During development, tooth germs undergo various morphological changes resulting from interactions between the oral epithelium and ectomesenchyme. These processes are influenced by the extracellular matrix, the composition of which, along with cell adhesion and signaling, is regulated by metalloproteinases. Notably, these include matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs). Our analysis of previously published scRNAseq datasets highlight that these metalloproteinases show dynamic expression patterns during tooth development, with expression in a wide range of cell types, suggesting multiple roles in tooth morphogenesis. To investigate this, Marimastat, a broad-spectrum inhibitor of MMPs, ADAMs, and ADAMTSs, was applied to ex vivo cultures of mouse molar tooth germs. The treated samples exhibited significant changes in tooth germ size and morphology, including an overall reduction in size and an inversion of the typical bell shape. The cervical loop failed to extend, and the central area of the inner enamel epithelium protruded. Marimastat treatment also disrupted proliferation, cell polarization, and organization compared with control tooth germs. In addition, a decrease in laminin expression was observed, leading to a disruption in continuity of the basement membrane at the epithelial-mesenchymal junction. Elevated hypoxia-inducible factor 1-alpha gene (Hif-1α) expression correlated with a disruption to blood vessel development around the tooth germs. These results reveal the crucial role of metalloproteinases in tooth growth, shape, cervical loop elongation, and the regulation of blood vessel formation during prenatal tooth development.NEW & NOTEWORTHY Inhibition of metalloproteinases during tooth development had a wide-ranging impact on molar growth affecting proliferation, cell migration, and vascularization, highlighting the diverse role of these proteins in controlling development.
- MeSH
- Hypoxia-Inducible Factor 1, alpha Subunit metabolism genetics MeSH
- Matrix Metalloproteinase Inhibitors pharmacology MeSH
- Hydroxamic Acids pharmacology MeSH
- Metalloproteases metabolism genetics MeSH
- Molar embryology growth & development metabolism enzymology MeSH
- Morphogenesis MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Odontogenesis * MeSH
- Cell Proliferation * MeSH
- Gene Expression Regulation, Developmental MeSH
- Tooth Germ embryology metabolism enzymology MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
G protein-coupled receptors (GPCRs) play a crucial role in cell function by transducing signals from the extracellular environment to the inside of the cell. They mediate the effects of various stimuli, including hormones, neurotransmitters, ions, photons, food tastants and odorants, and are renowned drug targets. Advancements in structural biology techniques, including X-ray crystallography and cryo-electron microscopy (cryo-EM), have driven the elucidation of an increasing number of GPCR structures. These structures reveal novel features that shed light on receptor activation, dimerization and oligomerization, dichotomy between orthosteric and allosteric modulation, and the intricate interactions underlying signal transduction, providing insights into diverse ligand-binding modes and signalling pathways. However, a substantial portion of the GPCR repertoire and their activation states remain structurally unexplored. Future efforts should prioritize capturing the full structural diversity of GPCRs across multiple dimensions. To do so, the integration of structural biology with biophysical and computational techniques will be essential. We describe in this review the progress of nuclear magnetic resonance (NMR) to examine GPCR plasticity and conformational dynamics, of atomic force microscopy (AFM) to explore the spatial-temporal dynamics and kinetic aspects of GPCRs, and the recent breakthroughs in artificial intelligence for protein structure prediction to characterize the structures of the entire GPCRome. In summary, the journey through GPCR structural biology provided in this review illustrates how far we have come in decoding these essential proteins architecture and function. Looking ahead, integrating cutting-edge biophysics and computational tools offers a path to navigating the GPCR structural landscape, ultimately advancing GPCR-based applications. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.
