IGFBP-6 Alters Mesenchymal Stromal Cell Phenotype Driving Dasatinib Resistance in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML), BCR-ABL1-positive, is classified as a myeloproliferative characterized by Philadelphia chromosome/translocation t(9;22) and proliferating granulocytes. Despite the clinical success of tyrosine kinase inhibitors (TKi) agents in the treatment of CML, most patients have minimal residual disease contained in the bone marrow microenvironment, within which stromal cells assume a pro-inflammatory phenotype that determines their transformation in cancer-associated fibroblasts (CAF) which, in turn can play a fundamental role in resistance to therapy. Insulin-like Growth Factor Binding Protein-6 (IGFBP-6) is expressed during tumor development, and is involved in immune-escape and inflammation as well, providing a potential additional target for CML therapy. Here, we aimed at investigating the role of IGFBP-6/SHH/TLR4 axis in TKi response. We used a CML cell line, LAMA84-s, and healthy bone marrow stromal cells, HS-5, in mono- or co-culture. The two cell lines were treated with Dasatinib and/or IGFBP-6, and the expression of inflammatory markers was tested by qRT-PCR; furthermore, expression of IGFBP-6, TLR4 and Gli1 were evaluated by Western blot analysis and immumocytochemistry. The results showed that both co-culture and Dasatinib exposure induce inflammation in stromal and cancer cells so that they modulate the expression of TLR4, and these effects were more marked following IGFBP-6 pre-treatment suggesting that this molecule may confer resistance through the inflammatory processes. This phenomenon was coupled with sonic hedgehog (SHH) signaling. Indeed, our data also demonstrate that HS-5 treatment with PMO (an inducer of SHH) induces significant modulation of TLR4 and overexpression of IGFPB-6 suggesting that the two pathways are interconnected with each other and with the TLR-4 pathway. Finally, we demonstrated that pretreatment with IGFBP-6 and/or PMO restored LAMA-84 cell viability after treatment with Dasatinib, suggesting that both IGFBP-6 and SHH are involved in the resistance mechanisms induced by the modulation of TLR-4, thus indicating that the two pathways may be considered as potential therapeutic targets.

The Role of Metals in the Neuroregenerative Action of BDNF, GDNF, NGF and Other Neurotrophic Factors.

Mature neurotrophic factors and their propeptides play key roles ranging from the regulation of neuronal growth and differentiation to prominent participation in neuronal survival and recovery after injury. Their signaling pathways sculpture neuronal circuits during brain development and regulate adaptive neuroplasticity. In addition, neurotrophic factors provide trophic support for damaged neurons, giving them a greater capacity to survive and maintain their potential to regenerate their axons. Therefore, the modulation of these factors can be a valuable target for treating or preventing neurologic disorders and age-dependent cognitive decline. Neuroregenerative medicine can take great advantage by the deepening of our knowledge on the molecular mechanisms underlying the properties of neurotrophic factors. It is indeed an intriguing topic that a significant interplay between neurotrophic factors and various metals can modulate the outcome of neuronal recovery. This review is particularly focused on the roles of GDNF, BDNF and NGF in motoneuron survival and recovery from injuries and evaluates the therapeutic potential of various neurotrophic factors in neuronal regeneration. The key role of metal homeostasis/dyshomeostasis and metal interaction with neurotrophic factors on neuronal pathophysiology is also highlighted as a novel mechanism and potential target for neuronal recovery. The progress in mechanistic studies in the field of neurotrophic factor-mediated neuroprotection and neural regeneration, aiming at a complete understanding of integrated pathways, offers possibilities for the development of novel neuroregenerative therapeutic approaches.

The Copper(II)-Assisted Connection between NGF and BDNF by Means of Nerve Growth Factor-Mimicking Short Peptides.

