stem and progenitor cells are thought to favor fermentation over oxidation, and most proliferating cells in vitro depend on lactate production. However, it has not been tested if fermentation and oxidation are the universal obligatory terminal fates for carbohydrates in vivo because the key enzymes, lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH), have not been simultaneously deleted in any […]
- by Lee, A. A., Mokhtari, D. A., Egan, E. D., Blacklow, S. C., Herschlag, D., Fordyce, P. M.Interpreting clinical and functional consequences of genetic variants remains challenging due to limited quantitative biochemical data at scale. We applied high-throughput microfluidic enzyme kinetics to profile 190 clinical variants of SHP2, a protein tyrosine phosphatase linked to developmental disorders and cancers. Through >300,000 reaction progress curves, we derived kinetic and thermodynamic parameters quantifying variant effects on catalysis, autoinhibition, stability, phosphopeptide binding, and drug responses. This multidimensional dataset reveals that dysregulated autoinhibition, rather than altered stability or catalysis, predominantly determines SHP2-associated […]
- by Gaurav, K., Baltz, L., Paez-Moscoso, D. J., Ketting, R. F., Stelzl, L. S.Phase separation of proteins gives rise to biomolecular condensates, which function as membraneless organelles that spatially and temporally organize cellular functions. Such condensates are often formed by intrinsically disordered regions of proteins (IDRs), whose multivalent and transient interactions govern condensate structure and dynamics. However, elucidating the molecular determinants of these interactions at atomistic resolution remains challenging. Here, we present a total of 10~s of atomistic molecular dynamics simulations of a phase-separated condensate formed by the foci-forming region (FFR) of MUT-16. […]
- by Banerjee, S., Curwen, D., Panwar, A. S., Martin, L.Antimicrobial peptides (AMPs) that also form functional amyloids exhibit remarkable environmental sensitivity, yet the physicochemical rules governing their structural switching remain unresolved. Here, we investigate how surfactant charge and assembly dynamics regulate the antimicrobial amyloidogenic transition of Uperin 3.5, a 17 residue amphibian AMP with pronounced conformational plasticity. Using an integrated approach combining all atom molecular dynamics simulations with circular dichroism and thioflavin T fluorescence assays, we systematically probe the effects of surfactant identity, concentration relative to the critical micelle […]
- by Furuike, Y., Akiyama, S.A primitive form of clock protein KaiC has diverged into autonomous or passive time-measuring system in prokaryotes under selective pressures of day-night environmental changes caused by the rotation of Earth. However, the timing of such functional diversification and its structural basis remain unknown. Here we traced molecular shape evolution of older KaiCs by using X-ray solution scattering and structure prediction techniques. The result shows that the oldest ancestral KaiC emerged approximately 3.1 billion years ago as a moderately expanded and […]
- by Fukui, K., Shibuya, A., Murakawa, T., Yano, T.GHKL ATPases share a unique Bergerat ATP-binding fold and regulate diverse biological processes through ATP-dependent conformational changes. An early step of ATP hydrolysis in this family has been attributed to a single highly conserved glutamate residue proposed to function as the general base. However, mutations of this residue impair both the ATPase activity and ATP binding, complicating interpretation of its catalytic role. Re-examination of the high-resolution crystal structures revealed a second conserved acidic residue positioned within a hydrogen-bonding distance from […]
- by Bai, J., Li, J., Zhang, Y., Chang, H., Zhang, N., Liu, Y., Cheng, J., Liu, X., Jiang, H.Paclitaxel biosynthesis is limited by the instability of taxadiene-4(5)-epoxide, which readily diverts to the non-productive byproduct 5(12)-oxa-3(11)-cyclotaxane (OCT) instead of rearranging to taxadiene-5-ol. Although FoTO1 suppresses OCT accumulation, its molecular function has been unclear. Here we identify FoTO1 as a dedicated epoxide isomerase that directs productive rearrangement. Biochemical characterization, site-directed mutagenesis, and QM/MM calculations reveal a pre-organized D68-D149 dyad that electrostatically activates epoxide ring opening and stereospecific rearrangement. Modular dissection of the C-terminal extension further reveals a functional partition between […]
