Self-assembly is a common theme in proteins of unrelated sequences or functions. The human papillomavirus E7 oncoprotein is an extended dimer with an intrinsically disordered domain, that can form ...large spherical oligomers. These are the major species in the cytosol of HPV transformed and cancerous cells. E7 binds to a large number of targets, some of which lead to cell transformation. Thus, the assembly process not only is of biological relevance, but represents a model system to investigate a widely distributed mechanism.
Using various techniques, we monitored changes in secondary, tertiary and quaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formation of a monomeric "Z-nucleus" after zinc removal, followed by an elongation phase consisting of sequential second-order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 µM protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a "conformation editing" mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5 kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsically disordered domain stabilizes the oligomer, preventing an insoluble amyloid route.
We described a controlled ordered mechanism with features in common with soluble amyloid precursors, chaperones, and other spherical oligomers, thus sharing determining factors for symmetry, size and shape. In addition, such a controlled and discrete polymerization reaction provides a valuable tool for nanotechnological applications. Finally, its increased immunogenicity related to its supramolecular structure is the basis for the development of a promising therapeutic vaccine candidate for treating HPV cancerous lesions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A psychrotolerant marine bacterial strain, designated JUB59T, was isolated from Antarctic surface seawater and classified as a new species of the genus BIZIONIA: Here, we present the first draft ...genome sequence for this genus, which suggests interesting features such as UV resistance, hydrolytic exoenzymes, and nitrogen metabolism.
Production of soluble recombinant proteins is crucial to the development of industry and basic research. However, the aggregation due to the incorrect folding of the nascent polypeptides is still a ...mayor bottleneck. Understanding the factors governing protein solubility is important to grasp the underlying mechanisms and improve the design of recombinant proteins. Here we show a quantitative study of the expression and solubility of a set of proteins from Bizionia argentinensis. Through the analysis of different features known to modulate protein production, we defined two parameters based on the %MinMax algorithm to compare codon usage clusters between the host and the target genes. We demonstrate that the absolute difference between all %MinMax frequencies of the host and the target gene is significantly negatively correlated with protein expression levels. But most importantly, a strong positive correlation between solubility and the degree of conservation of codons usage clusters is observed for two independent datasets. Moreover, we evince that this correlation is higher in codon usage clusters involved in less compact protein secondary structure regions. Our results provide important tools for protein design and support the notion that codon usage may dictate translation rate and modulate co-translational folding.
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•Natural samples of different maturation from Vaca Muerta formation are studied.•They are compared with kerogens of obtained by closed non-hydrous pyrolysis.•Evolution of molecular ...structure of both sets are followed by 13CNMR, XPS and FTIR.•Most structural parameters showed an excellent correlation between sets.•Differences in the branched content and heteroatoms speciation have been found.
Artificial maturation of kerogen is a widely used technique to assess the hydrocarbon potential of shale rocks and to observe thermal transformations of organic matter in the laboratory. However, the degree of reproducibility of natural geological transformation at the molecular level is still not fully established. In the present work, a set of kerogens isolated from Vaca Muerta Formation core samples at varying levels of thermal maturity were studied. Another set of samples was obtained by anhydrous pyrolysis of kerogen in a closed system. Molecular structures measured by solid-state techniques (13C NMR, XPS and FTIR) were compared in natural and artificially matured samples at equivalent levels of thermal maturation. We observed that heating in an anhydrous closed system accurately reproduces most of the molecular structural changes observed during natural thermal maturation, with exceptions related to the branching degree and oxygen containing groups. The evolution of some parameters, such as N and S content, are highly variable in the natural samples because of differences in their deposition environments. In these cases, artificial thermal maturation changes are smaller and follow more clearly defined trends because variables other than thermal changes are not present. This is the first work comparing natural and artificial thermal maturation in the Vaca Muerta Formation and add valuable data regarding the limitation of artificial maturation that can be extrapolated to other formations.
One of the key parameters in assessing the production potential of unconventional oil and gas reservoirs is the quantification of organic matter, which is generally determined by pyrolysis. ...Laboratory nuclear magnetic resonance (NMR) is widely used in the oil industry; being a nondestructive method, it enables the possibility to perform several analyses on the same sample, which is desirable due to the heterogeneity of these types of geological formations. 13C spectra provide much information, from speciation to maturity determination. The main drawback of this technique is the inherently low signal obtained for these nuclei, rendering extremely long experimental times. A solution is using cross-polarization schemes that transfer the high polarization of nearby 1H nuclei to 13C ones, with the penalty that quantification is a priori not feasible. In this work, we analyze the cross-polarization evolution and conclude that quantifying the total organic content (TOC) is possible for a wide range of organic matter content and maturities for rock samples corresponding to the Neuquén and Cuyo basins in Argentina.