Nerve growth factor (NGF) is a protein necessary for development and maintenance of the sympathetic and sensory nervous systems. We have previously shown that the NGF N-terminus peptide NGF(1-14) is sufficient to activate TrkA signaling pathways essential for neuronal survival and to induce an increase in brain-derived neurotrophic factor (BDNF) expression. Cu2+ ions played a critical role in the modulation of the biological activity of NGF(1-14). Using computational, spectroscopic, and biochemical techniques, here we report on the ability of a newly synthesized peptide named d-NGF(1-15), which is the dimeric form of NGF(1-14), to interact with TrkA. We found that d-NGF(1-15) interacts with the TrkA-D5 domain and induces the activation of its signaling pathways. Copper binding to d-NGF(1-15) stabilizes the secondary structure of the peptides, suggesting a strengthening of the noncovalent interactions that allow for the molecular recognition of D5 domain of TrkA and the activation of the signaling pathways. Intriguingly, the signaling cascade induced by the NGF peptides ultimately involves cAMP response element-binding protein (CREB) activation and an increase in BDNF protein level, in keeping with our previous result showing an increase of BDNF mRNA. All these promising connections can pave the way for developing interesting novel drugs for neurodegenerative diseases.

Cytotoxic phenanthroline derivatives alter metallostasis and redox homeostasis in neuroblastoma cells.

Copper homeostasis is generally investigated focusing on a single component of the metallostasis network. Here we address several of the factors controlling the metallostasis for neuroblastoma cells (SH-SY5Y) upon treatment with 2,9-dimethyl-1,10-phenanthroline-5,6-dione (phendione) and 2,9-dimethyl-1,10-phenanthroline (cuproindione). These compounds bind and transport copper inside cells, exert their cytotoxic activity through the induction of oxidative stress, causing apoptosis and alteration of the cellular redox and copper homeostasis network. The intracellular pathway ensured by copper transporters (Ctr1, ATP7A), chaperones (CCS, ATOX, COX 17, Sco1, Sco2), small molecules (GSH) and transcription factors (p53) is scrutinised.

New Insight in Copper-Ion Binding to Human Islet Amyloid: The Contribution of Metal-Complex Speciation To Reveal the Polypeptide Toxicity.

Type-2 diabetes (T2D) is considered to be a potential threat on a global level. Recently, T2D has been listed as a misfolding disease, such as Alzheimer's and Parkinson's diseases. Human islet amyloid polypeptide (hIAPP) is a molecule cosecreted in pancreatic ß cells and represents the main constituent of an aggregated amyloid found in individuals affected by T2D. The trace-element serum level is significantly influenced during the development of diabetes. In particular, the dys-homeostasis of Cu(2+) ions may adversely affect the course of the disease. Conflicting results have been reported on the protective role played by complex species formed by Cu(2+) ions with hIAPP or its peptide fragments in vitro. The histidine (His) residue at position 18 represents the main binding site for the metal ion, but contrasting results have been reported on other residues involved in metal-ion coordination, in particular those toward the N or C terminus. Sequences that encompass regions 17-29 and 14-22 were used to discriminate between the two models of the hIAPP coordination mode. Due to poor solubility in water, poly(ethylene glycol) (PEG) derivatives were synthesized. A peptide fragment that encompasses the 17-29 region of rat amylin (rIAPP) in which the arginine residue at position 18 was substituted by a histidine residue was also obtained to assess that the PEG moiety does not alter the peptide secondary structure. The complex species formed by Cu(2+) ions with Ac-PEG-hIAPP(17-29)-NH2 , Ac-rIAPP(17-29)R18H-NH2 , and Ac-PEG-hIAPP(14-22)-NH2 were studied by using potentiometric titrations coupled with spectroscopic methods (UV/Vis, circular dichroism, and EPR). The combined thermodynamic and spectroscopic approach allowed us to demonstrate that hIAPP is able to bind Cu(2+) ions starting from the His18 imidazole nitrogen atom toward the N-terminus domain. The stability constants of copper(II) complexes with Ac-PEG-hIAPP(14-22)-NH2 were used to simulate the different experimental conditions under which aggregate formation and oxidative stress of hIAPP has been reported. Speciation unveils: 1) the protective role played by increased amounts of Cu(2+) ions on the hIAPP fibrillary aggregation, 2) the effect of adventitious trace amounts of Cu(2+) ions present in phosphate-buffered saline (PBS), and 3) a reducing fluorogenic probe on H2 O2 production attributed to the polypeptide alone.

The neglected role of copper ions in wound healing.