- by Jaskovikaite, I., Offerhaus, H. S., Vinogradovas, M., Barkauskaite, U., Depken, M., Jones, S. K.Programmed with an RNA guide, Cas9 nuclease directs double-strand DNA cleavage via its two nuclease domains. However, Cas9 sometimes falsely identifies DNA targets by binding and cleaving DNA that does not match its PAM and guide RNA sequence requirements. Cas9's specificity is often affected by DNA topology, as DNA negative supercoiling can increase off-target activity while positive supercoiling can even prevent on-target activity. Such dramatic DNA topological changes routinely occur in cells as a result of transcription and replication, making […]
- by Vieira, V. A., Costa, G. S., Gonzales, G. S., Campos, R. G., dos Santos, C., da Costa, R. G., Sant'ana, M. R., Ramos, C. d. O., Cassiano, M. H. A., Alves, J. M., Pereira, N., Melo, P. H., Goncalves, T. T., Bonilha, I., Lopes, C. F. B., Gardinassi, L. G., Yoshinaga, M. Y., Alberici, L. C., Sposito, A. C., Cintra, D. E., Softic, S., Kahn, C. R., Kang, J. X., Malta, T., Mori, M. A., Tseng, Y.-H., Leiria, L. O.While cold exposure drives lipid flux from adipose tissue to the liver, this enhanced inter-organ crosstalk does not result in sustained hepatic steatosis during prolonged acclimation, indicating that factors beyond lipid flux shape metabolic outcome. To interrogate this adaptation, we performed integrated lipidomic and metabolic profiling across tissues and circulating lipoproteins over the course of cold acclimation. We showed that cold acclimation induces systemic reprogramming of lipid quality in mice, characterized by enrichment of n-3 highly unsaturated fatty acids (HUFAs) […]
- by Zhao, Q., Li, J., Tong, Y., Li, Y., Han, W., Li, Z., Wang, Y., Yin, Y., Fang, J., Jiang, W., Song, Q., Huang, S., Shen, Y., Cong, Y.The eukaryotic chaperonin TRiC/CCT is essential for folding complex proteins, yet how it folds substrates that exceed its closed chamber capacity remains a longstanding paradox. Here, we define the folding pathway of IFT172, the largest subunit (~200 kDa) of the intraflagellar transport (IFT) machinery, and uncover a "divide-and-conquer" mechanism. TRiC and HSP70 engage IFT172 concurrently but on distinct domains: TRiC captures the N-terminal WD40 {beta}-propellers within its chamber, whereas HSP70 independently stabilizes the C-terminal TPR domain in the cytosol. To […]
- by Geher, S. W. R., Falekun, S., Pita-Aquino, J. N., Swift, R. P., Okada, M., Jami-Alahmadi, Y., Wohlschlegel, J. A., Prigge, S. T., Sigala, P. A.Acyl carrier protein (ACP) and its 4-phosphopantetheine prosthetic group canonically function as the soluble scaffold for acyl chain assembly and elongation during type-II fatty acid synthesis (FASII). Plasmodium malaria parasites retain a FASII pathway in the apicoplast organelle that has been the subject of considerable scrutiny and confusion. Although apicoplast FASII is essential for P. falciparum growth within mosquitoes and the human liver, this pathway is dispensable and largely inactive in blood-stage parasites that can scavenge host fatty acids. In […]
- by Thang, N. X., Martiensen, E. L. B., Abdelhalim, M., Tran, T. T., Ledsaak, M., Rogne, M., Thiede, B., Eskeland, R.Osteosarcoma (OS) is an aggressive bone cancer that most commonly affects children and young adults. OS exhibits a high degree of genomic complexity, as well as cellular plasticity, and dynamic transcriptional regulation is suggested to contribute to treatment resistance and metastasis. Cell lines are well characterized as models to advance our knowledge on OS biology. HOS and U2OS cells have increased invasiveness and higher migratory ability compared with MG63. In this study, we employed a tandem array of consensus transcription […]
- by Musheev, M. U., Siefert, J., Bauten, W., Butepage, M., Luscher, B., Niehrs, C., Feijs, K. L., Zaja, R.ADP-ribosylation is known as a protein modification, yet recent studies have expanded the range of ADP-ribosyltransferase (ART) substrates to include nucleic acids. tRNA 2'-phosphotransferase 1 (TRPT1) and several PARP family members can modify the 5'-phosphate of single-stranded RNA. Here, we show that PARP10 and PARP15 extend this activity beyond the 5'-phosphate terminus and generate N3-ADP-ribosyl uracil and N1-ADP-ribosyl guanine bases. The base-linked ADP-ribosylation is reversed selectively by the macrodomain-containing hydrolase TARG1. In TARG1 knockout cells, N1-ADP-ribosyl guanine can be detected. […]
- by Bilsen, A., Ottati, S., Parise, C., Abba, S., Vanden Broeck, J., Galetto, L., Santos, D.Growing evidence shows that eukaryotic genomes contain DNA sequences of non-retroviral RNA virus origin, yet the mechanisms underlying the generation of this virus-derived complementary DNA (vDNA) remain poorly understood, particularly in insects. Here, we demonstrate that infection with diverse non-retroviral RNA viruses induces the production of reverse-transcribed vDNA across multiple lepidopteran and hemipteran species. We further show that a Drosophila melanogaster-derived cell line actively secretes reverse-transcribed vDNA associated with extracellular vesicles (EVs), widely conserved nanoscale mediators of intercellular communication. Together, […]