- MeSH
- Protein Conformation MeSH
- Humans MeSH
- Receptors, G-Protein-Coupled * chemistry metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Glioblastomas are aggressive brain tumors for which effective therapy is still lacking, resulting in dismal survival rates. These tumors display significant phenotypic plasticity, harboring diverse cell populations ranging from tumor core cells to dispersed, highly invasive cells. Neuron navigator 3 (NAV3), a microtubule-associated protein affecting microtubule growth and dynamics, is downregulated in various cancers, including glioblastoma, and has thus been considered a tumor suppressor. In this study, we challenge this designation and unveil distinct expression patterns of NAV3 across different invasion phenotypes. Using glioblastoma cell lines and patient-derived glioma stem-like cell cultures, we disclose an upregulation of NAV3 in invading glioblastoma cells, contrasting with its lower expression in cells residing in tumor spheroid cores. Furthermore, we establish an association between low and high NAV3 expression and the amoeboid and mesenchymal invasive phenotype, respectively, and demonstrate that overexpression of NAV3 directly stimulates glioblastoma invasive behavior in both 2D and 3D environments. Consistently, we observed increased NAV3 expression in cells migrating along blood vessels in mouse xenografts. Overall, our results shed light on the role of NAV3 in glioblastoma invasion, providing insights into this lethal aspect of glioblastoma behavior.
- MeSH
- Phenotype * MeSH
- Glioblastoma * pathology genetics metabolism MeSH
- Neoplasm Invasiveness * genetics MeSH
- Humans MeSH
- Membrane Proteins MeSH
- Microtubules metabolism MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Brain Neoplasms * pathology genetics metabolism MeSH
- Cell Movement genetics physiology MeSH
- Nerve Tissue Proteins metabolism genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
3-methylglutaconic aciduria (3-MGCA) is a biochemical finding in a diverse group of inherited metabolic disorders. Conditions manifesting 3-MGCA are classified into two major categories, primary and secondary. Primary 3-MGCAs involve two inherited enzymatic deficiencies affecting leucine catabolism, whereas secondary 3-MGCAs comprise a larger heterogeneous group of conditions that have in common compromised mitochondrial energy metabolism. Here, we report 3-MGCA in two siblings presenting with sensorineural hearing loss and neurological abnormalities associated with a novel, homozygous missense variant (c.1999C>G, p.Leu667Val) in the YME1L1 gene which encodes a mitochondrial ATP-dependent metalloprotease. We show that the identified variant results in compromised YME1L1 function, as evidenced by abnormal proteolytic processing of substrate proteins, such as OPA1 and PRELID1. Consistent with the aberrant processing of the mitochondrial fusion protein OPA1, we demonstrate enhanced mitochondrial fission and fragmentation of the mitochondrial network in patient-derived fibroblasts. Furthermore, our results indicate that YME1L1L667V is associated with attenuated activity of rate-limiting Krebs cycle enzymes and reduced mitochondrial respiration, which may explain the build-up of 3-methylglutaconic and 3-methylglutaric acid due to the diversion of acetyl-CoA, not efficiently processed in the Krebs cycle, towards the formation of 3-methylglutaconyl-CoA, the precursor of these metabolites. In summary, our findings classify YME1L1 deficiency as a new type of secondary 3-MGCA, thus expanding the genetic landscape and facilitating the diagnosis of inherited metabolic disorders featuring this biochemical phenotype.
- MeSH
- Child MeSH
- Fibroblasts metabolism MeSH
- Glutarates MeSH
- Humans MeSH
- Metalloendopeptidases * genetics metabolism MeSH
- Mutation, Missense MeSH
- Mitochondrial Dynamics MeSH
- Mitochondrial Proteins * genetics MeSH
- Mitochondria metabolism MeSH
- Hearing Loss, Sensorineural genetics MeSH
- Siblings MeSH
- Metabolism, Inborn Errors * genetics MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
N-Methyl-d-aspartate receptors (NMDARs) play a crucial role in excitatory neurotransmission, with numerous pathogenic variants identified in the GluN subunits, including their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs, but this was tested in a limited number of studies. Using microscopy and electrophysiology in the human embryonic kidney 293 (HEK293) cells, we found that surface expression of GluN1/GluN2A receptors containing a set of alanine substitutions within the LBDs correlated with the measured EC50 values for glycine (GluN1 subunit mutations) while not correlating with the measured EC50 values for l-glutamate (GluN2A subunit mutations). The mutant cycle of GluN1-S688 residue, including the pathogenic GluN1-S688Y and GluN1-S688P variants, showed a correlation between relative surface expression of the GluN1/GluN2A receptors and the measured EC50 values for glycine, as well as with the calculated ΔGbinding values for glycine obtained from molecular dynamics simulations. In contrast, the mutant cycle of GluN2A-S511 residue did not show any correlation between the relative surface expression of the GluN1/GluN2A receptors and the measured EC50 values for l-glutamate or calculated ΔGbinding values for l-glutamate. Coexpression of both mutated GluN1 and GluN2A subunits led to additive or synergistic alterations in the surface number of GluN1/GluN2A receptors. The synchronized ER release by ARIAD technology confirmed the altered early trafficking of GluN1/GluN2A receptors containing the mutated LBDs. The microscopical analysis from embryonal rat hippocampal neurons (both sexes) corroborated our conclusions from the HEK293 cells.
