Martijn F. B. G. Gebbink

Amyloids - A functional coat for microorganisms

Amyloids are filamentous protein structures ∼10 nm wide and 0.1–10 μm long that share a structural motif, the cross-β structure. These fibrils are usually associated with degenerative diseases in mammals. However, recent research has shown that these proteins are also expressed on bacterial and fungal cell surfaces. Microbial amyloids are important in mediating mechanical invasion of abiotic and biotic substrates. In animal hosts, evidence indicates that these protein structures also contribute to colonization by activating host proteases that are involved in haemostasis, inflammation and remodelling of the extracellular matrix. Activation of proteases by amyloids is also implicated in modulating blood coagulation, resulting in potentially life-threatening complications.

Misfolded proteins activate Factor XII in humans, leading to kallikrein formation without initiating coagulation

When blood is exposed to negatively charged surface materials such as glass, an enzymatic cascade known as the contact system becomes activated. This cascade is initiated by autoactivation of Factor XII and leads to both coagulation (via Factor XI) and an inflammatory response (via the kallikrein-kinin system). However, while Factor XII is important for coagulation in vitro, it is not important for physiological hemostasis, so the physiological role of the contact system remains elusive. Using patient blood samples and isolated proteins, we identified a novel class of Factor XII activators. Factor XII was activated by misfolded protein aggregates that formed by denaturation or by surface adsorption, which specifically led to the activation of the kallikrein-kinin system without inducing coagulation. Consistent with this, we found that Factor XII, but not Factor XI, was activated and kallikrein was formed in blood from patients with systemic amyloidosis, a disease marked by the accumulation and deposition of misfolded plasma proteins. These results show that the kallikrein-kinin system can be activated by Factor XII, in a process separate from the coagulation cascade, and point to a protective role for Factor XII following activation by misfolded protein aggregates.

Molecular and cellular aspects of protein misfolding and disease

Proteins are essential elements for life. They are building blocks of all organisms and the operators of cellular functions. Humans produce a repertoire of at least 30,000 different proteins, each with a different role. Each protein has its own unique sequence and shape (native conformation) to fulfill its specific function. The appearance of incorrectly shaped (misfolded) proteins occurs on exposure to environmental changes. Protein misfolding and the subsequent aggregation is associated with various, often highly debilitating, diseases for which no sufficient cure is available yet. In the first part of this review we summarize the structural composition of proteins and the current knowledge of underlying forces that lead proteins to lose their native structure. In the second and third parts we describe the molecular and cellular mechanisms that are associated with protein misfolding in disease. Finally, in the last part we portray recent efforts to develop treatments for protein misfolding diseases.—Herczenik, E., and Gebbink, M. F. B. G. Molecular and cellular aspects of protein misfolding and disease. FASEB J. 22, 2115–2133 (2008)

A Role for Protein Misfolding in Immunogenicity of Biopharmaceuticals

For largely unknown reasons, biopharmaceuticals evoke potentially harmful antibody formation. Such antibodies can inhibit drug efficacy and, when directed against endogenous proteins, cause life-threatening complications. Insight into the mechanisms by which biopharmaceuticals break tolerance and induce an immune response will contribute to finding solutions to prevent this adverse effect. Using a transgenic mouse model, we here demonstrate that protein misfolding, detected with the use of tissue-type plasminogen activator and thioflavin T, markers of amyloid-like properties, results in breaking of tolerance. In wild-type mice, misfolding enhances protein immunogenicity. Several commercially available biopharmaceutical products were found to contain misfolded proteins. In some cases, the level of misfolded protein was found to increase upon storage under conditions prescribed by the manufacturer. Our results indicate that misfolding of therapeutic proteins is an immunogenic signal and a risk factor for immunogenicity. These findings offer novel possibilities to detect immunogenic protein entities with tPA and reduce immunogenicity of biopharmaceuticals.

Tissue-Type Plasminogen Activator Is a Multiligand Cross-β Structure Receptor

Abstract Tissue-type plasminogen activator (tPA) regulates fibrin clot lysis by stimulating the conversion of plasminogen into the active protease plasmin [1]. Fibrin is required for efficient tPA-mediated plasmin generation and thereby stimulates its own proteolysis. Several fibrin regions can bind to tPA [1], but the structural basis for this interaction is unknown. Amyloid β (Aβ) is a peptide aggregate that is associated with neurotoxicity in brains afflicted with Alzheimers disease [2]. Like fibrin, it stimulates tPA-mediated plasmin formation [3–5]. Intermolecular stacking of peptide backbones in β sheet conformation underlies cross-β structure in amyloid peptides [6]. We show here that fibrin-derived peptides adopt cross-β structure and form amyloid fibers. This correlates with tPA binding and stimulation of tPA-mediated plasminogen activation. Prototype amyloid peptides, including Aβ and islet amyloid polypeptide (IAPP) (associated with pancreatic β cell toxicity in type II diabetes [7]), have no sequence similarity to the fibrin peptides but also bind to tPA and can substitute for fibrin in plasminogen activation by tPA. Moreover, the induction of cross-β structure in an otherwise globular protein (endostatin) endows it with tPA-activating potential. Our results classify tPA as a multiligand receptor and show that cross-β structure is the common denominator in tPA binding ligands.

Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor.

