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Acta Biochimica Polonica Jan 2021Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate,...
Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate, regulates glucose homeostasis by controlling the glyconeogenic pathway. FBPase requires divalent cations, such as Mg2+, Mn2+, or Zn2+, for its catalytic activity; however, calcium ions inhibit the muscle isoform of FBPase by interrupting the movement of the catalytic loop. It has been shown that residue E69 in this loop plays a key role in the sensitivity of muscle FBPase towards calcium ions. The study presented here is based on five crystal structures of wild-type human muscle FBPase and its E69Q mutant in complexes with the substrate and product of the enzymatic reaction, namely F1,6BP and F6P. The ligands are bound in the active site of the studied proteins in the same manner and have excellent definition in the electron density maps. In all studied crystals, the homotetrameric enzyme assumes the same cruciform quaternary structure, with the κ angle, which describes the orientation of the upper dimer with respect to the lower dimer, of -85o. This unusual quaternary arrangement of the subunits, characteristic of the R-state of muscle FBPase, is also observed in solution by small-angle X-ray scattering (SAXS).
Topics: Biocatalysis; Catalytic Domain; Crystallization; Fructose-Bisphosphatase; Fructosephosphates; Humans; Hydrogen Bonding; Hydrolysis; Ligands; Models, Molecular; Molecular Weight; Muscles; Mutant Proteins; Protein Multimerization; Protein Structure, Quaternary; Protein Subunits; Scattering, Small Angle; X-Ray Diffraction
PubMed: 33502838
DOI: 10.18388/abp.2020_5554 -
The Journal of Clinical Investigation Sep 2021Herculean efforts by the Wellcome Sanger Institute, the National Cancer Institute, and the National Human Genome Research Institute to sequence thousands of tumors... (Review)
Review
Herculean efforts by the Wellcome Sanger Institute, the National Cancer Institute, and the National Human Genome Research Institute to sequence thousands of tumors representing all major cancer types have yielded more than 700 genes that contribute to neoplastic growth when mutated, amplified, or deleted. While some of these genes (now included in the COSMIC Cancer Gene Census) encode proteins previously identified in hypothesis-driven experiments (oncogenic transcription factors, protein kinases, etc.), additional classes of cancer drivers have emerged, perhaps none more surprisingly than RNA-binding proteins (RBPs). Over 40 RBPs responsible for virtually all aspects of RNA metabolism, from synthesis to degradation, are recurrently mutated in cancer, and just over a dozen are considered major cancer drivers. This Review investigates whether and how their RNA-binding activities pertain to their oncogenic functions. Focusing on several well-characterized steps in RNA metabolism, we demonstrate that for virtually all cancer-driving RBPs, RNA processing activities are either abolished (the loss-of-function phenotype) or carried out with low fidelity (the LoFi phenotype). Conceptually, this suggests that in normal cells, RBPs act as gatekeepers maintaining proper RNA metabolism and the "balanced" proteome. From the practical standpoint, at least some LoFi phenotypes create therapeutic vulnerabilities, which are beginning to be exploited in the clinic.
Topics: Active Transport, Cell Nucleus; Databases, Genetic; Humans; Metabolic Networks and Pathways; MicroRNAs; Models, Biological; Mutant Proteins; Mutation; Neoplasm Proteins; Neoplasms; Phenotype; Protein Biosynthesis; RNA Processing, Post-Transcriptional; RNA Splicing; RNA, Neoplasm; RNA-Binding Proteins; Transcription, Genetic
PubMed: 34523614
DOI: 10.1172/JCI151627 -
Nature Communications Jun 2023Natural proteins exhibit rich structural diversity based on the folds of an invariably linear chain. Macromolecular catenanes that cooperatively fold into a single...
Natural proteins exhibit rich structural diversity based on the folds of an invariably linear chain. Macromolecular catenanes that cooperatively fold into a single domain do not belong to the current protein universe, and their design and synthesis open new territories in chemistry. Here, we report the design, synthesis, and properties of a single-domain green fluorescent protein catenane via rewiring the connectivity of GFP's secondary motifs. The synthesis could be achieved in two steps via a pseudorotaxane intermediate or directly via expression in cellulo. Various proteins-of-interest may be inserted at the loop regions to give fusion protein catenanes where the two subunits exhibit enhanced thermal resilience, thermal stability, and mechanical stability due to strong conformational coupling. The strategy can be applied to other proteins with similar fold, giving rise to a family of single-domain fluorescent proteins. The results imply that there may be multiple protein topological variants with desirable functional traits beyond their corresponding linear protein counterparts, which are now made accessible and fully open for exploration.
Topics: Green Fluorescent Proteins; Anthracenes; Catenanes; Coloring Agents; Mutant Proteins
PubMed: 37311944
DOI: 10.1038/s41467-023-39233-7 -
Cell Reports Jan 2023Huntington's disease (HD) is caused by expanded CAG repeats in the huntingtin gene (HTT) resulting in expression of mutant HTT proteins (mHTT) with extended...
