Human RNase 1 can extensively oligomerize through 3D domain swapping thanks to the crucial contribution of its C-terminus.

Human ribonuclease (RNase) 1 and bovine RNase A are the proto-types of the secretory "pancreatic-type" (pt)-RNase super-family. RNase A can oligomerize through the 3D domain swapping (DS) mechanism upon acetic acid (HAc) lyophilisation, producing enzymatically active oligomeric conformers by swapping both N- and C-termini. Also some RNase 1 mutants were found to self-associate through 3D-DS, however forming only N-swapped dimers. Notably, enzymatically active dimers and larger oligomers of wt-RNase 1 were collected here, in higher amount than RNase A, from HAc lyophilisation. In particular, RNase 1 self-associates through the 3D-DS of its N-terminus and, at a higher extent, of the C-terminus. Since RNase 1 is four-residues longer than RNase A, we further analyzed its oligomerization tendency in a mutant lacking the last four residues. The C-terminus role has been investigated also in amphibian onconase (ONC®), a pt-RNase that can form only a N-swapped dimer, since its C-terminus, that is three-residues longer than RNase A, is locked by a disulfide bond. While ONC mutants designed to unlock or cut this constraint were almost unable to dimerize, the RNase 1 mutant self-associated at a higher extent than the wt, suggesting a specific role of the C-terminus in the oligomerization of different RNases. Overall, RNase 1 reaches here the highest ability, among pt-RNases, to extensively self-associate through 3D-DS, paving the way for new investigations on the structural and biological properties of its oligomers.

Protein Oligomerization.

Protein self-association is a biologically remarkable event that involves and affects the structural and functional properties of proteins [...].

Slow Evolution toward "Super-Aggregation" of the Oligomers Formed through the Swapping of RNase A N-Termini: A Wish for Amyloidosis?

Natively monomeric RNase A can oligomerize upon lyophilization from 40% acetic acid solutions or when it is heated at high concentrations in various solvents. In this way, it produces many dimeric or oligomeric conformers through the three-dimensional domain swapping (3D-DS) mechanism involving both RNase A N- or/and C-termini. Here, we found many of these oligomers evolving toward not negligible amounts of large derivatives after being stored for up to 15 months at 4 °C in phosphate buffer. We call these species super-aggregates (SAs). Notably, SAs do not originate from native RNase A monomer or from oligomers characterized by the exclusive presence of the C-terminus swapping of the enzyme subunits as well. Instead, the swapping of at least two subunits' N-termini is mandatory to produce them. Through immunoblotting, SAs are confirmed to derive from RNase A even if they retain only low ribonucleolytic activity. Then, their interaction registered with Thioflavin-T (ThT), in addition to TEM analyses, indicate SAs are large and circular but not "amyloid-like" derivatives. This confirms that RNase A acts as an "auto-chaperone", although it displays many amyloid-prone short segments, including the 16-22 loop included in its N-terminus. Therefore, we hypothesize the opening of RNase A N-terminus, and hence its oligomerization through 3D-DS, may represent a preliminary step favoring massive RNase A aggregation. Interestingly, this process is slow and requires low temperatures to limit the concomitant oligomers' dissociation to the native monomer. These data and the hypothesis proposed are discussed in the light of protein aggregation in general, and of possible future applications to contrast amyloidosis.

Role of the Ribonuclease ONCONASE in miRNA Biogenesis and tRNA Processing: Focus on Cancer and Viral Infections.

The majority of transcribed RNAs do not codify for proteins, nevertheless they display crucial regulatory functions by affecting the cellular protein expression profile. MicroRNAs (miRNAs) and transfer RNA-derived small RNAs (tsRNAs) are effectors of interfering mechanisms, so that their biogenesis is a tightly regulated process. Onconase (ONC) is an amphibian ribonuclease known for cytotoxicity against tumors and antiviral activity. Additionally, ONC administration in patients resulted in clinical effectiveness and in a well-tolerated feature, at least for lung carcinoma and malignant mesothelioma. Moreover, the ONC therapeutic effects are actually potentiated by cotreatment with many conventional antitumor drugs. This review not only aims to describe the ONC activity occurring either in different tumors or in viral infections but also to analyze the molecular mechanisms underlying ONC pleiotropic and cellular-specific effects. In cancer, data suggest that ONC affects malignant phenotypes by generating tRNA fragments and miRNAs able to downregulate oncogenes expression and upregulate tumor-suppressor proteins. In cells infected by viruses, ONC hampers viral spread by digesting the primer tRNAs necessary for viral DNA replication. In this scenario, new therapeutic tools might be developed by exploiting the action of ONC-elicited RNA derivatives.