- MeSH
- Glycine metabolism MeSH
- HEK293 Cells MeSH
- Hippocampus cytology metabolism MeSH
- Rats MeSH
- Glutamic Acid metabolism MeSH
- Humans MeSH
- Ligands MeSH
- Mutation genetics MeSH
- Protein Domains MeSH
- Nerve Tissue Proteins MeSH
- Receptors, N-Methyl-D-Aspartate * metabolism genetics chemistry MeSH
- Protein Transport physiology genetics MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Phase separation forms membraneless compartments, including heterochromatin "domains" and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination. In Drosophila cells, "safe" homologous recombination (HR) repair of these sequences requires their relocalization to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation is unclear. Here, we show that Nup98 nucleoporin is recruited to repair sites before relocalization by Sec13 or Nup88, and downstream of the Smc5/6 complex and heterochromatin protein 1 (HP1). Remarkably, Nup98 condensates are immiscible with HP1 condensates, and they are required and sufficient to mobilize repair sites and exclude Rad51, thus preventing aberrant recombination while promoting HR repair. Disrupting this pathway results in heterochromatin repair defects and widespread chromosome rearrangements, revealing an "off-pore" role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote.
- MeSH
- Chromosomal Proteins, Non-Histone metabolism genetics MeSH
- Drosophila melanogaster * genetics metabolism MeSH
- DNA Breaks, Double-Stranded MeSH
- Heterochromatin * genetics metabolism MeSH
- Chromobox Protein Homolog 5 MeSH
- Nuclear Pore Complex Proteins * metabolism genetics MeSH
- Cell Cycle Proteins metabolism genetics MeSH
- Drosophila Proteins * metabolism genetics MeSH
- Recombinational DNA Repair * MeSH
- Rad51 Recombinase * metabolism genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
INTRODUCTION: The immunosuppressive roles of galectin-3 (Gal-3) in carcinogenesis make this lectin an attractive target for pharmacological inhibition in immunotherapy. Although current clinical immunotherapies appear promising in the treatment of solid tumors, their efficacy is significantly weakened by the hostile immunosuppressive tumor microenvironment (TME). Gal-3, a prominent TME modulator, efficiently subverts the elimination of cancer, either directly by inducing apoptosis of immune cells or indirectly by binding essential effector molecules, such as interferon-gamma (IFNγ). METHODS: N-(2-Hydroxypropyl)methacrylamide (HPMA)-based glycopolymers bearing poly-N-acetyllactosamine-derived tetrasaccharide ligands of Gal-3 were designed, synthesized, and characterized using high-performance liquid chromatography, dynamic light scattering, UV-Vis spectrophotometry, gel permeation chromatography, nuclear magnetic resonance, high-resolution mass spectrometry and CCK-8 assay for evaluation of glycopolymer non-toxicity. Pro-immunogenic effects of purified glycopolymers were tested by apoptotic assay using flow cytometry, competitive ELISA, and in vitro cell-free INFγ-based assay. RESULTS: All tested glycopolymers completely inhibited Gal-3-induced apoptosis of monocytes/macrophages, of which the M1 subtype is responsible for eliminating cancer cells during immunotherapy. Moreover, the glycopolymers suppressed Gal-3-induced capture of glycosylated IFNγ by competitive inhibition to Gal-3 carbohydrate recognition domain (CRD), which enables further inherent biological activities of this effector, such as differentiation of monocytes into M1 macrophages and repolarization of M2-macrophages to the M1 state. CONCLUSION: The prepared glycopolymers are promising inhibitors of Gal-3 and may serve as important supportive anti-cancer nanosystems enabling the infiltration of proinflammatory macrophages and the reprogramming of unwanted M2 macrophages into the M1 subtype.
