miR-34c may protect lung cancer cells from paclitaxel-induced apoptosis.

MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that negatively regulate the expression of their target genes. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and are considered as promising new therapeutic targets for cancer. However, the identity of miRNAs involved in apoptosis and their respective targets remain largely unknown. Given the elevated complexity of miRNA regulation of gene expression, we performed a functional screening as an alternative strategy to identify those miRNAs that in lung cancer cells may interfere with the apoptotic process. To this aim, we generated a derivative of the non-small cell lung carcinoma A549 cell line in which caspase-8, a critical upstream initiator of apoptosis, can be activated by administration of the small dimerizer drug AP20187. We found a number of miRNAs that may rescue cell viability from caspase-8 activation. They included miRNAs already described as oncogenic such as miR-17, miR-135 and miR-520, but also some miRNAs such as miR-124-1 and miR-34c for which a tumor-suppressive role has instead been described or expected. Among them, miR-34c-5p markedly increased resistance to paclitaxel-induced apoptosis. We demonstrate that Bmf (Bcl-2-modifying factor) is a target of miR-34c-5p, and that its silencing, together with that of c-myc, a known target of miR-34c-5p, contributes to resistance to apoptosis induced by paclitaxel through p53 downregulation.

Nucleic acids in human glioma treatment: innovative approaches and recent results.

Gliomas are the most common primary central nervous system tumors with a dismal prognosis. Despite recent advances in surgery, radiotherapy, and chemotherapy, current treatment regimens have a modest survival benefit. A crucial challenge is to deliver drugs effectively to invasive glioma cells residing in a sanctuary within the central nervous system. New therapies are essential, and oligonucleotide-based approaches, including antisense, microRNAs, small interfering RNAs, and nucleic acid aptamers, may provide a viable strategy. Thanks to their unique characteristics (low size, good affinity for the target, no immunogenicity, chemical structures that can be easily modified to improve their in vivo applications), these molecules may represent a valid alternative to antibodies particularly to overcome challenges presented by the blood-brain barrier. Here we will discuss recent results on the use of oligonucleotides that will hopefully provide new effective treatment for gliomas.

Nucleic acid aptamers against protein kinases.

Deregulation of kinase function has been implicated in several important diseases, including cancer, neurological and metabolic disorders. Because of their key role in causing disease, kinases have become one of the most intensively pursued classes of drug targets. To date, several monoclonal antibodies (mAbs) and small-molecule inhibitors have been approved for the treatment of cancer. Aptamers are short structured single stranded RNA or DNA ligands that bind at high affinity to their target molecules and are now emerging as promising molecules to target specific cancer epitopes in clinical diagnosis and therapy. Further, because of their high specificity and low toxicity aptamers will likely reveal among the most promising molecules for in vivo targeted recognition as therapeutics or delivery agents for nanoparticles, small interfering RNAs bioconjugates, chemotherapeutic cargos and molecular imaging probes. In this article, we discuss recent advances in the development of aptamers targeting kinase proteins.

The tyrosine phosphatase Shp-2 mediates intracellular signaling initiated by Ret mutants.

The Src homology 2-containing tyrosine phosphatase, Shp-2, is a crucial enzyme that mediates intracellular signaling and is implicated in cell proliferation and differentiation. Here we investigated the involvement of the Shp-2 tyrosine phosphatase in determining the downstream signaling pathways initiated by the Ret oncogene, carrying either the cysteine 634 to tyrosine or the methionine 918 to threonine substitutions. These mutations convert the receptor tyrosine kinase, Ret, into a dominant transforming protein and induce constitutive activation of its intrinsic tyrosine kinase activity leading to congenital and sporadic cancers in neuroendocrine organs. Using the PC12, rat pheochromocytoma cell line, as model system, we show that Shp-2 mediates immediate-early gene expression if induced by either of the mutant alleles. Furthermore, we show that Shp-2 activity is required for RetM918T-induced Akt activation. The results indicate that Shp-2 is a downstream mediator of the mutated receptors RetC634Y and RetM918T, thus suggesting that it may act as a limiting factor in Ret-associated endocrine tumors, in the neoplastic syndromes multiple endocrine neoplasia types 2A and 2B.