- by Roberson, A. B., Marks, J., Pitts, R., Tamilselvam, B., Grieb, B., Tansey, W. P., Meydan, S.5-Azacytidine (5-AzaC) is a cytidine analog and is widely used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Although its therapeutic activity is primarily attributed to hypomethylation resulting from DNA incorporation, the majority of 5-AzaC is incorporated into RNA. However, the functional consequences of 5-AzaC incorporation into RNA have been unknown. Here, we show that 5-AzaC treatment of cells leads to inhibition of protein synthesis. Ribo-seq, Disome-seq, and RNA-seq in cells treated with 5-AzaC exhibit a time-dependent C-to-G […]
- by Fatayer, R., Sammut, S.-J., Senthil Murugan, G.Tumour biomarkers such as CA125, CA15-3, CA19-9, AFP and CEA are routinely used in the oncology clinic to diagnose cancer, monitor response to therapy, and detect relapse. However, their quantification depends on immunoassay-based methods that are time-consuming, reagent-dependent, and poorly suited to resource-limited settings. Here, we present a machine learning-assisted ATR-FTIR spectroscopy approach for label-free tumour biomarker analysis to enable simple and rapid quantification at the bedside. Using principal component analysis (PCA), we first demonstrate that these five clinically relevant […]
- by Mullick, S., Suresh Kumar, C., Dey, S., Koushik, P. B., Ganie, R., Mahanty, S., Sirajuddin, M., Gadadhar, S.Tubulin glycylation, a cilia-specific posttranslational modification is emerging as a potentially key regulator of ciliary axonemal microtubules. However, insights into the functional consequences of glycylation have remained limited. Here, using in vitro reconstitution assays with unmodified or custom-glycylated tubulin, we provide a systematic mechanistic analysis of glycylation-dependent regulation of motors and microtubule-associated proteins. Our studies highlight that glycylation selectively enhances ciliary kinesin-2 motility while reducing kinesin-1 activity, suggesting a role in promoting efficient intraflagellar transport along axonemal microtubules. Moreover, glycylation […]
- by Bitz, L., Pihlava, J.-M., Marnila, P., Blasco, L., Paavilainen, V. O., Hartikainen, M., Nukari, A., Tranter, D., Tenhola-Roininen, T.The genetically authenticated Finnish hop genotype LUKE 2541 obtained from wild was evaluated for antibacterial, anti-inflammatory, and anticancer activities. Water extracts from hop cones inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus, with MIC values of 0.094- 0.188mg/mL, whereas Gram-negative strains showed limited sensitivity. In LPS-primed THP-1 cells, both IPA and IPA-Control extracts reduced reactive oxygen species formation in a dose-dependent manner, exhibiting similar IC50 values (50.41{micro}g/mL and 35.41{micro}g/mL). This hop genotype also displayed clear tissue- and solvent-dependent antiproliferative […]
- by Watson, M. A., Soygur, B., King, C. D., Devrukhkar, P., Shanes, E. D., Melov, S., Pavone, M. G., Duncan, F., Schilling, B.The human ovary is among the first organs to show age-related functional decline, resulting in menopause. Beyond this transition, the postmenopausal ovary is often regarded as quiescent and remains poorly characterized. We analyzed the proteomes of healthy, non-pathological ovaries using mass spectrometry (data-independent acquisitions) from 28 postmenopausal women (50-75 years old), stratified into three age groups (50-59, 60-69, [≥]70). We quantified 5,812 protein groups and observed progressive age-associated shifts with 117 proteins significantly altered in the [≥]70 vs 50-59 age […]
- by Schramm, T., Gillet, L., Reber, V., de Souza, N., Gstaiger, M., Picotti, P.Peptide-level analyses are becoming increasingly popular in mass spectrometry-based proteomics and are being applied, for example, in immunopeptidomics, structural proteomics, and analyses of post-translational modifications. In such analyses, peptides that are not biologically meaningful but instead arise as artifacts prior to mass spectrometry analysis pose the risk of data misinterpretation. Here, we describe an approach based on retention time analysis and precise chromatographic peak matching to identify peptides generated by in-source fragmentation (ISF), which occurs between chromatographic separation of peptide […]
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