- MeSH
- Acrylamides chemistry pharmacology MeSH
- Apoptosis drug effects MeSH
- Galectin 3 * antagonists & inhibitors MeSH
- Galectins MeSH
- Interferon-gamma * metabolism MeSH
- Blood Proteins MeSH
- Humans MeSH
- Macrophages drug effects MeSH
- Monocytes * drug effects MeSH
- Tumor Microenvironment drug effects MeSH
- Polymers * chemistry pharmacology MeSH
- Antineoplastic Agents * pharmacology chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Interferon‐induced transmembrane proteins (IFITMs) are frequently overexpressed in cancer cells, including cervical carcinoma cells, and play a role in the progression of various cancer types. However, their mechanisms of action remain incompletely understood. In the present study, by employing a combination of surface membrane protein isolation and quantitative mass spectrometry, it was comprehensively described how the IFITM1 protein influences the composition of the cervical cancer cell surfaceome. Additionally, the effects of interferon‐γ on protein expression and cell surface exposure were evaluated in the presence and absence of IFITM1. The IFITM1‐regulated membrane and membrane‐associated proteins identified are involved mainly in processes such as endocytosis and lysosomal transport, cell‐cell and cell‐extracellular matrix adhesion, antigen presentation and the immune response. To complement the proteomic data, gene expression was analyzed using reverse transcription‐quantitative PCR to distinguish whether the observed changes in protein levels were attributable to transcriptional regulation or differential protein dynamics. Furthermore, the proteomic and gene expression data are supported by functional studies demonstrating the impact of the IFITM1 and IFITM3 proteins on the adhesive, migratory and invasive capabilities of cervical cancer cells, as well as their interactions with immune cells.
- MeSH
- Cell Adhesion MeSH
- Antigens, Differentiation * metabolism genetics MeSH
- Phenotype MeSH
- Interferon-gamma pharmacology metabolism MeSH
- Humans MeSH
- Membrane Proteins * metabolism genetics MeSH
- Cell Line, Tumor MeSH
- Uterine Cervical Neoplasms * pathology genetics metabolism immunology MeSH
- Cell Movement MeSH
- RNA-Binding Proteins * metabolism genetics MeSH
- Proteome * MeSH
- Proteomics methods MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.
- MeSH
- DNA-Binding Proteins MeSH
- Endopeptidases metabolism chemistry genetics MeSH
- Escherichia coli metabolism genetics MeSH
- Cardiolipins * metabolism chemistry MeSH
- Membrane Proteins * metabolism chemistry genetics MeSH
- Protein Engineering * MeSH
- Escherichia coli Proteins * metabolism chemistry genetics MeSH
- Molecular Dynamics Simulation MeSH
- Protein Binding MeSH
- Publication type
- Journal Article MeSH
Cíl: Observační studie sledující dynamiku tvorby protilátek IgG proti S proteinu SARS-CoV-2 u osob očkovaných dvěma dávkami mRNA vakcíny Pfizer-BioNTech BNT162b2 (Comirnaty) byla provedena v období let 2021–2022 u skupiny dobrovolníků bez známek předcházející nákazy SARS-CoV-2 a u skupiny s anamnézou přirozené infekce covidem-19. Hlavním cílem bylo monitorovat hladiny protilátek až do 12 měsíců po druhé dávce a zjistit podíl očkovaných, u kterých došlo k sérokonverzi. Studie také hodnotila případy selhání vakcíny v rámci třinácti měsíců po očkování. Metodika: Před zahájením očkování byly u účastníků studie provedeny laboratorní testy na protilátky IgG proti S proteinu SARS-CoV-2 a shromážděna anamnestická data týkající se onemocnění covidem-19. Na základě negativních výsledků byla vytvořena kohorta imunologicky naivních osob, které byly následně očkovány dvěma dávkami vakcíny BNT162b2 (Comirnaty). Vzorky venózní krve byly odebírány v šesti časových bodech: před první dávkou, dva až tři týdny po první dávce, měsíc po druhé dávce, tři až čtyři měsíce po druhé dávce, půl roku a jeden rok po druhé dávce. Doplňkovou kohortu tvořili dobrovolníci s pozitivním nálezem protilátek nebo potvrzeným onemocněním covidem-19. U části z nich byla rovněž sledována dynamika vývoje protilátek po očkování. Účastníci měsíčně vyplňovali dotazníky o symptomech respiračních infekcí, zaměřené na