- by Musheev, M., Schomacher, L., Schott, J. M., Basu, A., Moeckel, M. M., Heinen, S., Frosch, L., Guo, P., Yang, G., Huang, Q., Niehrs, C.Recent evidence indicates that mono- and poly-ADP ribosylation (MARylation and PARylation) are not limited to proteins but extend to DNA. Notably, in vitro base PARylation by PARP1 in single stranded DNA (ssDNA) was demonstrated at N1-deoxyadenosine (N1-dA). Here, we report that PARP1 catalyzes N3-specific ADP-ribosylation of deoxycytidine (N3-dC) in single-stranded DNA. Analogous to N1-dA PARylation, which is prone to spontaneous adenine-to-inosine deamination, N3-dC PARylation promotes cytosine deamination, yielding N3-PARylated-deoxyuridine. These deamination products yield diagnostic PARylation signatures in LC-MS/MS, namely N1-ribosyl-deoxyinosine […]
- by Wang, X., Meng, L., Li, H., ZHANG, Y., Li, M., Xiang, B., Zhu, J., Li, C., Yang, C., YU, S., Pang, X., Wei, J., Zhang, M., Bakken, L. R., Frostegard, A., Zhang, L., Zhang, X.Denitrifying bacteria are major contributors to nitrous oxide (N2O) emissions, which rise with global soil acidification, likely because low pH impairs maturation of the periplasmic enzyme N2O reductase (NosZ) in most bacteria. While some bacteria can reduce N2O under acidic conditions, the underlying mechanism remain unclear. Here, we isolated bacteria from acidic soils and identified strains that efficiently reduce N2O at low pH. They carry two novel proteins, NosP and NosQ, along with NosZ clade I. Functional analyses of deletion […]
- by Pedroza-Davila, U., Camacho-Villasana, Y., Vazquez-Acevedo, M., Lutikurti, M., Gonzalez-Halphen, D., Cabrera-Orefice, A., Perez-Martinez, X.Cytochrome c oxidase (CIV) is the last electron acceptor of the mitochondrial respiratory chain. In yeast, it is composed of 12 subunits, three of which are encoded in the mitochondrial genome. CIV assembly is a modular and highly regulated process that requires several specific factors. In this work, we characterized the role of Aim11 in CIV biogenesis. By high-throughput analysis, it was previously detected that Aim11 interacted with some CIV subunits, but the physiological relevance of these interactions was unknown. […]
- by GITON, F., DER VARTANIAN, A., WYCKENS, N., FERECATU, I., CHESTER, M., HERAUD, C., ISIK, A., MATHIS, C., GUIGON, C. J.Understanding the tissue-specific distribution and metabolism of sex steroids is critical for elucidating their physiological roles and pathological alterations. However, existing in vivo approaches often require surgical castration to suppress endogenous hormones, disrupting systemic endocrine balance and limiting physiological relevance. Here, we present a novel mass spectrometry method combining systemic administration of deuterium-labeled sex steroids with high-sensitivity gas chromatography-tandem mass spectrometry (GC-MS/MS) to simultaneously quantify exogenous, endogenous, and metabolized steroid species in serum and multiple tissues of non-castrated mice. This […]
- by Peroutkova, P., Jirasko, R., Taylor, A., Holcapek, M., Peterka, O.Supercritical fluid chromatography is traditionally employed for nonpolar and moderately polar analytes, while the analysis of ionic compounds remains a recognized limitation of the technique. Here, we introduce a novel ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method using a bioinert column, enabling the separation of lipids with a broad polarity range from nonpolar to ionic species. The UHPSFC/MS method was optimized using 79 lipid species across 41 lipid subclasses, achieving a total run time of 7.5 min, including the column […]
- by Han, C., Weng, Y., Zheng, Q., Qu, Q., Erramilli, S. K., Su, Z., Duan, Y., Han, Y., Zhai, X., Kossiakoff, A. A., Pan, M., Zhao, M., Liu, L., Yu, Y.Lysine 27 (K27)-linked polyubiquitination plays critical yet incompletely defined roles in proteostasis, innate immunity, and disease progression; however, investigations into this process have long been hindered by its extremely low abundance and the lack of conformation-specific enrichment tools. Herein, we describe the development of a long-sought conformation-specific antibody, K27-IgG, which can selectively recognize-among all ubiquitin chain types-the unique architecture of K27-linked polyubiquitin (K27-polyUb) characterized by a distinct buried K27-isopeptide bond, with high affinity (KD = 4.66 nM). This antibody was […]
- by Boyle, E. P., Aruscavage, P. J., Consalvo, C. D., Bass, B. L.In C. elegans, antiviral RNA interference (RNAi) relies on the coordinated activity of Dicer (DCR-1), the helicase DRH-1, and the double-stranded RNA (dsRNA)-binding protein, RDE-4, yet the domain- specific contributions of RDE-4 remain unclear. Here, we reconstituted the antiviral complex from independently purified DCR-1{middle dot}DRH-1 and RDE-4 to define how RDE-4 stabilizes and activates the complex. Addition of recombinant RDE-4 restored ATP hydrolysis and dsRNA cleavage to levels previously observed with the pre-assembled complex, and time-course assays revealed that RDE-4 […]