Huntington's disease (HD) is caused by expanded CAG repeats in the huntingtin gene (HTT) resulting in expression of mutant HTT proteins (mHTT) with extended polyglutamine tracts, including in striatal neurons and astrocytes. It is unknown whether pathophysiology in vivo can be attenuated by lowering mHTT in either cell type throughout the brain, and the relative contributions of neurons and astrocytes to HD remain undefined. We use zinc finger protein (ZFP) transcriptional repressors to cell-selectively lower mHTT in vivo. Astrocytes display loss of essential functions such as cholesterol metabolism that are partly driven by greater neuronal dysfunctions, which encompass neuromodulation, synaptic, and intracellular signaling pathways. Using transcriptomics, proteomics, electrophysiology, and behavior, we dissect neuronal and astrocytic contributions to HD pathophysiology. Remarkably, brain-wide delivery of neuronal ZFPs results in strong mHTT lowering, rescue of HD-associated behavioral and molecular phenotypes, and significant extension of lifespan, findings that support translational development.
Topics: Animals; Huntington Disease; Astrocytes; Huntingtin Protein; Neurons; Transcription Factors; Zinc Fingers; Mutant Proteins; Disease Models, Animal
PubMed: 36640336
DOI: 10.1016/j.celrep.2022.111953 -
International Journal of Molecular... Dec 2019The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of... (Review)
Review
The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of p53 as a tumour suppressor has been thoroughly investigated, but the functions of all isoforms and mutants and the interplay between them are still poorly understood. Mutant p53 proteins lose p53 function, display dominant-negative (DN) activity and display gain-of-function (GOF) to varying degrees. GOF was originally attributed to mutant p53's inhibitory function over the p53 family members p63 and p73. It has become apparent that this is not the only way in which mutant p53 operates as a large number of transcription factors that are not related to p53 are activated on mutant p53 binding. This raises the question to what extent mutant p53 binding to p63 and p73 plays a role in mutant p53 GOF. In this review, we discuss the literature around the interaction between mutant p53 and family members, including other binding partners, the functional consequences and potential therapeutics.
Topics: Humans; Mutant Proteins; Mutation; Neoplasms; Protein Binding; Protein Isoforms; Transcription Factors; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins
PubMed: 31817935
DOI: 10.3390/ijms20246188 -
Journal of Extracellular Vesicles Feb 2023Extracellular vesicles (EVs) contain more than 100 proteins. Whether there are EVs proteins that act as an 'organiser' of protein networks to generate a new or different...
Extracellular vesicles (EVs) contain more than 100 proteins. Whether there are EVs proteins that act as an 'organiser' of protein networks to generate a new or different biological effect from that identified in EV-producing cells has never been demonstrated. Here, as a proof-of-concept, we demonstrate that EV-G12D-mutant KRAS serves as a leader that forms a protein complex and promotes lung inflammation and tumour growth via the Fn1/IL-17A/FGF21 axis. Mechanistically, in contrast to cytosol derived G12D-mutant KRAS complex from EVs-producing cells, EV-G12D-mutant KRAS interacts with a group of extracellular vesicular factors via fibronectin-1 (Fn1), which drives the activation of the IL-17A/FGF21 inflammation pathway in EV recipient cells. We show that: (i), depletion of EV-Fn1 leads to a reduction of a number of inflammatory cytokines including IL-17A; (ii) induction of IL-17A promotes lung inflammation, which in turn leads to IL-17A mediated induction of FGF21 in the lung; and (iii) EV-G12D-mutant KRAS complex mediated lung inflammation is abrogated in IL-17 receptor KO mice. These findings establish a new concept in EV function with potential implications for novel therapeutic interventions in EV-mediated disease processes.
Topics: Mice; Animals; Interleukin-17; Proto-Oncogene Proteins p21(ras); Mutant Proteins; Extracellular Vesicles; Lung Neoplasms; Pneumonia
PubMed: 36754903
DOI: 10.1002/jev2.12307 -
Cells Oct 2022In this review, we shed light on recent advances regarding the characterization of biochemical pathways of cellular mechanosensing and mechanotransduction with... (Review)
Review
In this review, we shed light on recent advances regarding the characterization of biochemical pathways of cellular mechanosensing and mechanotransduction with particular attention to their role in neurodegenerative disease pathogenesis. While the mechanistic components of these pathways are mostly uncovered today, the crosstalk between mechanical forces and soluble intracellular signaling is still not fully elucidated. Here, we recapitulate the general concepts of mechanobiology and the mechanisms that govern the mechanosensing and mechanotransduction processes, and we examine the crosstalk between mechanical stimuli and intracellular biochemical response, highlighting their effect on cellular organelles' homeostasis and dysfunction. In particular, we discuss the current knowledge about the translation of mechanosignaling into biochemical signaling, focusing on those diseases that encompass metabolic accumulation of mutant proteins and have as primary characteristics the formation of pathological intracellular aggregates, such as Alzheimer's Disease, Huntington's Disease, Amyotrophic Lateral Sclerosis and Parkinson's Disease. Overall, recent findings elucidate how mechanosensing and mechanotransduction pathways may be crucial to understand the pathogenic mechanisms underlying neurodegenerative diseases and emphasize the importance of these pathways for identifying potential therapeutic targets.