Wound healing is a complex biological process that aims to repair damaged tissue. Even though many biological and biochemical mechanisms associated with the steps of physiological wound healing are known, there is still significant morbidity and mortality due to dysregulation of physiological mechanisms. It might be useful to revise the activity of old players and their links with new, often neglected, molecular entities. This review revises new findings supporting the hypothesis that copper ions regulate the activity and/or the expression of proteins crucially involved in the wound repair process. A better understanding of these interactions might suggest potential new targets for therapeutic intervention on scars or non-healing wounds.

Semax, an ACTH4-10 peptide analog with high affinity for copper(II) ion and protective ability against metal induced cell toxicity.

Heptapeptide Semax, encompassing the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone (ACTH) and a C-terminal Pro-Gly-Pro tripeptide, belongs to a short regulatory peptides family. This compound has been found to affect learning processes and to exert marked neuroprotective activities on cognitive brain functions. Dys-homeostasis of metal ions is involved in several neurodegenerative disorders and growing evidences have showed that brain is a specialized organ able to concentrate metal ions. In this work, the metal binding ability and protective activity of Semax and its metal complexes were studied. The equilibrium study clearly demonstrated the presence of three complex species. Two minor species [CuL] and [CuLH-1]- co-exist together with the [CuLH-2]2- in the pH range from 3.6 to 5. From pH5 the [CuLH-2]2- species becomes predominant with the donor atoms around copper arranged in a 4N planar coordination mode. Noteworthy, a reduced copper induced cytotoxicity was observed in the presence of Semax by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay on a SHSY5Y neuroblastoma and RBE4 endothelial cell lines.

The role of copper(II) in the aggregation of human amylin.

Amylin is a 37-residue peptide hormone produced by the islet ß-cells of pancreas and the formation of amylin aggregates is strongly associated with ß-cell degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Aß and α-synuclein and there is evidence that amylin self-assembly is also largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure, which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non-fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported.

Copper, BDNF and Its N-terminal domain: inorganic features and biological perspectives.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin that influences development, maintenance, survival, and synaptic plasticity of central and peripheral nervous systems. Altered BDNF signaling is involved in several neurodegenerative disorders including Alzheimer's disease. Metal ions may influence the BDNF activity and it is well known that the alteration of Cu(2+) homeostasis is a prominent factor in the development of neurological pathologies. The N-terminal domain of BDNF represents the recognition site of its specific receptor TrkB, and metal ions interaction with this protein domain may influence the protein/receptor interaction. In spite of this, no data inherent the interaction of BDNF with Cu(2+) ions has been reported up to now. Cu(2+) complexes of the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of N-terminal domain of human BDNF protein were characterized by means of potentiometry, spectroscopic methods (UV/Vis, CD, EPR), parallel tempering simulations and DFT-geometry optimizations. Coordination features of the acetylated form, Ac-BDNF(1-12), were also characterized to understand the involvement of the terminal amino group. Whereas, an analogous peptide, BDNF(1-12)D3N, in which the aspartate residue was substituted by an asparagine, was synthesized to provide evidence on the possible role of carboxylate group in Cu(2+) coordination. The results demonstrated that the amino group is involved in metal binding and the metal coordination environment of the predominant complex species at physiological pH consisted of one amino group, two amide nitrogen atoms, and one carboxylate group. Noteworthy, a strong decrease of the proliferative activity of both BDNF(1-12) and the whole protein on a SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). The effect of metal addition is opposite to that observed for the analogous fragment of nerve growth factor (NGF) protein, highlighting the role of specific domains, and suggesting that Cu(2+) may drive different pathways for the BDNF and NGF in physiological as well as pathological conditions.

The inorganic perspectives of neurotrophins and Alzheimer's disease.

The recent metal hypothesis represents an attempt of a new interpretation key of Alzheimer's disease (AD) to overcome the limits of amyloid cascade. Neurons need to maintain metal ions within a narrow range of concentrations to avoid a detrimental alteration of their homeostasis, guaranteed by a network of specific metal ion transporters and chaperones. Indeed, it is well known that transition metal ions take part in neuromodulation/neurotrasmission. In addition, they are prominent factors in the development and exacerbation of neurodegeneration. Neurotrophins are proteins involved in development, maintenance, survival and synaptic plasticity of central and peripheral nervous systems. A neurotrophin hypothesis of AD has been proposed, whereas the link between neurotrophic factor, the amyloid cascade and biometals has not been taken into account. As a matter of fact, there is a significant overlap between brain areas featured by metal ion dys-homeostasis, and those where the neurotrophins exert their biological activity. Metal ions can directly modulate their activities, through conformational changes, and/or indirectly by activating their downstream signaling in a neurotrophin-independent mode. The focus of this review is on the molecular aspects of Zn(2+) and Cu(2+) interactions with neurotrophins, with the aim to shed light on the intricate mechanisms involving metallostasis and proteostasis in AD.