Human frataxin (FXN) is a highly conserved mitochondrial protein involved in iron homeostasis and activation of the iron-sulfur cluster assembly. FXN deficiency causes the neurodegenerative disease ...Friedreich's Ataxia. Here, we investigated the effect of alterations in loop-1, a stretch presumably essential for FXN function, on the conformational stability and dynamics of the native state. We generated four loop-1 variants, carrying substitutions, insertions and deletions. All of them were stable and well-folded proteins. Fast local motions (ps-ns) and slower long-range conformational dynamics (μs-ms) were altered in some mutants as judged by NMR. Particularly, loop-1 modifications impact on the dynamics of a distant region that includes residues from the β-sheet, helix α1 and the C-terminal. Remarkably, all the mutants retain the ability to activate cysteine desulfurase, even when two of them exhibit a strong decrease in iron binding, revealing a differential sensitivity of these functional features to loop-1 perturbation. Consequently, we found that even for a small and relatively rigid protein, engineering a loop segment enables to alter conformational dynamics through a long-range effect, preserving the native-state structure and important aspects of function.
•Loop-1 mutants of frataxin were designed to alter conformational dynamics.•Tertiary structure is mainly unaffected by the mutations.•Mutations result in both local and long-range effects on dynamics.•Iron binding and cysteine desulfurase activation are differentially affected.
The Pfam PF04536 TPM_phosphatase family is a broadly conserved family of domains found across prokaryotes, plants and invertebrates. Despite having a similar protein fold, members of this family have ...been implicated in diverse cellular processes and found in varied subcellular localizations. Very recently, the biochemical characterization of two evolutionary divergent TPM domains has shown that they are able to hydrolyze phosphate groups from different substrates. However, there are still incorrect functional annotations and uncertain relationships between the structure and function of this family of domains. BA41 is an uncharacterized single-pass transmembrane protein from the Antarctic psychrotolerant bacterium Bizionia argentinensis with a predicted compact extracytoplasmic TPM domain and a C-terminal cytoplasmic low complexity region. To shed light on the structural properties that enable TPM domains to adopt divergent roles, we here accomplish a comprehensive structural and functional characterization of the central TPM domain of BA41 (BA41-TPM). Contrary to its predicted function as a beta-propeller methanol dehydrogenase, light scattering and crystallographic studies showed that BA41-TPM behaves as a globular monomeric protein and adopts a conserved Rossmann fold, typically observed in other TPM domain structures. Although the crystal structure reveals the conservation of residues involved in substrate binding, no putative catalytic or intramolecular metal ions were detected. Most important, however, extensive biochemical studies demonstrated that BA41-TPM has hydrolase activity against ADP, ATP, and other di- and triphosphate nucleotides and shares properties of cold-adapted enzymes. The role of BA41 in extracellular ATP-mediated signaling pathways and its occurrence in environmental and pathogenic microorganisms is discussed.
The specific roles of Notch in progressive adulthood neurodegenerative disorders have begun to be unraveled in recent years. A number of independent studies have shown significant increases of Notch ...expression in brains from patients at later stages of sporadic Alzheimer’s disease (AD). However, the impact of Notch canonical signaling activation in the pathophysiology of AD is still elusive. To further investigate this issue, 2-month-old wild-type (WT) and hemizygous McGill-R-Thy1-APP rats (Tg(+/−)) were injected in CA1 with lentiviral particles (LVP) expressing the transcriptionally active fragment of Notch, known as Notch Intracellular Domain (NICD), (LVP-NICD), or control lentivirus particles (LVP-C). The Tg(+/−) rat model captures presymptomatic aspects of the AD pathology, including intraneuronal amyloid beta (Aβ) accumulation and early cognitive deficits. Seven months after LVP administration, Morris water maze test was performed, and brains isolated for biochemical and histological analysis. Our results showed a learning impairment and a worsening of spatial memory in LVP-NICD- as compared to LVP-C-injected Tg(+/−) rats. In addition, immuno histochemistry, ELISA multiplex, Western blot, RT-qPCR, and
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H-NMR spectrometry of cerebrospinal fluid (CSF) indicated that chronic expression of NICD promoted hippocampal vessel thickening with accumulation of Aβ in brain microvasculature, alteration of blood-brain barrier (BBB) permeability, and a decrease of CSF glucose levels. These findings suggest that, in the presence of early Aβ pathology, expression of NICD may contribute to the development of microvascular abnormalities, altering glucose transport at the BBB with impact on early decline of spatial learning and memory.
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