Upregulation of miR-34a-5p, miR-20a-3p and miR-29a-3p by Onconase in A375 Melanoma Cells Correlates with the Downregulation of Specific Onco-Proteins.

Onconase (ONC) is an amphibian secretory ribonuclease displaying cytostatic and cytotoxic activities against many mammalian tumors, including melanoma. ONC principally damages tRNA species, but also other non-coding RNAs, although its precise targets are not known. We investigated the ONC ability to modulate the expression of 16 onco-suppressor microRNAs (miRNAs) in the A375 BRAF-mutated melanoma cell line. RT-PCR and immunoblots were used to measure the expression levels of miRNAs and their regulated proteins, respectively. In silico study was carried out to verify the relations between miRNAs and their mRNA targets. A375 cell transfection with miR-20a-3p and miR-34a-5p mimics or inhibitors was performed. The onco-suppressors miR-20a-3p, miR-29a-3p and miR-34a-5p were highly expressed in 48-h ONC-treated A375 cells. The cytostatic effect of ONC in A375 cells was mechanistically explained by the sharp inhibition of cyclins D1 and A2 expression level, as well as by downregulation of retinoblastoma protein and cyclin-dependent-kinase-2 activities. Remarkably, the expression of kinases ERK1/2 and Akt, as well as of the hypoxia inducible factor-1α, was inhibited by ONC. All these proteins control pro-survival pathways. Finally, many crucial proteins involved in migration, invasion and metastatic potential were downregulated by ONC. Results obtained from transfection of miR-20a-3p and miR-34a-5p inhibitors in the presence of ONC show that these miRNAs may participate in the antitumor effects of ONC in the A375 cell line. In conclusion, we identified many intracellular downregulated proteins involved in melanoma cell proliferation, metabolism and progression. All mRNAs coding these proteins may be targets of miR-20a-3p, miR-29a-3p and/or miR-34a-5p, which are in turn upregulated by ONC. Data suggest that several known ONC anti-proliferative and anti-metastatic activities in A375 melanoma cells might depend on the upregulation of onco-suppressor miRNAs. Notably, miRNAs stability depends on the upstream regulation by long-non-coding-RNAs or circular-RNAs that can, in turn, be damaged by ONC ribonucleolytic activity.

Dimerization of Human Angiogenin and of Variants Involved in Neurodegenerative Diseases.

Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies.

The crystal structure of the domain-swapped dimer of onconase highlights some catalytic and antitumor activity features of the enzyme.

Onconase (ONC) is a monomeric amphibian "pancreatic-type" RNase endowed with remarkable anticancer activity. ONC spontaneously forms traces of a dimer (ONC-D) in solution, while larger amounts can be formed when ONC is lyophilized from mildly acidic solutions. Here, we report the crystal structure of ONC-D and analyze its catalytic and antitumor activities in comparison to ONC. ONC-D forms via the three-dimensional swapping of the N-terminal α-helix between two monomers, but it displays a significantly different quaternary structure from that previously modeled [Fagagnini A et al., 2017, Biochem J 474, 3767-81], and based on the crystal structure of the RNase A N-terminal swapped dimer. ONC-D presents a variable quaternary assembly deriving from a variable open interface, while it retains a catalytic activity that is similar to that of ONC. Notably, ONC-D displays antitumor activity against two human melanoma cell lines, although it exerts a slightly lower cytostatic effect than the monomer. The inhibition of melanoma cell proliferation by ONC or ONC-D is associated with the reduction of the expression of the anti-apoptotic B cell lymphoma 2 (Bcl2), as well as of the total expression and phosphorylation of the Signal Transducer and Activator of Transcription (STAT)-3. Phosphorylation is inhibited in both STAT3 Tyr705 and Ser727 key-residues, as well as in its upstream tyrosine-kinase Src. Consequently, both ONC species should exert their anti-cancer action by inhibiting the pro-tumor pleiotropic STAT3 effects deriving either by its phospho-tyrosine activation or by its non-canonical signaling pathways. Both ONC species, indeed, increase the portion of A375 cells undergoing apoptotic cell death. This study expands the variety of RNase domain-swapped dimeric structures, underlining the unpredictability of the open interface arrangement upon domain swapping. Structural data also offer valuable insights to analyze the differences in the measured ONC or ONC-D biological activities.

RNase A Domain-Swapped Dimers Produced Through Different Methods: Structure-Catalytic Properties and Antitumor Activity.

