Giampietro Ramponi

Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.

A range of human degenerative conditions, including Alzheimers disease, light-chain amyloidosis and the spongiform encephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3′-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.

Rationalization of the effects of mutations on peptide and protein aggregation rates.

In order for any biological system to function effectively, it is essential to avoid the inherent tendency of proteins to aggregate and form potentially harmful deposits. In each of the various pathological conditions associated with protein deposition, such as Alzheimers and Parkinsons diseases, a specific peptide or protein that is normally soluble is deposited as insoluble aggregates generally referred to as amyloid. It is clear that the aggregation process is generally initiated from partially or completely unfolded forms of the peptides and proteins associated with each disease. Here we show that the intrinsic effects of specific mutations on the rates of aggregation of unfolded polypeptide chains can be correlated to a remarkable extent with changes in simple physicochemical properties such as hydrophobicity, secondary structure propensity and charge. This approach allows the pathogenic effects of mutations associated with known familial forms of protein deposition diseases to be rationalized, and more generally enables prediction of the effects of mutations on the aggregation propensity of any polypeptide chain.

Reactive oxygen species as essential mediators of cell adhesion: the oxidative inhibition of a FAK tyrosine phosphatase is required for cell adhesion

Signal transduction by reactive oxygen species (ROS; “redox signaling”) has recently come into focus in cellular biology studies. The signaling properties of ROS are largely due to the reversible oxidation of redox-sensitive target proteins, and especially of protein tyrosine phosphatases, whose activity is dependent on the redox state of a low pKa active site cysteine. A variety of mitogenic signals, including those released by receptor tyrosine kinase (RTKs) ligands and oncogenic H-Ras, involve as a critical downstream event the intracellular generation of ROS. Signaling by integrins is also essential for the growth of most cell types and is constantly integrated with growth factor signaling. We provide here evidence that intracellular ROS are generated after integrin engagement and that these oxidant intermediates are necessary for integrin signaling during fibroblast adhesion and spreading. Moreover, we propose a synergistic action of integrins and RTKs for redox signaling. Integrin-induced ROS are required to oxidize/inhibit the low molecular weight phosphotyrosine phosphatase, thereby preventing the enzyme from dephosphorylating and inactivating FAK. Accordingly, FAK phosphorylation and other downstream events, including MAPK phosphorylation, Src phosphorylation, focal adhesion formation, and cell spreading, are all significantly attenuated by inhibition of redox signaling. Hence, we have outlined a redox circuitry whereby, upon cell adhesion, oxidative inhibition of a protein tyrosine phosphatase promotes the phosphorylation/activation and the downstream signaling of FAK and, as a final event, cell adhesion and spreading onto fibronectin.

Kinetic partitioning of protein folding and aggregation

We have systematically studied the effects of 40 single point mutations on the conversion of the denatured form of the α/β protein acylphosphatase (AcP) into insoluble aggregates. All the mutations that significantly perturb the rate of aggregation are located in two regions of the protein sequence, residues 16–31 and 87–98, each of which has a relatively high hydrophobicity and propensity to form β-sheet structure. The measured changes in aggregation rate upon mutation correlate with changes in the hydrophobicity and β-sheet propensity of the regions of the protein in which the mutations are located. The two regions of the protein sequence that determine the aggregation rate are distinct from those parts of the sequence that determine the rate of protein folding. Dissection of the protein into six peptides corresponding to different regions of the sequence indicates that the kinetic partitioning between aggregation and folding can be attributed to the intrinsic conformational preferences of the denatured polypeptide chain.

Intracellular Reactive Oxygen Species Activate Src Tyrosine Kinase during Cell Adhesion and Anchorage-Dependent Cell Growth

ABSTRACT Src tyrosine kinases are central components of adhesive responses and are required for cell spreading onto the extracellular matrix. Among other intracellular messengers elicited by integrin ligation are reactive oxygen species, which act as synergistic mediators of cytoskeleton rearrangement and cell spreading. We report that after integrin ligation, the tyrosine kinase Src is oxidized and activated. Src displays an early activation phase, concurrent with focal adhesion formation and driven mainly by Tyr527 dephosphorylation, and a late phase, concomitant with reactive oxygen species production, cell spreading, and integrin-elicited kinase oxidation. In addition, our results suggest that reactive oxygen species are key mediators of in vitro and in vivo v-Src tumorigenic properties, as both antioxidant treatments and the oxidant-insensitive C245A and C487A Src mutants greatly decrease invasivity, serum-independent and anchorage-independent growth, and tumor onset. Therefore we propose that, in addition to the known phosphorylation/dephosphorylation circuitry, redox regulation of Src activity is required during both cell attachment to the extracellular matrix and tumorigenesis.

Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases

Protein aggregation and the formation of highly insoluble amyloid structures is associated with a range of debilitating human conditions, which include Alzheimers disease, Parkinsons disease, and the Creutzfeldt–Jakob disease. Muscle acylphosphatase (AcP) has already provided significant insights into mutational changes that modulate amyloid formation. In the present paper, we have used this system to investigate the effects of mutations that modify the charge state of a protein without affecting significantly the hydrophobicity or secondary structural propensities of the polypeptide chain. A highly significant inverse correlation was found to exist between the rates of aggregation of the protein variants under denaturing conditions and their overall net charge. This result indicates that aggregation is generally favored by mutations that bring the net charge of the protein closer to neutrality. In light of this finding, we have analyzed natural mutations associated with familial forms of amyloid diseases that involve alteration of the net charge of the proteins or protein fragments associated with the diseases. Sixteen mutations have been identified for which the mechanism of action that causes the pathological condition is not yet known or fully understood. Remarkably, 14 of these 16 mutations cause the net charge of the corresponding peptide or protein that converts into amyloid deposits to be reduced. This result suggests that charge has been a key parameter in molecular evolution to ensure the avoidance of protein aggregation and identifies reduction of the net charge as an important determinant in at least some forms of protein deposition diseases.

Mutational analysis of the propensity for amyloid formation by a globular protein

Acylphosphatase can be converted in vitro, by addition of trifluoroethanol (TFE), into amyloid fibrils of the type observed in a range of human diseases. The propensity to form fibrils has been investigated for a series of mutants of acylphosphatase by monitoring the range of TFE concentrations that result in aggregation. We have found that the tendency to aggregate correlates inversely with the conformational stability of the native state of the protein in the different mutants. In accord with this, the most strongly destabilized acylphosphatase variant forms amyloid fibrils in aqueous solution in the absence of TFE. These results show that the aggregation process that leads to amyloid deposition takes place from an ensemble of denatured conformations under conditions in which non‐covalent interactions are still favoured. These results support the hypothesis that the stability of the native state of globular proteins is a major factor preventing the in vivo conversion of natural proteins into amyloid fibrils under non‐pathological conditions. They also suggest that stabilizing the native states of amyloidogenic proteins could aid prevention of amyloidotic diseases.

Redox Regulation of β-Actin during Integrin-mediated Cell Adhesion

Redox sensitivity of actin toward an exogenous oxidative stress has recently been reported. We report here the first evidence of in vivo actin redox regulation by a physiological source of reactive oxygen species, specifically those species generated by integrin receptors during cell adhesion. Actin oxidation takes place via the formation of a mixed disulfide between cysteine 374 and glutathione; this modification is essential for spreading and for cytoskeleton organization. Impairment of actin glutathionylation, either through GSH depletion or expression of the C374A redox-insensitive mutant, greatly affects cell spreading and the formation of stress fibers, leading to inhibition of the disassembly of the actinomyosin complex. These data suggest that actin glutathionylation is essential for cell spreading and cytoskeleton organization and that it plays a key role in disassembly of actinomyosin complex during cell adhesion.

Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival

Proper attachment to the extracellular matrix (ECM) is essential for cell survival. The loss of integrin-mediated cell–ECM contact results in an apoptotic process termed anoikis. However, mechanisms involved in regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we demonstrate that reactive oxygen species (ROS) produced through the involvement of the small GTPase Rac-1 upon integrin engagement exert a mandatory role in transducing a pro-survival signal that ensures that cells escape from anoikis. In particular, we show that ROS are responsible for the redox-mediated activation of Src that trans-phosphorylates epidermal growth factor receptor (EGFR) in a ligand-independent manner. The redox-dependent phosphorylation of EGFR activates both extracellular signal-regulated protein kinase and Akt downstream signalling pathways, culminating in degradation of the pro-apoptotic protein Bim. Hence, our results shed new light on the mechanism granting the adhesion-dependent antiapoptotic effect, highlighting a fundamental role of ROS-mediated Src regulation in ensuring anoikis protection.

PDGF receptor as a specific in vivo target for low M r phosphotyrosine protein phosphatase

Low M r phosphotyrosine protein phosphatase (LMWPTP) is a 18 kDa cytosolic enzyme widely distributed in eukaryotic cells. LMW‐PTP catalyses the hydrolysis of phosphotyrosine residues and overexpression of the enzyme in normal and transformed cells inhibits cell proliferation. Site directed mutagenesis, together with crystallographic studies, have contributed to clarify the catalytic mechanism, which involves the active site signature sequence C12XXXXXR18, a main feature of all PTPase family members. In order to identify the LMW‐PTP substratels we have expressed in NIH‐3T3 cells a catalytically inert Cys12 to Ser phosphatase mutant which has preserved its capacity for substrate binding. Overexpression of the mutant phosphatase leads to enhanced cell proliferation and serum induced mitogenesis, indicating that the mutation results in the production of a dominant negative protein. Analysis of mutant LMW‐PTP expressing cells has enabled us to demonstrate an association between LMW‐PTP and platelet derived growth factor receptor that appears to be highly specific. Our data suggest a catalytic action of LMW‐PTP on the phosphorylated platelet derived growth factor receptor.