detekci selhání vakcíny. Výsledky: Studie zahrnula 166 účastníků, kteří před očkováním neměli protilátky proti S proteinu SARS-CoV-2. Medián věku byl 52 let, převažovaly ženy (71,1 %). Po první a druhé dávce vakcíny hladiny protilátek vykázaly významný vzestup, poté následoval postupný pokles. U všech účastníků, kromě jednoho imunosuprimovaného, proběhla sérokonverze. Podíl selhání vakcíny proti onemocnění covidem-19 do 12 měsíců byl 13,3 %. Druhá kohorta zahrnula 60 účastníků s předchozí infekcí SARS-CoV-2. Po očkování došlo u této skupiny k výraznému nárůstu hladin protilátek, který byl vyšší než u osob bez předchozí infekce. Druhá dávka u těchto osob již nezvýšila hladiny protilátek statisticky významně. Závěry: Studie potvrdila, že očkování mRNA vakcínou BNT162b2 vyvolává silnou protilátkovou odpověď, s většinovou sérokonverzí již po první dávce. Starší osoby vykazovaly nižší protilátkovou odpověď, což zdůrazňuje význam posilujících dávek. U osob s prožitou infekcí SARS-CoV-2 byly po první dávce vakcíny protilátky výrazně vyšší než u naivních jedinců. Výsledky přispívají k porozumění dynamiky protilátkové odpovědi a naznačují potřebu dalšího výzkumu zaměřeného na optimalizaci očkovacích schémat.
Objective: An observational study was conducted from 2021 to 2022 to track the dynamics of the production of IgG antibody against the SARS-CoV-2 S protein in individuals vaccinated with two doses of the Pfizer-BioNTech mRNA vaccine BNT162b2 (Comirnaty). The study included a group of volunteers without any previous signs of SARS-CoV-2 infection as well as a group with a history of natural COVID-19 infection. The primary objective was to monitor antibody levels up to 12 months after the second dose and determine the proportion of vaccinated individuals who underwent seroconversion. The study also evaluated cases of vaccine failure within 13 months post-vaccination. Methods: Before the vaccination began, participants had laboratory tests for IgG antibodies against the SARS-CoV-2 S protein, and their medical history related to COVID-19 was taken. Based on negative test results, a cohort of immunologically na?ve individuals was formed and subsequently vaccinated with two doses of BNT162b2 (Comirnaty). Venous blood samples were collected at six time points: before the first dose, 2–3 weeks after the first dose, one month after the second dose, 3–4 months after the second dose, 6 months after the second dose, and 12 months after the second dose. A supplementary cohort included volunteers with positive antibody findings or confirmed COVID-19 infection. In some of these individuals, the dynamics of post-vaccination antibody response was also monitored. Participants filled out monthly questionnaires about respiratory infection symptoms to detect vaccine failure. Results: The study included 166 participants who did not have SARS-CoV-2 S protein antibodies prior to vaccination. The median age was 52 years, with a higher proportion of women (71.1%). After the first and second doses of the vaccine, antibody levels showed a significant increase, followed by a gradual decline over 12 months. Seroconversion occurred in all participants except for one immunosuppressed individual. The vaccine failure rate against COVID-19 within 12 months was 13.3%. The second cohort included 60 participants with prior SARS-CoV-2 infection. In this group, post-vaccination antibody levels increased significantly, more than in individuals without prior infection. The second dose did not result in further statistically significant increase in antibody levels for this cohort. Conclusions: The study confirmed that the BNT162b2 mRNA vaccine induces a strong antibody response, with the majority of participants experiencing seroconversion after the first dose. Older individuals exhibited a lower antibody response, highlighting the importance of booster doses. In individuals with prior SARS-CoV-2 infection, antibody levels were significantly higher after the first vaccine dose than in na?ve individuals. These findings add to understanding antibody response dynamics and suggest the need for further research focused on optimizing vaccination schedules.