Upon oligomerization, RNase A can acquire important properties, such as cytotoxicity against leukemic cells. When lyophilized from 40% acetic acid solutions, the enzyme self-associates through the so-called three-dimensional domain swapping (3D-DS) mechanism involving both N- and/or C-terminals. The same species are formed if the enzyme is subjected to thermal incubation in various solvents, especially in 40% ethanol. We evaluated here if significant structural modifications might occur in RNase A N- or C-swapped dimers and/or in the residual monomer(s), as a function of the oligomerization protocol applied. We detected that the monomer activity vs. ss-RNA was partly affected by both protocols, although the protein does not suffer spectroscopic alterations. Instead, the two N-swapped dimers showed differences in the fluorescence emission spectra but almost identical enzymatic activities, while the C-swapped dimers displayed slightly different activities vs. both ss- or ds-RNA substrates together with not negligible fluorescence emission alterations within each other. Besides these results, we also discuss the reasons justifying the different relative enzymatic activities displayed by the N-dimers and C-dimers. Last, similarly with data previously registered in a mouse model, we found that both dimeric species significantly decrease human melanoma A375 cell viability, while only N-dimers reduce human melanoma MeWo cell growth.

NMR Characterization of Angiogenin Variants and tRNAAla Products Impacting Aberrant Protein Oligomerization.

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.

Biological Activities of Secretory RNases: Focus on Their Oligomerization to Design Antitumor Drugs.

Ribonucleases (RNases) are a large number of enzymes gathered into different bacterial or eukaryotic superfamilies. Bovine pancreatic RNase A, bovine seminal BS-RNase, human pancreatic RNase 1, angiogenin (RNase 5), and amphibian onconase belong to the pancreatic type superfamily, while binase and barnase are in the bacterial RNase N1/T1 family. In physiological conditions, most RNases secreted in the extracellular space counteract the undesired effects of extracellular RNAs and become protective against infections. Instead, if they enter the cell, RNases can digest intracellular RNAs, becoming cytotoxic and having advantageous effects against malignant cells. Their biological activities have been investigated either in vitro, toward a number of different cancer cell lines, or in some cases in vivo to test their potential therapeutic use. However, immunogenicity or other undesired effects have sometimes been associated with their action. Nevertheless, the use of RNases in therapy remains an appealing strategy against some still incurable tumors, such as mesothelioma, melanoma, or pancreatic cancer. The RNase inhibitor (RI) present inside almost all cells is the most efficacious sentry to counteract the ribonucleolytic action against intracellular RNAs because it forms a tight, irreversible and enzymatically inactive complex with many monomeric RNases. Therefore, dimerization or multimerization could represent a useful strategy for RNases to exert a remarkable cytotoxic activity by evading the interaction with RI by steric hindrance. Indeed, the majority of the mentioned RNases can hetero-dimerize with antibody derivatives, or even homo-dimerize or multimerize, spontaneously or artificially. This can occur through weak interactions or upon introducing covalent bonds. Immuno-RNases, in particular, are fusion proteins representing promising drugs by combining high target specificity with easy delivery in tumors. The results concerning the biological features of many RNases reported in the literature are described and discussed in this review. Furthermore, the activities displayed by some RNases forming oligomeric complexes, the mechanisms driving toward these supramolecular structures, and the biological rebounds connected are analyzed. These aspects are offered with the perspective to suggest possible efficacious therapeutic applications for RNases oligomeric derivatives that could contemporarily lack, or strongly reduce, immunogenicity and other undesired side-effects.

Onconase Restores Cytotoxicity in Dabrafenib-Resistant A375 Human Melanoma Cells and Affects Cell Migration, Invasion and Colony Formation Capability.

