Literatura académica sobre el tema "Oligomerization, ribonucleases"

Bovine pancreatic RNase A (ribonuclease A) aggregates to form various types of catalytically active oligomers during lyophilization from aqueous acetic acid solutions. Each oligomeric species is present in at least two conformational isomers. The structures of two dimers and one of the two trimers have been solved, while plausible models have been proposed for the structures of a second trimer and two tetrameric conformers. In this review, these structures, as well as the general conditions for RNase A oligomerization, based on the well known 3D (three-dimensional) domain-swapping mechanism, are described and discussed. Attention is also focused on some functional properties of the RNase A oligomers. Their enzymic activities, particularly their ability to degrade double-stranded RNAs and polyadenylate, are summarized and discussed. The same is true for the remarkable antitumour activity of the oligomers, displayed in vitro and in vivo, in contrast with monomeric RNase A, which lacks these activities. The RNase A multimers also show an aspermatogenic action, but lack any detectable embryotoxicity. The fact that both activity against double-stranded RNA and the antitumour action increase with the size of the oligomer suggests that these activities may share a common structural requirement, such as a high number or density of positive charges present on the RNase A oligomers.

Protein oligomerization is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Human Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads to abnormal protein oligomerization, resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson’s disease. While a score of disease linked h-ANG mutants has been studied by X-ray diffraction, some elude crystallization. There is also a debate regarding the structure that RNA fragments adopt after cleavage by h-ANG. Here, to better understand the beginning of the process that leads to aberrant protein oligomerization, the solution secondary structure and residue-level dynamics of WT h-ANG and two mutants i.e., H13A and R121C, are characterized by multidimensional heteronuclear NMR spectroscopy under near-physiological conditions. All three variants are found to adopt well folded and highly rigid structures in the solution, although the elements of secondary structure are somewhat shorter than those observed in crystallography studies. R121C alters the environment of nearby residues only. By contrast, the mutation H13A affects local residues as well as nearby active site residues K40 and H114. The conformation characterization by CD and 1D 1H NMR spectroscopies of tRNAAla before and after h-ANG cleavage reveals a retention of the duplex structure and little or no G-quadruplex formation.

The N1/T1 RNase superfamily comprises enzymes with well-established antitumor effects, such as ribotoxins secreted by fungi, primarily byAspergillusandPenicilliumspecies, and bacterial RNase secreted byB. pumilus(binase) andB. amyloliquefaciens(barnase). RNase is regarded as an alternative to classical chemotherapeutic agents due to its selective cytotoxicity towards tumor cells. New RNase with a high degree of structural similarity with binase (73%) and barnase (74%) was isolated and purified fromBacillus licheniformis(balifase, calculated molecular weight 12421.9 Da, pI 8.91). The protein sample with enzymatic activity of 1.5 × 106units/A280was obtained. The physicochemical properties of balifase are similar to those of barnase. However, in terms of its gene organization and promoter activity, balifase is closer to binase. The unique feature of balifase gene organization consists in the fact that genes of RNase and its inhibitor are located in one operon. Similarly to biosynthesis of binase, balifase synthesis is induced under phosphate starvation; however, in contrast to binase, balifase does not form dimers under natural conditions. We propose that the highest stability of balifase among analyzed RNase types allows the protein to retain its structure without oligomerization.

Inflammasomes are cytosolic innate immune sensors of pathogen infection and cellular damage that induce caspase-1–mediated inflammation upon activation. Although inflammation is protective, uncontrolled excessive inflammation can cause inflammatory diseases and can be detrimental, such as in coronavirus disease (COVID-19). However, the underlying mechanisms that control inflammasome activation are incompletely understood. Here we report that the leucine-rich repeat (LRR) protein ribonuclease inhibitor (RNH1), which shares homology with LRRs of NLRP (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing) proteins, attenuates inflammasome activation. Deletion of RNH1 in macrophages increases interleukin (IL)-1β production and caspase-1 activation in response to inflammasome stimulation. Mechanistically, RNH1 decreases pro-IL-1β expression and induces proteasome-mediated caspase-1 degradation. Corroborating this, mouse models of monosodium urate (MSU)-induced peritonitis and lipopolysaccharide (LPS)-induced endotoxemia, which are dependent on caspase-1, respectively, show increased neutrophil infiltration and lethality in Rnh1−/− mice compared with wild-type mice. Furthermore, RNH1 protein levels were negatively related with disease severity and inflammation in hospitalized COVID-19 patients. We propose that RNH1 is a new inflammasome regulator with relevance to COVID-19 severity.