- by Adsersen, B. I., Moeller-Jensen, C. M., Jacobsen, C. P., Domeyer, T., Loeffler, J. R., Fernandez-Quintero, M. L., Tomberli, I., Jensen, A. B., Jakobsen, M. S., Bangaru, S., Ward, A., Rivera-de-Torre, E., Gunnarsson, S. B., Hald, A., Jenkins, T. P.Growth-factor antagonists regulate key developmental and pathological processes, yet the molecular principles governing their control remain poorly understood. Gremlin-1 (GREM1) is a secreted antagonist of bone morphogenetic proteins (BMPs) whose dysregulation is implicated in fibrosis and cancer. Here, we report a de novo designed protein that binds GREM1 with sub-nanomolar affinity and selectively releases BMPs for downstream signalling. By integrating generative deep-learning-based protein design with molecular-dynamics-derived flexibility descriptors, we identify a predictive relationship between interface rigidity, desolvation energy, and binding […]
- by West, R. M., Nicolescu, R. C. B., Brear, P., Wagstaff, J. M., Blaszczyk, B. K., Deingruber, T., Sanders, M. G., Perez-Areales, F. J., Spring, D. R., Hyvönen, M.Bruton's Tyrosine Kinase (BTK) is a validated target for haematological malignancies, with numerous FDA approved inhibitors on the market. Current therapies target the highly conserved ATP binding site and hence limit the therapeutic index given the site's highly conserved nature across the kinome. We explore a novel approach for BTK inhibition, by targeting the PH domain-mediated membrane recruitment and activation of BTK. We have identified a fragment which covalently labels a lysine in the inositol phosphate (PIP3) binding site. Fragment […]
- by Shadman, H., Ziebarth, J. D., Cheng, Q., Laradji, M., Wang, Y.Linking the sequences of intrinsically disordered regions (IDRs) to their structural ensembles and biological functions remains a central challenge in understanding how disorder encodes activity. A recent study has shown that human IDRs with different levels of compactness, as measured by Florys exponent, are associated with specific cellular functions and localizations, and Florys exponent can be predicted from sequence features with reasonable accuracy. However, IDRs are known to sample highly heterogenous conformations that can be masked by ensemble-averaged metrics such […]
- by Das, R., Biswas, S., Mukhopadhyay, D.Discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase (RTK) family member, plays a pivotal role in collagen-mediated signaling, influencing several key cellular processes. In Alzheimer's disease (AD), DDR2 expression and activity are significantly upregulated. While collagens, the endogenous ligands of DDR2, either remain unchanged or are downregulated in AD-like conditions, Amyloid-beta 42(A{beta}42) has been found to activate DDR2 in the absence of collagen non-canonically. Additionally, DDR2 naturally translocates to the nucleus in the neuroblastoma cell line SH-SY5Y, and the […]
- by Auger, C., Nishida, H., Yuan, B., Silva, G. M., Fujimoto, M., Li, M., Katoh, D., Wang, D., Granath-Panelo, M., Shin, J., Verkerke, A., Banks, A., Hui, S. T., Sun, L., Yook, J.-S., Witte, R., Kajimura, S.Metabolic adaptation to environmental changes, such as fasting and cold exposure, involves a dynamic shift in fuel utilization from glucose to fatty acid oxidation, a process that relies on carnitine-mediated fatty acid oxidation in mitochondria. While dietary sources of animal origin (e.g., red meat) contribute to the carnitine pool, de novo carnitine synthesis from trimethyllysine (TML) is essential, particularly for those whose dietary sources are vegetables and fruits that contain negligible amounts of carnitine. However, the molecular pathway of de […]
- by Chakraborty, S., Jiang, J., Camallonga, J. V., Gazgalis, D., Gero, T. W., Tavares, M. T., Dittemore, G., Tkacik, E., Jang, D. M., Ha, B. H., Gottlieb, F. H., Levitz, T., Metivier, R. J., Schmoker, A. M., Scott, D. A., Jeon, H., Janne, P. A., Eck, M. J.Aberrant activation of the RAS/RAF/MEK/ERK pathway is a frequent cause of cancer. Allosteric MEK inhibitors block this pathway by binding RAF-MEK complexes to prevent activation of MEK by RAF. However, how MEK inhibitor potency varies across the three RAF isoforms remains poorly understood. We profiled seven allosteric MEK inhibitors and observed a striking hierarchy of sensitivity: all most potently inhibited CRAF-driven MEK activation while relatively sparing ARAF-driven activation. We identified point mutations in ARAF and CRAF proximate to the MEK […]
