Nowadays, the list of proteins and peptides that are known to be able to adopt the amyloid-like cross-β structure conformation is tremendous. This has led to the idea that refolding of polypeptides from a native fold to an amyloid-like structure is an inherent property, independent of the amino-acid sequence of the polypeptides. We found that tissue-type plasminogen activator (tPA) and factor XII are specifically activated by many polypeptides, once they have adopted the cross-β structure conformation. This led us to recognize that a “cross-β structure pathway” exists that regulates the recognition and clearance of unwanted proteins.1 Polypeptides can refold spontaneously at the end of their life cycle, or refolding can be induced by environmental factors such as pH, glycation, oxidative stress, heat, irradiation, mechanical stress, proteolysis and so on, at least part of the polypeptide refolds and adopts the amyloid-like cross-β structure conformation. This conformation is then the signal that triggers a cascade of events that induces clearance and breakdown of the obsolete particle. When clearance is inadequate unwanted polypeptides can aggregate and form toxic structures ranging from soluble oligomers up to precipitating fibrils and amorphous plaques. Such cross-β structure conformation comprising aggregates underlie various diseases, such as Alzheimer's disease, Huntington's disease, diabetes mellitus type 2, systemic amyloidoses or Creutzfeldt-Jakob's disease, depending on the underlying polypeptide that accumulates and on the part of the body where accumulation occurs.
The presence of cross-β structures in proteins triggers multiple responses. As mentioned, cross-β structure comprising proteins can activate tPA and FXII, thereby initiating the fibrinolytic system and the contact system of hemostasis. Besides activation of the coagulation system through FXII, the cross-β structure conformation may induce coagulation, platelet aggregation and blood clotting via direct platelet activation and/or the release of tissue factor (Tf) by activated endothelial cells (described in more detail in a co-pending patent application). In addition, the complement system is another example of a proteolytic cascade that is activated by cross-β structure conformation. This system can be activated by the amyloid-β peptide associated with Alzheimer's Disease or by zirconium or aluminum or titanium. The latter being compounds that can induce cross-β structure conformation in proteins. The innate and adaptive immune systems are yet another example. Amyloid-β activates the innate and adaptive immune response.2 β2-glycoprotein I is an auto-immune antigen only upon contact with a negatively charged lipid surface, such as cardiolipin.3 We have now shown that cardiolipin induces cross-β structure conformation in β2-glycoprotein I (described in more detail in a co-pending patent application). Moreover, we have shown that ligands for Toll-like receptors that are implicated in the regulation of immunity induce cross-β structure conformation in proteins. These ligands include lipopolysaccharide and CpG oligodeoxynucleotides (ODN) (described in more detail in a co-pending patent application).
FXII can be activated by negatively charged agents. For example, when blood is drawn into a glass tube it rapidly clots, due to activation of FXII. However, when the tube is made of plastic clotting is delayed. This mechanism of this contact system of coagulation is termed the intrinsic pathway because all clotting factors are present in plasma; in contrast to the extrinsic pathway, which requires the presence of tissue factor on the surface of cells, that is not exposed to the circulation during homeostasis. Interestingly, the nature of the FXII activator in vivo is still unknown. We now found that cross-β structure, that is formed when globular proteins unfold due to any denaturing trigger, is a trigger for FXII and contact activation. Since negatively charged surfaces, such as glass, induce denaturation of proteins, it may well be possible that activation of FXII is secondary to formation of cross-β structure by negatively charged surfaces. We have tested whether activation of FXII by dextran sulphate 500,000 Da (DXS500k) and kaolin is accompanied and mediated by cross-β structure, and our results indeed show that this is occurring. We have determined that plasma exposure to a surface of DXS500k or kaolin indeed induces cross-β structure conformation by staining with Thioflavin T (ThT) and by binding of a recombinant finger domain. In addition, we test whether the amyloid binding reagents Congo Red, ThT, recombinant finger domains of tPA, FXII, HGFA and fibronectin, or full-length tPA, FXII, HGFA, fibronectin, serum amyloid P component (SAP), anti-cross-β structure antibodies and/or a soluble fragment of receptor for advanced glycation endproducts (sRAGE) inhibit activation of FXII induced by DXS500k, kaolin, any other activating surface, or by denatured polypeptides comprising the cross-β structure conformation.
tPA is a serine protease involved in fibrin clot lysis. tPA stimulates activation of plasminogen into plasmin. Fibrin serves as an efficient cofactor in stimulating tPA mediated plasmin formation. Besides fibrin and fibrin fragments a large number of other proteins or protein fragments have been found that stimulate tPA activity, though that exhibit no apparent amino-acid sequence homology. Therefore, the anticipated common structural basis underlying the acquired tPA binding remained elusive. We recently found that the amyloid-like cross-β structure (conformation), the structural element found in amyloid deposits in diseases such as Alzheimer's disease, is a prerequisite and the common denominator in tPA-binding ligands.1, 4 FXII shows close homology with tPA and is known to be activated by amyloid-β (Aβ) and by bacteria with an amyloid core.5 The domain structure of FXII includes, like tPA, a finger domain and its sequence shows the closest homologies with tPA. FXII also binds fibrin (Sanchez et al. 2003, ISTH XIX Congress; surface deposited fibrin activates FXII and the intrinsic coagulation pathway) and FXII can also, like tPA, mediate the conversion of plasminogen to plasmin.6 We found that FXII, like tPA, is activated by polypeptides with amyloid-like cross-β structure conformation in general. Moreover, we established that well-known activators of FXII, DXS500k and kaolin, induce amyloid-like cross-β structure conformation in proteins and that DXS500k is only then an effective activator of FXII when an excess of protein cofactor over the amount of FXII present is added to the reaction mixture. Thus, in contrast to direct activation by binding to negatively charged surfaces, FXII is activated by (plasma) proteins that denature and form amyloid on negatively charged surfaces, or denature by any other means, e.g., pH change, exposure to radicals, proteolysis, glycation, oxidation, change in temperature. It is thus stated that the cross-β structure conformation regulates contact activation and fibrinolysis.
At present, it is assumed that activation of FXII directly involves binding to negatively charged surfaces. Based on our findings, we show that negatively charged surfaces induce amyloid cross-β structure formation and that this structure element triggers FXII activation. This finding renews the view on contact-mediated activation of blood coagulation.
In conclusion, tPA and factor XII are cross-β structure binding proteins. Moreover, cross-β structure comprising proteins can activate these proteins.
To be able to further study the role of the cross-β structure in (patho)physiology it is necessary that (more) compounds capable of binding to a protein comprising a cross-β structure, amongst others cross-β structure binding compounds, are identified. Such compounds are not only useful to be able to better understand cross-β structures, but are also very useful in respect of understanding the refolding from a native state, assembly and toxicity and are also useful for the development of diagnostic and therapeutic agents or useful as component of a diagnostic or therapeutic agent.
USPTO Backgrounds
USPTO Backgrounds