Poly(A)-specific ribonuclease (PARN) is a deadenylase that degrades the poly(A) tail of eukaryotic mRNA. PARN also interacts with the 5’-cap structure of the mRNA. The binding of the cap structure enhances the deadenylation rate. PARN has previously been described as a dimer. We have studied PARN with size exclusion chromatography to investigate the oligomeric composition and revealed oligomeric compositions of PARN that are larger than dimeric PARN. Deadenylation assays have been used to measure the cap stimulated activity of PARN. The deadenylation assays showed that the cap stimulated activity of PARN correlated with the abundance of oligomers corresponding in size to tetrameric PARN. We present a model for tetrameric PARN and propose a mechanistic model for how the cap stimulates PARN mediated deadenylation.

The three dimensional domain swapping (3D-DS) mechanism represents a useful strategy that proteins can follow to self-associate. This mechanism requires the presence of a flexible portion that generally links the N- and/or C-terminus of the protein to its core. This portion, called hinge loop, can adopt different conformations as a function of the environmental conditions and allows the mentioned N- and/or C-terminal domains to be detached from the protein core and to be swapped with an identical domain of another protomer. This induces the formation of dimer(s) or larger oligomer(s) that often display, or enforce, biological properties that are absent, or attenuated, in the native monomer. The 3D-DS mechanism is shared by some proteins involved in amyloidosis, such as the human prion protein, β2-microglobulin, or some cystatins. Although not being amyloidogenic, the “pancreatic-type” RNases, i.e. resembling the features of the well-known pancreatic bovine RNase A, have become milestones to comprehend the determinants ruling out the 3D-DS mechanism. This is true especially for RNase A and for its natively dimeric homolog bovine seminal (BS)-RNase, whose structural determinants characterizing their self-association through 3D-DS have been deeply studied in the recent past. Moreover, also other ribonucleases included in the same RNase A super-family can oligomerize through this mechanism, as for example onconase (ONC), that can be induced to dimerize through the 3D-DS of its N-terminal domain. In this thesis, my aim was to elucidate some structural determinants that settle the tendency of some pancreatic-type RNases to self-associate, keeping the known features of RNase A as a reference. Therefore, we firstly investigated if the different methods that can be used to obtain the oligomers of RNase A might influence their properties. We treated RNase A with two different protocols: in particular, i. lyophilization of acetic acid solutions of the protein, or, ii. thermal incubations performed at 60°C in 20 or 40% aqueous ethanol. We observed that the enzymatic activity of the monomers and of the N- and C-swapped dimers (ND and CD, respectively) are slightly affected by the particular method used to induce their self-association. Then, I focused my attention to onconase (ONC), an amphibian member of the “pancreatic-type” RNase super-family lead by RNase A. In particular my aim was to unlock its C-terminal domain, that in the wild-type is blocked by a disulfide bond involving its C-terminal Cys104 residue, in order to investigate if this RNase variant may dimerize also through the C-terminal-end swapping. Unfortunately, none of numerous ONC mutant that I produced could form either the C-dimer, or also larger oligomers. In addition, all these variants formed less N-dimer than the wild type, confirming the actual mutual influence existing between the N- and C-termini of the protein, as it occurs for other RNases. We also supposed that the impossibility for ONC to form a C-dimer could be ascribable to the elongation of its C-terminal domain, with respect to RNase A. Therefore, we analyzed also the aggregation propensity of human pancreatic RNase, a variant that displays a C-terminus elongation of four AA residues in comparison with RNase A, similarly to ONC. Both the wt and a mutant obtained by the deleting the four mentioned residues displayed SEC profiles qualitatively very similar to the one of RNase A. Therefore, the human variant actually displayed that it can extensively oligomerize, but we could also deduce that the C-terminal elongation influences only marginally this process. The last project I developed was focused on the analysis of the oligomerization tendency of human angiogenin (ANG), a 14 kDa RNase variant characterized by a low ribonucleolytic activity (10-5/10-6 fold less than of RNase A), however necessary for its crucial angiogenic effects. ANG is involved in tumorigenesis but it exerts also a survival-promoting effect on the central nervous system (CNS) neuronal progenitors. However, some ANG variants are involved in neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS) and Parkinson Disease (PD). ALS is a multifactor disease, but one hypothesis to explain the pathogenic effect could be related to a possible oligomerization and precipitation of the mutants in the CNS. Among the numerous pathogenic ANG variants existing, one candidate retained by some scientists prone to undergo self-association is the S28N mutant. In our hands, this variant showed to dimerize at a slightly higher extent than the wild type, that in turn dimerized as well. Then, other ANG pathogenic mutants, in particular H13A and Q117G, also showed to dimerize, but with less reproducible results. Anyway, we detected for the first time that also ANG can dimerize through the 3D-DS mechanism, as many other RNases do. In conclusion, all the results of this thesis can be considered a step forward to comprehend the determinants settling the 3D-DS dimerization, or oligomerization tendency of many pancreatic-type RNases, and to compare the determinants that induce the differences emerging within them.