Melanoma is a lethal tumor because of its severe metastatic potential, and serine/threonine-protein kinase B-raf inhibitors (BRAFi) are used in patients harboring BRAF-mutation. Unfortunately, BRAFi induce resistance. Therefore, we tested the activity of onconase (ONC), a cytotoxic RNase variant, against BRAFi-resistant cells to re-establish the efficacy of the chemotherapy. To do so, an A375 dabrafenib-resistant (A375DR) melanoma cell subpopulation was selected and its behavior compared with that of parental (A375P) cells by crystal violet, 5-Bromo-2'-deoxyuridine incorporation, and cleaved poly(ADP-ribose) polymerase 1 (PARP1) western blot measurements. Then, nuclear p65 Nuclear Factor kappaB (NF-κB) and IκB kinases-α/ß (IKK) phosphorylation levels were measured. Gelatin zymography was performed to evaluate metalloproteinase 2 (MMP2) activity. In addition, assays to measure migration, invasion and soft agar colony formation were performed to examine the tumor cell dissemination propensity. ONC affected the total viability and the proliferation rate of both A375P and A375DR cell subpopulations in a dose-dependent manner and also induced apoptotic cell death. Among its pleiotropic effects, ONC reduced nuclear p65 NF-κB amount and IKK phosphorylation level, as well as MMP2 activity in both cell subpopulations. ONC decreased cell colony formation, migration, and invasion capability. Notably, it induced apoptosis and inhibited colony formation and invasiveness more extensively in A375DR than in A375P cells. In conclusion, ONC successfully counteracts melanoma malignancy especially in BRAFi-resistant cells and could become a tool against melanoma recurrence.

Influence of onconase in the therapeutic potential of PARP inhibitors in A375 malignant melanoma cells.

Human malignant melanoma is one of the most aggressive cancers, accompanied with poor prognosis, metastatic evolution and high mortality. Many strategies have been developed using BRAF and MEK inhibitors in spite of the classic therapy with alkylating agents, but failure related to the ability of the tumor to activate alternative proliferation pathways occurred after promising initial successes. Poly(ADP-ribose) polymerase (PARP) enzymes are well known to be crucial for DNA damage response, and PARP inhibition results in the accumulation of DNA strand breaks that induce cell injury. For this reason, PARP-inhibitors (PARPi) have become promising tools to counteract many cancer types. One of the most used by clinicians is olaparib, that, however, showed again cancer resistance in patients. Thus, new generation molecules have been designed mainly to counteract this problem. Among them, we chose to test AZD2461 on the particularly aggressive human melanoma A375 cell line. This drug is a PARPi significantly less prone than olaparib to undergo the P-glycoprotein-mediated efflux mechanism, one of those responsible for resistance, that in turn is the main adversity in melanoma therapy. Then, we analysed AZD2461 also together with the enzyme onconase (ONC) on the same A375 cells, to investigate if the combination of drugs could possibly increase the in vitro antitumor activity. ONC is a small amphibian "pancreatic-type" ribonuclease that is able to exert a remarkable antitumor activity against many cancers, either in vitro or in vivo, principally because it can evade the ubiquitous ribonuclease cytosolic inhibitor thanks to its structural determinants. Hence, ONC became relevant in the use of protein-drug strategies against incurable cancers. The studies performed in this work showed that both drugs definitely affect A375 cells viability by inducing cytostatic and pro-apoptotic effects in a time- and dose-dependent manner, either if administered alone or in combination. Although we registered low synergistic effects with the combination of the two drugs, we found that AZD2461 did not induce resistance in A375 after two months treatment with high concentration of this molecule. Moreover, we underline that A375 cells treated for a prolonged time with AZD2461 were definitely more susceptible than parental A375 cells to the pro-apoptotic action of ONC. Considering also the different inhibitory effects of the two drugs on TNF-α gene expression and NF-κB DNA-binding, the tuning of their combined delivery to the A375 tumor cell line might open a promising scenario for future therapeutic applications devoted to defeat human melanoma.

Asthma and poly(ADP-ribose) polymerase inhibition: a new therapeutic approach.

Asthma is a chronic lung disease affecting people of all ages worldwide, and it frequently begins in childhood. Because of its chronic nature, it is characterized by pathological manifestations, including airway inflammation, remodeling, and goblet cell hyperplasia. Current therapies for asthma, including corticosteroids and beta-2 adrenergic agonists, are directed toward relieving the symptoms of the asthmatic response, with poor effectiveness against the underlying causes of the disease. Asthma initiation and progression depends on the T helper (Th) 2 type immune response carried out by a complex interplay of cytokines, such as interleukin (IL) 4, IL5, and IL13, and the signal transducer and activator of transcription 6. Much of the data resulting from different laboratories support the role of poly(ADP-ribose) polymerase (PARP) 1 and PARP14 activation in asthma. Indeed, PARP enzymes play key roles in the regulation and progression of the inflammatory asthma process because they affect the expression of genes and chemokines involved in the immune response. Consistently, PARP inhibition achievable either upon genetic ablation or by using pharmacological agents has shown a range of therapeutic effects against the disease. Indeed, in the last two decades, several preclinical studies highlighted the protective effects of PARP inhibition in various animal models of asthma. PARP inhibitors showed the ability to reduce the overall lung inflammation acting with a specific effect on immune cell recruitment and through the modulation of asthma-associated cytokines production. PARP inhibition has been shown to affect the Th1-Th2 balance and, at least in some aspects, the airway remodeling. In this review, we summarize and discuss the steps that led PARP inhibition to become a possible future therapeutic strategy against allergic asthma.

