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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 -
Methods in Molecular Biology (Clifton,... 2022Normal functions of cell-surface proteins are dependent on their proper trafficking from the site of synthesis to the cell surface. Transport proteins mediating solute...
Normal functions of cell-surface proteins are dependent on their proper trafficking from the site of synthesis to the cell surface. Transport proteins mediating solute transfer across the plasma membrane constitute an important group of cell-surface proteins. There are several diseases resulting from mutations in these proteins that interfere with their transport function or trafficking, depending on the impact of the mutations on protein folding and structure. Recent advances in successful treatment of some of these diseases with small molecules which correct the mutations-induced folding and structural changes underline the need for detailed structural and biophysical characterization of membrane proteins. This requires methods to express and purify these proteins using heterologous expression systems. Here, using the solute carrier (SLC) transporter NaCT (Na-coupled citrate transporter) as an example, we describe experimental strategies for this approach. We chose this example because several mutations in NaCT, distributed throughout the protein, cause a severe neurologic disease known as early infantile epileptic encephalopathy-25 (EIEE-25). NaCT was modified with various peptide tags, including a RGS-His, a Twin-Strep, the SUMOstar domain, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. When transiently expressed in HEK293 cells, recombinant NaCT proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited citrate transport activity similar to the nontagged protein. Surface NaCT expression was enhanced by the presence of SUMOstar on the N-terminus. The dual-purpose peptide epitopes RGS-His and Twin-Strep facilitated detection of NaCT by immunohistochemistry and western blot and may serve useful tags for affinity purification. This approach sets the stage for future analyses of mutant NaCT proteins that may alter protein folding and trafficking. It also demonstrates the capability of a transient mammalian cell expression system to produce human NaCT of sufficient quality and quantity to augment future biophysical and structural studies and drug discovery efforts.
Topics: Animals; Biological Transport; Cell Membrane; HEK293 Cells; Humans; Mammals; Membrane Proteins; Mutant Proteins; Peptides; Symporters
PubMed: 35773591
DOI: 10.1007/978-1-0716-2368-8_18 -
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 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 -
Biochemical and Biophysical Research... Dec 2022Autoimmune diseases afflict nearly 10% of the world's population and have a serious impact on survival and quality of life. Unfortunately, the specific pathogenesis of... (Review)
Review
Autoimmune diseases afflict nearly 10% of the world's population and have a serious impact on survival and quality of life. Unfortunately, the specific pathogenesis of almost all autoimmune diseases is still unclear, with more research findings identifying some key pathogenic genes at the genetic level and several pathogenic inflammatory factor phenotypes. ERAP1 has been suggested as a potential therapeutic target for several autoimmune diseases, especially MHC-Ⅰ related. How the structure and antigenic peptide processing function of ERAP1 affect the pathogenesis of these autoimmune diseases needs to be elucidated more clearly. Genetic studies on single nucleotide polymorphism of ERAP1 provide a good bridge to better understand the relationship and pattern between ERAP1 structure, function, and disease. However, existing reviews have focused on the genetic association of ERAP1 SNPs with autoimmune diseases, and no one has specifically addressed how ERAP1 gene polymorphisms embodied at the protein level specifically mediate antigenic peptide editing and the development of multiple autoimmune diseases. In this paper, we present a comprehensive review of these ERAP1 SNPs associated with multiple autoimmune diseases, in particular the polymorphisms affecting their protein structure and enzyme function, and attempt to unravel the underlying structural and biochemical mechanisms by which ERAP1 affects the pathogenesis of multiple autoimmune diseases through the SNP-protein structure-function-disease relationship. This study will provide theoretical help and ideas for understanding the relationship between ERAP1 and autoimmune diseases and for drug design targeting wild-type and mutant proteins with different polymorphisms.
Topics: Humans; Aminopeptidases; Autoimmune Diseases; Genetic Predisposition to Disease; Minor Histocompatibility Antigens; Mutant Proteins; Peptides; Polymorphism, Single Nucleotide
PubMed: 36228519
DOI: 10.1016/j.bbrc.2022.09.086 -
Cells Nov 2020Understanding the link between agonist-induced phosphorylation of the mu-opioid receptor (MOR) and the associated physiological effects is critical for the development... (Review)
Review
Understanding the link between agonist-induced phosphorylation of the mu-opioid receptor (MOR) and the associated physiological effects is critical for the development of novel analgesic drugs and is particularly important for understanding the mechanisms responsible for opioid-induced tolerance and addiction. The family of G protein receptor kinases (GRKs) play a pivotal role in such processes, mediating phosphorylation of residues at the C-tail of opioid receptors. Numerous strategies, such as phosphosite specific antibodies and mass spectrometry have allowed the detection of phosphorylated residues and the use of mutant knock-in mice have shed light on the role of GRK regulation in opioid receptor physiology. Here we review our current understanding on the role of GRKs in the actions of opioid receptors, with a particular focus on the MOR, the target of most commonly used opioid analgesics such as morphine or fentanyl.
Topics: Animals; G-Protein-Coupled Receptor Kinases; Humans; Mutant Proteins; Phosphorylation; Receptors, Opioid, mu; Signal Transduction
PubMed: 33147802
DOI: 10.3390/cells9112400