In the work I report and analyze the strong influence of RNase A deamidation on the enzyme tendency to oligomerize, the structural and functional differences induced by three oligomerization methods on RNase A monomer and dimers, and the structural features of a dimeric species produced by induced ONC oligomerization

"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B.L. et al. (2007) Proteins 66, 183-195], were analyzed, and tested for their possible in vitro cytotoxic activity. Results: (i) Besides dimers, also trimers and higher oligomers can be identified among the products of the covalently linking reaction. (ii) The "zero-length" dimers prepared by us appear not to be a unique species, as was instead reported by Simons et al. The product is heterogeneous, as shown by the involvement in the amide bond of amino and carboxyl groups others than only those belonging to Lys66 and Glu9. This is demonstrated by results obtained with two RNase A mutants, E9A and K66A. (iii) The "zero-length" dimers degrade poly(A).poly(U) (dsRNA) and yeast RNA (ssRNA): while the activity against poly(A).poly(U) increases with the increase of the oligomer's basicity, the activity towards yeast RNA decreases with the increase of oligomers' basicity, in agreement with many previous data, but in contrast with the results reported by Simons et al. (iv) No cytotoxicity against various tumor cells lines could be evidenced in RNase A "zero-length" dimers. (v) They instead become cytotoxic if cationized by conjugation with polyethylenimine [Futami, J. et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, polyethylenimine derivatives of RNase A "zero-length" dimers and native, monomeric RNase A are equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, (vi) cytotoxicity seems not to be specific for tumor cells: polyethylenimine-cationized native RNase A is also cytotoxic towards human monocytes.
"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B.L. et al. (2007) Proteins 66, 183-195], were analyzed, and tested for their possible in vitro cytotoxic activity. Results: (i) Besides dimers, also trimers and higher oligomers can be identified among the products of the covalently linking reaction. (ii) The "zero-length" dimers prepared by us appear not to be a unique species, as was instead reported by Simons et al. The product is heterogeneous, as shown by the involvement in the amide bond of amino and carboxyl groups others than only those belonging to Lys66 and Glu9. This is demonstrated by results obtained with two RNase A mutants, E9A and K66A. (iii) The "zero-length" dimers degrade poly(A).poly(U) (dsRNA) and yeast RNA (ssRNA): while the activity against poly(A).poly(U) increases with the increase of the oligomer's basicity, the activity towards yeast RNA decreases with the increase of oligomers' basicity, in agreement with many previous data, but in contrast with the results reported by Simons et al. (iv) No cytotoxicity against various tumor cells lines could be evidenced in RNase A "zero-length" dimers. (v) They instead become cytotoxic if cationized by conjugation with polyethylenimine [Futami, J. et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, polyethylenimine derivatives of RNase A "zero-length" dimers and native, monomeric RNase A are equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, (vi) cytotoxicity seems not to be specific for tumor cells: polyethylenimine-cationized native RNase A is also cytotoxic towards human monocytes.