Onconase dimerization through 3D domain swapping: structural investigations and increase in the apoptotic effect in cancer cells.

Onconase® (ONC), a protein extracted from the oocytes of the Rana pipiens frog, is a monomeric member of the secretory 'pancreatic-type' RNase superfamily. Interestingly, ONC is the only monomeric ribonuclease endowed with a high cytotoxic activity. In contrast with other monomeric RNases, ONC displays a high cytotoxic activity. In this work, we found that ONC spontaneously forms dimeric traces and that the dimer amount increases about four times after lyophilization from acetic acid solutions. Differently from RNase A (bovine pancreatic ribonuclease) and the bovine seminal ribonuclease, which produce N- and C-terminal domain-swapped conformers, ONC forms only one dimer, here named ONC-D. Cross-linking with divinylsulfone reveals that this dimer forms through the three-dimensional domain swapping of its N-termini, being the C-terminus blocked by a disulfide bond. Also, a homology model is proposed for ONC-D, starting from the well-known structure of RNase A N-swapped dimer and taking into account the results obtained from spectroscopic and stability analyses. Finally, we show that ONC is more cytotoxic and exerts a higher apoptotic effect in its dimeric rather than in its monomeric form, either when administered alone or when accompanied by the chemotherapeutic drug gemcitabine. These results suggest new promising implications in cancer treatment.

A comparison study on RNase A oligomerization induced by cisplatin, carboplatin and oxaliplatin.

Cisplatin (CDDP) can form interprotein cross-links, leading to the formation of platinated oligomers. A dimer, a trimer and higher oligomers of bovine pancreatic ribonuclease (RNase A) obtained upon reaction with CDDP in 1:10 protein to metal ratio at 37°C have been previously characterized. Here, we verify the ability of carboplatin and oxaliplatin to induce RNase A oligomerization under the same experimental conditions. The amount of formed RNase A oligomers was compared with that obtained in the reaction of the protein with CDDP. Among the three anticancer agents, CDDP is the most reactive and the most effective in inhibiting the ribonucleolytic activity of the protein. Oxaliplatin is the least potent oligomerization agent. Biophysical characterizations of structure and stability of platinated dimers formed in the presence of carboplatin and oxaliplatin suggest that they have a similar thermal stability and are more prone to dissociation than the corresponding dimer obtained with CDDP. Oligomers obtained in the presence of carboplatin are the most active. X-ray structures of the monomeric adducts that RNase A forms with the three drugs provide a rational basis to explain the different effects of the three anticancer agents on enzymatic activity and protein aggregation. Although platinated oligomers of RNase A formed upon reaction with CDDP, carboplatin and oxaliplatin retain a residual ribonuclease activity, they do not show cytotoxic action, suggesting that protein aggregation processes induced by Pt-based drugs can represent a collateral drawback, which affects the functional state of protein targets and reduces the efficacy of Pt-based drug treatment.

Extensive deamidation of RNase A inhibits its oligomerization through 3D domain swapping.

Bovine pancreatic ribonuclease A (RNase A) is the monomeric prototype of the so-called secretory 'pancreatic-type' RNase super-family. Like the naturally domain-swapped dimeric bovine seminal variant, BS-RNase, and its glycosylated RNase B isoform, RNase A forms N- and C-terminal 3D domain-swapped oligomers after lyophilization from acid solutions, or if subjected to thermal denaturation at high protein concentration. All mentioned RNases can undergo deamidation at Asn67, forming Asp or isoAsp derivatives that modify the protein net charge and consequently its enzymatic activity. In addition, deamidation slightly affects RNase B self-association through the 3D domain swapping (3D-DS) mechanism. We report here the influence of extensive deamidation on RNase A tendency to oligomerize through 3D-DS. In particular, deamidation of Asn67 alone slightly decreases the propensity of the protein to oligomerize, with the Asp derivative being less affected than the isoAsp one. Contrarily, the additional Asp and/or isoAsp conversion of residues other than N67 almost nullifies RNase A oligomerization capability. In addition, Gln deamidation, although less kinetically favorable, may affect RNase A self-association. Using 2D and 3D NMR we identified the Asn/Gln residues most prone to undergo deamidation. Together with CD spectroscopy, NMR also indicates that poly-deamidated RNase A generally maintains its native tertiary structure. Again, we investigated in silico the effect of the residues undergoing deamidation on RNase A dimers structures. Finally, the effect of deamidation on RNase A oligomerization is discussed in comparison with studies on deamidation-prone proteins involved in amyloid formation.