The chromosomal protein sso7d of the crenarchaeon Sulfolobus solfataricus rescues aggregated proteins in an ATP hydrolysis-dependent manner.

In this work, we show that the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus displays a cation-dependent ATPase activity with a pH optimum around neutrality and a temperature optimum of 70 degrees C. Measurements of tryptophan fluorescence and experiments that used 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that ATP hydrolysis induces a conformational change in the molecule and that the binding of the nucleotide triggers the ATP hydrolysis-induced conformation of the protein to return to the native conformation. We found that Sso7d rescues previously aggregated proteins in an ATP hydrolysis-dependent manner; the native conformation of Sso7d forms a complex with the aggregates, while the ATP hydrolysis-induced conformation is incapable of this interaction. Sso7d is believed to be the first protein isolated from an archaeon capable of rescuing aggregates.

An archaeal chaperonin-based reactor for renaturation of denatured proteins.

We describe an original chaperonin-based reactor that yields folded and active proteins from denatured materials. We used the 920-kDa chaperonin of the archaeon Sulfolobus solfataricus, which does not require any protein partner for its full activity and assists in vitro folding with low substrate specificity. The reactor consists of an ultrafiltration cell equipped with a membrane that retains the chaperonin in a functional state for folding in solution and permits the flowthrough of the folded substrates. By studying the ATP-dependent functional cycle of the chaperonin, we were able to use the reactor for repeated refolding processes. The scale-up of the reactor is made possible by the overproduction of chaperonin in Sulfolobus solfataricus cells that acquired thermotolerance upon appropriate heat shock.

Prevention of in vitro protein thermal aggregation by the Sulfolobus solfataricus chaperonin. Evidence for nonequivalent binding surfaces on the chaperonin molecule.

We have studied the effects of the Sulfolobus solfataricus chaperonin on the aggregation and inactivation upon heating of four model enzymes: chicken egg white lysozyme (one 14.4-kDa chain), yeast alpha-glucosidase (one 68.5-kDa chain), chicken liver malic enzyme (four 65-kDa subunits), and yeast alcohol dehydrogenase (four 37.5-kDa subunits). When the proteins were heated in the presence of an equimolar amount of chaperonin, 1) the aggregation was prevented in all solutions; 2) the inactivation profiles of the single-chain enzymes were comparable with those detected in the absence of the chaperonin, and enzyme activities were regained in the solutions heated in the presence of the chaperonin upon ATP hydrolysis (78 and 55% activity regains for lysozyme and alpha-glucosidase, respectively); 3) the inactivation of the tetrameric enzymes was completely prevented, whereas the activities decreased in the absence of the chaperonin. We demonstrate by gel filtration chromatography that the chaperonin interacted with the structures occurring during thermal denaturation of the model proteins and that the interaction with the single-chain proteins (but not that with the tetrameric proteins) was reversed upon ATP hydrolysis. The chaperonin had nonequivalent surfaces for the binding of the model proteins upon heating: the thermal denaturation intermediates of the single-chain proteins share Surfaces I, while the thermal denaturation intermediates of the tetrameric proteins share Surfaces II. ATP binding to the chaperonin induced a conformation that lacked Surfaces I and carried Surfaces II. These data support the concept that chaperonins protect native proteins against thermal aggregation by two mechanistically distinct strategies (an ATP-dependent strategy and an ATP-independent strategy), and provide the first evidence that a chaperonin molecule bears functionally specialized surfaces for the binding of the protein substrates.

The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro.

We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 degrees C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium-dependent ATPase activity with an optimum temperature at 80 degrees C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride-denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible inactivations at 90 degrees C of several thermophilic enzymes by the binding of the denaturation intermediate; the time-courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 degrees C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.

Isolation of a thermostable enzyme catalyzing disulfide bond formation from the archaebacterium Sulfolobus solfataricus.

A disulfide bond-forming enzyme was purified from the cytosol of the archaebacterium Sulfolobus solfataricus, strain MT-4. The enzyme, assayed by its ability to oxidize and reactivate reductively denatured ribonuclease A, had a small molecular size and displayed a high thermostability. The N-terminal amino acid sequence is reported.