Protective effects of L- and D-carnosine on alpha-crystallin amyloid fibril formation: implications for cataract disease.

Mildly denaturing conditions induce bovine alpha-crystallin, the major structural lens protein, to self-assemble into fibrillar structures in vitro. The natural dipeptide l-carnosine has been shown to have potential protective and therapeutic significance in many diseases. Carnosine derivatives have been proposed as potent agents for ophthalmic therapies of senile cataracts and diabetic ocular complications. Here we report the inhibitory effect induced by the peptide (l- and d-enantiomeric form) on alpha-crystallin fibrillation and the almost complete restoration of the chaperone activity lost after denaturant and/or heat stress. Scanning force microscopy (SFM), thioflavin T, and a turbidimetry assay have been used to determine the morphology of alpha-crystallin aggregates in the presence and absence of carnosine. DSC and a near-UV CD assay evidenced that the structural precursors of amyloid fibrils are polypeptide chain segments that lack stable structural elements. Moreover, we have found a disassembling effect of carnosine on alpha-crystallin amyloid fibrils. Finally, we show the ability of carnosine to restore most of the lens transparency in organ-cultured rat lenses exposed to similar denaturing conditions that were used for the in vitro experiments.

Carnosine interaction with nitric oxide and astroglial cell protection.

The neuropeptide carnosine (beta-amyloid peptide aggregation has been demonstrated. Carnosine protection against peroxynitrite damage is particularly relevant, but until now there has been no evidence of any direct interaction with nitric oxide. In this study we examined the protection that carnosine provides against nitric oxide (NO)-induced cell death in primary rat astroglial cell cultures treated with lipopolysaccharide (LPS) and interferon gamma (INFgamma), a well-known neurotoxic proinflammatory condition. A correlation was found between cell protection and NO free-radical scavenging activity of carnosine. Moreover, by competitive spectrophotometric measurement and electrospray mass spectrometry analysis in cell-free experiments, we demonstrated a direct interaction of the dipeptide with NO. A comparison of carnosine with its homologues or derivatives (homocarnosine and carcinine) as well as with its amino acid constituents (L-histidine and beta-alanine) highlighted that only histidine showed significant scavenging activity. Therefore, carnosine shows direct NO-trapping ability and may be a valuable multifunctional molecule in the treatment of neurodegenerative disorders.

Trehalose effects on alpha-crystallin aggregates.

alpha-Crystallin in its native state is a large, heterogeneous, low-molecular weight (LMW) aggregate that under certain conditions may progressively became part of insoluble high-molecular weight (HMW) systems. These systems are supposed to play a relevant role in eye lens opacification and vision impairment. In this paper, we report the effects of trehalose on alpha-crystallin aggregates. The role of trehalose in alpha-crystallin stress tolerance, chaperone activity and thermal stability is studied. The results show that trehalose stabilizes the alpha-crystallin native structure, inhibits alpha-crystallin aggregation, and disaggregates preformed LMW systems not affecting its chaperone activity.

Role of PARP under stress conditions: cell death or protection?

A great deal of increasing evidence designs PARP as a multifunctional protein implicated in many cellular functions. Much interest is emerging to understand the precise mechanisms by which PARP mediates genome stabilization and protection against damage, as well as its involvement in cell death, either apoptotic or necrotic. Aside from the clearly established role of PARP hyperactivation in necrotic cell death, after excessive DNA damage and energy failure, it appears to be actively involved in the phenomenon of apoptosis. However, its exact role is still controversial. The identification of several enzymes sharing the poly(ADP-ribose) polymerase catalytic domain (PARPs), but with different features and subcellular localization, has opened a new perspective in the field of poly(ADP-ribosyl)ation. The picture of the role of PARP in the control of cell homeostasis became even more complex after demonstration of its implication in the regulation of gene transcription. The notion that energy failure is the sole mechanism by which PARP promotes cell death is therefore under reevaluation.