Intermolecular disulfide bond influences unphosphorylated STAT3 dimerization and function.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor activated by the phosphorylation of tyrosine 705 in response to many cytokines and growth factors. Recently, the roles for unphosphorylated STAT3 (U-STAT3) have been described in response to cytokine stimulation, in cancers, and in the maintenance of heterochromatin stability. It has been reported that U-STAT3 dimerizes, shuttles between the cytoplasm and nucleus, and binds to DNA, thereby driving genes transcription. Although many reports describe the active role of U-STAT3 in oncogenesis in addition to phosphorylated STAT3, the U-STAT3 functional pathway remains elusive.In this report, we describe the molecular mechanism of U-STAT3 dimerization, and we identify the presence of two intermolecular disulfide bridges between Cys367 and Cys542 and Cys418 and Cys426, respectively. Recently, we reported that the same cysteines contribute to the redox regulation of STAT3 signaling pathway both in vitro and in vivo The presence of these disulfides is here demonstrated to largely contribute to the structure and the stability of U-STAT3 dimer as the dimeric form rapidly dissociates upon reduction in the S-S bonds. In particular, the Cys367-Cys542 disulfide bridge is shown to be critical for U-STAT3 DNA-binding activity. Mutation of the two Cys residues completely abolishes the DNA-binding capability of U-STAT3. Spectroscopic investigations confirm that the noncovalent interactions are sufficient for proper folding and dimer formation, but that the interchain disulfide bonds are crucial to preserve the functional dimer. Finally, we propose a reaction scheme of U-STAT3 dimerization with a first common step followed by stabilization through the formation of interchain disulfide bonds.

Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine.

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ~one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer-monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ~95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation.

Platinated oligomers of bovine pancreatic ribonuclease: Structure and stability.

The reaction between cis-diamminedichloroplatinum(II) (CDDP), cisplatin, a common anticancer drug, and bovine pancreatic ribonuclease (RNase A), induces extensive protein aggregation, leading to the formation of one dimer, one trimer and higher oligomers whose yields depend on cisplatin/protein ratio. Structural and functional properties of the purified platinated species, together with their spontaneous dissociation and thermally induced denaturation, have been characterized. Platinated species preserve a significant, although reduced, ribonuclease activity. The high resistance of the dimers against dissociation and the different thermal unfolding profiles suggest a quaternary structure different from those of the well-known swapped dimers of RNase A.

Onconase induces autophagy sensitizing pancreatic cancer cells to gemcitabine and activates Akt/mTOR pathway in a ROS-dependent manner.

Onconase® (ONC) is a member of the RNase super-family that is secreted in oocytes and early embryos of Rana pipiens. Over the last years, research interest about this small and basic frog RNase, also called ranpirnase, constantly increased because of its high cytotoxicity and anticancer properties. Onconase is currently used in clinical trials for cancer therapy; however, the precise mechanisms determining cytotoxicity in cancer cells have not yet been fully investigated. In the present manuscript, we evaluate the antitumoral property of onconase in pancreatic adenocarcinoma cells and in non-tumorigenic cells as a control. We demonstrate that ONC stimulates a strong antiproliferative and proapoptotic effect in cancer cells by reporting for the first time that ONC triggers Beclin1-mediated autophagic cancer cell death. In addition, ONC inhibits the expression of mitochondrial uncoupling protein 2 (UCP2) and of manganese-dependent superoxide dismutase (MnSOD) triggering mitochondrial superoxide ion production. ONC-induced reactive oxygen species (ROS) are responsible for Akt/mTOR pathway stimulation determining the sensitivity of cancer cells to mTOR inhibitors and lessening autophagic stimulation. This indicates ROS/Akt/mTOR axis as a strategy adopted by cancer cells to reduce ONC-mediated cytotoxic autophagy stimulation. In addition, we demonstrate that ONC can sensitize pancreatic cancer cells to the standard chemotherapeutic agent gemcitabine allowing a reduction of drug concentration when used in combination settings, thus suggesting a lowering of chemotherapy-related side effects. Altogether, our results shed more light on the mechanisms lying at the basis of ONC antiproliferative effect in cancer cells and support its potential use to develop new anticancer strategies.