- by Tkacik, E., Jang, D. M., Boxer, K., Ha, B. H., Eck, M. J.RAF kinases are central regulators of the RAS/MAP kinase pathway and important targets in cancer therapy. Paradoxically, RAF inhibitors can activate wild-type RAF signaling. Negative allostery is a central feature of the prevailing model for this phenomenon, wherein inhibitors induce RAF dimers in which inhibitor binding to one protomer promotes an active but inhibitor-resistant conformation in the other protomer. Here we systematically examined paradoxical activation of ARAF, BRAF, and CRAF using biochemical assays with isolated RAF/MEK kinase domain complexes. We […]
- by Iyer, A., Buetikofer, P.Phospholipases D (PLDs) are ubiquitous enzymes of the PLD superfamily that catalyze phosphodiester bond cleavage and transphosphatidylation reactions. They are characterized by the presence of two conserved HXK(X)4D(X)6G(X)2N or HKD motifs. While these motifs are essential for catalysis in diverse PLD family members, their functional significance in the bacterial-type cardiolipin synthase of the protozoan parasite Trypanosoma brucei (TbCls) has not yet been investigated. TbCls is essential for parasite survival in culture and contains two conserved HKD motifs in its amino […]
- by Ashour, M. E., Krekeler, E., Bhowmik, M. C., Tsao, N., Herrera-Montavez, C., Tsai, M.-S., Kantar, H., Zaher, M., Galletto, R., Mosammaparast, N.Alkylation chemotherapy is commonly used against tumors such as glioblastoma, yet resistance often develops through downregulation of mismatch repair (MMR). Previous work has established that loss of MMR prevents the excision of the thymine-containing strand across O6meG-T mismatches. Thus, MMR dysfunction is advantageous because it prevents a vicious cycle of attempted repair that leads to cell death. Here, we provide an alternative explanation to this prevailing mechanism of alkylation chemoresistance by MMR loss. We find that the MMR protein MutL […]
- by Bosman, G. P., Ehlers, A., Buckley, T., Dobruchowska, J., Torano, J. S., Bentlage, A. E. H., Vidarsson, G., Boons, G.-J.Glycosylation heterogeneity in therapeutic monoclonal antibodies (mAbs) necessitates precise glycoengineering to optimize effector functions, such as those mediated by Fc{gamma}-receptors (Fc{gamma}Rs). Current enzymatic remodeling methods are limited in scope, producing only a narrow spectrum of glycoforms and struggling to achieve complete control over the synthesis of defined asymmetrical structures, which are often critical for enhanced mAb efficacy. We report a multi-step, fully enzymatic platform for the solution-phase synthesis of a comprehensive library of biantennary mAb glycoforms with complete control over […]
- by Tuovinen, A., Pääkkönen, J., Maksimainen, M. M., Hirschen, L., Hentilä, H. I., Tauscher, M., Luscher, B., Vela-Rodriguez, C., Korn, P., Lehtiö, L.PARP proteins are enzymes catalyzing ADP-ribosylation, a well-conserved post-translational modification. In addition to the catalytic domain present in all PARPs, these proteins have a wide selection of other domains possessing different functions. Additional domains present in human PARP15 are macrodomains, capable of binding ADP-ribose. PARP15 is the least studied of the macrodomain-containing human PARPs, and it is linked to different diseases from infections to cancer. We show that the full-length canonical isoform 1 of PARP15 auto-ADP-ribosylates robustly on glutamate and […]
- by Wang, T., Binion, B., Alves, J. M. P., Ridlon, J. M.Epitestosterone (epiT) is the isomer of the androgen testosterone. Historically, the role of epiT has remained unclear. Recently, it has been reported that epiT promotes AR-dependent prostate cancer cell proliferation. The gut bacterium Clostridium scindens VPI 12708 converts androstenedione (AD) to epiT. The bacterial enzymatic pathways involved in epiT formation have been reported, where the desF gene that encodes 17-hydroxysteroid dehydrogenase converts AD to epiT using NADPH as a cofactor. In this study, we quantitatively characterized DesF kinetic parameters and […]
- by Lippens, G., Li, Y., Jacob-Dubuisson, F., Dubiley, S.Multinuclear nonheme iron-dependent oxidases (MNIOs) constitute one of the largest families of enzymes involved in natural product biosynthesis. Distinct MNIO subfamilies utilize molecular oxygen to catalyze a wide variety of complex peptide rearrangements, including {beta}-carbon excision and heterocyclization. Highly homologous MNIOs have been proposed to install either oxazolone-thioamides or 5-thiooxazoles as cysteine post-translational modifications in the closely related bufferin and EGKCG families of peptide chalkophores. These alternative structures prompted discussion of the subtle mechanistic features of MNIO enzymes that might […]
Mass spectrometry websites, social media, journal feeds, and other links and items of interest to the mass spectrometry community.