Topics: Alzheimer Disease; Humans; Mechanotransduction, Cellular; Mutant Proteins; Neurodegenerative Diseases; Parkinson Disease
PubMed: 36231055
DOI: 10.3390/cells11193093 -
Biomolecules Apr 2022is a tumor suppressor gene that encodes a sequence-specific DNA-binding transcription factor activated by stressful stimuli; it upregulates target genes involved in... (Review)
Review
is a tumor suppressor gene that encodes a sequence-specific DNA-binding transcription factor activated by stressful stimuli; it upregulates target genes involved in growth suppression, cell death, DNA repair, metabolism, among others. is the most frequently mutated gene in tumors, with mutations not only leading to loss-of-function (LOF), but also gain-of-function (GOF) that promotes tumor progression, and metastasis. The tumor-specific status of mutant p53 protein has suggested it is a promising target for cancer therapy. We summarize the current progress of targeting wild-type and mutant p53 for cancer therapy through biotherapeutic and biopharmaceutical methods for (1) boosting p53 activity in cancer, (2) p53-dependent and p53-independent strategies for targeting p53 pathway functional restoration in p53-mutated cancer, (3) targeting p53 in immunotherapy, and (4) combination therapies targeting p53, p53 checkpoints, or mutant p53 for cancer therapy.
Topics: Cell Death; Humans; Mutant Proteins; Mutation; Neoplasms; Tumor Suppressor Protein p53
PubMed: 35454137
DOI: 10.3390/biom12040548 -
Cells Jan 2022Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by five mutations in the gene. Autophagy and late endocytic trafficking...
Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by five mutations in the gene. Autophagy and late endocytic trafficking were already characterized in CMT2B. Indeed, impairment of autophagy and an increase in lysosomal degradative activity were found in cells expressing the mutant proteins. Recently, we described a novel mutation associated with predominantly motor CMT2 and impaired EGFR trafficking. With the aim to analyze the autophagy process and lysosomal activity in CMT2B fibroblasts carrying the p.K126R RAB7 novel mutation and to investigate further the causes of the different phenotype, we have performed Western blot, immunofluorescence and cytometric analyses monitoring autophagic markers and endocytic proteins. Moreover, we investigated lipophagy by analyzing accumulation of lipid droplets and their co-localization with endolysosomal degradative compartments. We found that cells expressing the RAB7 mutant protein were characterized by impairment of autophagy and lipophagy processes and by a moderate increase in lysosomal activity compared to the previously studied cells carrying the RAB7 mutation. Thus, we concluded that EGFR trafficking alterations and a moderate increase in lysosomal activity with concomitant impairment of autophagy could induce the specific predominantly motor phenotype observed in K126R patients.
Topics: Autophagy; Charcot-Marie-Tooth Disease; ErbB Receptors; Fibroblasts; Humans; Laminopathies; Lysosomes; Mutant Proteins; Mutation; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins
PubMed: 35159308
DOI: 10.3390/cells11030496 -
Cells May 2020Annexin A2 (AnxA2) is a cytosolic Ca regulated membrane binding protein that can induce lipid domain formation and plays a role in exocytosis and endocytosis. To better...
Annexin A2 (AnxA2) is a cytosolic Ca regulated membrane binding protein that can induce lipid domain formation and plays a role in exocytosis and endocytosis. To better understand the mode of annexin-membrane interaction, we analyzed membrane-bound AnxA2 assemblies by employing a novel 3-armed chemical crosslinker and specific AnxA2 mutant proteins. Our data show that AnxA2 forms crosslinkable oligomers upon binding to membranes containing negatively charged phospholipids. AnxA2 mutants with amino acid substitutions in residues predicted to be involved in lateral protein-protein interaction show compromised oligomer formation, albeit still being capable of binding to negatively charged membranes in the presence of Ca. These results suggest that lateral protein-protein interactions are involved in the formation of AnxA2 clusters on a biological membrane.
Topics: Alkylation; Annexin A2; Biotin; Cell Membrane; Cross-Linking Reagents; Humans; Models, Molecular; Mutant Proteins; Protein Binding; Protein Multimerization
PubMed: 32397265
DOI: 10.3390/cells9051169