RESUMO
The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.
Assuntos
Endonucleases , Elementos Nucleotídeos Longos e Dispersos , DNA Polimerase Dirigida por RNA , Transcrição Reversa , Humanos , Microscopia Crioeletrônica , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Cristalografia por Raios X , DNA/biossíntese , DNA/genética , Imunidade Inata , Interferons/biossínteseRESUMO
During their proliferation and the host's concomitant attempts to suppress it, LINE-1 (L1) retrotransposons give rise to a collection of heterogeneous ribonucleoproteins (RNPs); their protein and RNA compositions remain poorly defined. The constituents of L1-associated macromolecules can differ depending on numerous factors, including, for example, position within the L1 life cycle, whether the macromolecule is productive or under suppression, and the cell type within which the proliferation is occurring. This chapter describes techniques that aid the capture and characterization of protein and RNA components of L1 macromolecules from tissues that natively express them. The protocols described have been applied to embryonal carcinoma cell lines that are popular model systems for L1 molecular biology (e.g., N2102Ep, NTERA-2, and PA-1 cells), as well as colorectal cancer tissues. N2102Ep cells are given as the use case for this chapter; the protocols should be applicable to essentially any tissue exhibiting endogenous L1 expression with minor modifications.
Assuntos
Elementos Nucleotídeos Longos e Dispersos , Retroelementos , Substâncias Macromoleculares , Células-Tronco de Carcinoma Embrionário , RNARESUMO
A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.
Cells are constantly perceiving and responding to changes in their surroundings, and challenging conditions such as extreme heat or toxic chemicals can put cells under stress. When this happens, protein production can be affected. Proteins are long chains of chemical building blocks called amino acids, and they can only perform their roles if they fold into the right shape. Some proteins fold easily and remain folded, but others can be unstable and often become misfolded. Unfolded proteins can become a problem because they stick to each other, forming large clumps called aggregates that can interfere with the normal activity of cells, causing damage. The causes of stress that have a direct effect on protein folding are called proteotoxic stresses, and include, for example, high temperatures, which make proteins more flexible and unstable, increasing their chances of becoming unfolded. To prevent proteins becoming misfolded, cells can make 'protein chaperones', a type of proteins that help other proteins fold correctly and stay folded. The production of protein chaperones often increases in response to proteotoxic stress. However, there are other types of stress too, such as genotoxic stress, which damages DNA. It is unclear what effect genotoxic stress has on protein folding. Huiting et al. studied protein folding during genotoxic stress in human cells grown in the lab. Stress was induced by either blocking the proteins that repair DNA or by 'trapping' the proteins that release DNA tension, both of which result in DNA damage. The analysis showed that, similar to the effects of proteotoxic stress, genotoxic stress increased the number of proteins that aggregate, although certain proteins formed aggregates even without stress, particularly if they were common and relatively unstable proteins. Huiting et al.'s results suggest that aggregation increases in cells under genotoxic stress because the cells fail to produce enough chaperones to effectively fold all the proteins that need it. Indeed, Huiting et al. showed that aggregates contain many proteins that rely on chaperones, and that increasing the number of chaperones in stressed cells reduced protein aggregation. This work shows that genotoxic stress can affect protein folding by limiting the availability of chaperones, which increases protein aggregation. Remarkably, there is a substantial overlap between proteins that aggregate in diseases that affect the brain such as Alzheimer's disease and proteins that aggregate after genotoxic stress. Therefore, further research could focus on determining whether genotoxic stress is involved in the progression of these neurological diseases.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA Topoisomerases/metabolismo , Chaperonas Moleculares/metabolismo , Dano ao DNA , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Peptídeos/metabolismo , Agregados Proteicos , Dobramento de Proteína , Proteoma/metabolismo , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Fast photochemical oxidation of proteins (FPOP) is a MS-based method that has proved useful in studies of protein structures, interactions, conformations, and protein folding. The success of this method relies on the irreversible labeling of solvent-exposed amino acid side chains by hydroxyl radicals. FPOP generates these radicals through laser-induced photolysis of hydrogen peroxide. The data obtained provide residue-level resolution of protein structures and interactions on the microsecond timescale, enabling investigations of fast processes such as protein folding and weak protein-protein interactions. An extensive comparison between FPOP and other footprinting techniques gives insight on their complementarity as well as the robustness of FPOP to provide unique structural information once unattainable. The versatility of this method is evidenced by both the heterogeneity of samples that can be analyzed by FPOP and the myriad of applications for which the method has been successfully used: from proteins of varying size to intact cells. This review discusses the wide applications of this technique and highlights its high potential. Applications including, but not limited to, protein folding, membrane proteins, structure elucidation, and epitope mapping are showcased. Furthermore, the use of FPOP has been extended to probing proteins in cells and in vivo These promising developments are also presented herein.
Assuntos
Proteínas/química , Proteômica/métodos , Cromatografia Líquida de Alta Pressão , Radical Hidroxila/química , Lasers , Espectrometria de Massas , Oxirredução , Dobramento de Proteína , Proteínas/metabolismoRESUMO
Despite significant affinity to carbonyl oxygens, thermal hydrogen atoms attach to unmodified polypeptides at a very low rate, while the hydrogen-hydrogen exchange rate is high. Here, using the novel omnitrap setup, we found that attachment to polypeptides is much more facile when radical site is already present, but the rate decreases for larger radical ions. The likely explanation is the intramolecular hydrogen atom rearrangement in hydrogen-deficient radicals to a more stable or less accessible site.
Assuntos
Radicais Livres/química , Gases/química , Hidrogênio/química , Peptídeos/química , Cátions , Modelos MolecularesRESUMO
In this paper, the first study of cationic cyanine dye Astrazon Orange-R by combined spectroscopic and theoretical investigation is presented. It is shown that molecular modeling of Astrazon Orange-R is in very good agreement with experiment, allowing us to gain insight into its complicated photophysics. A solvent viscosity controlled relaxation of excited states, involving cyanine isomerization, is also outlined.