Summary.  Plasmin and other components of the plasminogen activation system play an important role in tissue repair by regulating extracellular matrix remodeling, including fibrin degradation. Thrombin‐activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase that, after activation, can attenuate plasmin‐mediated fibrin degradation by removing the C‐terminal lysine residues from fibrin, which play a role in the binding and activation of plasminogen. To test the hypothesis that TAFI is an important determinant in the control of tissue repair, we investigated the effect of TAFI deficiency on the healing of cutaneous wounds and colonic anastomoses. Histological examination revealed inappropriate organization of skin wound closure in the TAFI knockout mice, including an altered pattern of epithelial migration. The time required to completley heal the cutaneous wounds was slightly delayed in TAFI‐deficient mice. Healing of colonic anastomoses was also impaired, as reflected by decreased strength of the tissue at the site of the suture, and by bleeding complications in 3 of 14 animals. Together, these abnormalities resulted in increased mortality in TAFI‐deficient mice after colonic anastomoses. Although our study shows that tissue repair, including re‐epithelialization and scar formation, occurs in TAFI‐deficient mice, TAFI appears to be important for appropriate organization of the healing process.

Immune responses against domain I of β2-glycoprotein I are driven by conformational changes: Domain I of β2-glycoprotein I harbors a cryptic immunogenic epitope

OBJECTIVE The presence of autoantibodies against a cryptic epitope in domain I of β(2)-glycoprotein I (β(2)GPI) is strongly associated with thrombotic events in patients with the antiphospholipid syndrome. We hypothesized that a conformational change could be a trigger for the formation of antibodies against domain I of β(2)GPI. Therefore, we investigated whether immune responses against β(2)GPI are related to its conformation. METHODS Conformational changes in β(2)GPI were studied using various techniques, either upon binding to cardiolipin or after disruption of the internal disulfide bonds. The immunogenicity of β(2)GPI in different conformations as well as the individual domains of β(2)GPI were studied in vivo by monitoring the generation of antibodies after intravenous administration of β(2)GPI to mice. Furthermore, plasma samples from these mice were assessed for lupus anticoagulant activity and thrombin-antithrombin complex levels. RESULTS We observed that the interaction of β(2)GPI with cardiolipin induced a conformational change in β(2)GPI: electron microscopy revealed that β(2)GPI assembled into polymeric meshworks. We next investigated the immunogenicity of both human and murine β(2)GPI in mice. Both human and murine β(2)GPI combined with cardiolipin and misfolded β(2)GPI triggered antibody formation against the native protein as well as against domain I of β(2)GPI, while native β(2)GPI was not immunogenic. In addition, we observed that anti-domain I antibodies developed in mice injected with domain I of β(2)GPI, and that antibodies did not develop in mice injected with domains II-V. The induced anti-domain I antibodies prolonged the dilute Russells viper venom plasma clotting time. The plasma of mice with anti-domain I antibodies had increased levels of circulating thrombin-antithrombin complexes. CONCLUSION The results of our studies indicate that the exposure of cryptic epitopes due to conformational changes in β(2)GPI can induce autoantibody formation.

Activation of Human Platelets by Misfolded Proteins

Objective—Protein misfolding diseases result from the deposition of insoluble protein aggregates that often contain fibrils called amyloid. Amyloids are found in Alzheimer disease, atherosclerosis, diabetes mellitus, and systemic amyloidosis, which are diseases where platelet activation might be implicated. Methods and Results—We induced amyloid properties in 6 unrelated proteins and found that all induced platelet aggregation in contrast to fresh controls. Amyloid-induced platelet aggregation was independent of thromboxane A2 formation and ADP secretion but enhanced by feedback stimulation through these pathways. Treatments that raised cAMP (iloprost), sequestered Ca2+ (BAPTA-AM) or prevented amyloid-platelet interaction (sRAGE, tissue-type plasminogen activator [tPA]) induced almost complete inhibition. Modulation of the function of CD36 (CD36−/− mice), p38MAPK (SB203580), COX-1 (indomethacin), and glycoprotein Ib&agr; (Nk-protease, 6D1 antibody) induced ≈50% inhibition. Interference with fibrinogen binding (RGDS) revealed a major contribution of &agr;IIb&bgr;3-independent aggregation (agglutination). Conclusions—Protein misfolding resulting in the appearance of amyloid induces platelet aggregation. Amyloid activates platelets through 2 pathways: one is through CD36, p38MAPK, thromboxane A2–mediated induction of aggregation; the other is through glycoprotein Ib&agr;–mediated aggregation and agglutination. The platelet stimulating properties of amyloid might explain the enhanced platelet activation observed in many diseases accompanied by the appearance of misfolded proteins with amyloid.

Recombinant endostatin forms amyloid fibrils that bind and are cytotoxic to murine neuroblastoma cells in vitro

Endostatin is a fragment of collagen XVIII that acts as an endogenous inhibitor of tumor angiogenesis and tumor growth. Anti‐tumor effects have been described using both soluble and insoluble recombinant endostatin. However, differences in endostatin structure are likely to cause differences in bioactivity. In the present study we have investigated the structure and cellular effects of insoluble endostatin. We found that insoluble endostatin shows all the hallmarks of amyloid aggregates. Firstly, it binds Congo red and shows the characteristic apple‐green birefringe when examined under polarized light. Secondly, electron microscopy shows that endostatin forms short unbranched fibrils. Thirdly, X‐ray analysis shows the abundant presence of cross‐β sheets, the tertiary structure that underlies fibrillogenesis. None of these properties was observed when examining soluble endostatin. Soluble endostatin can be triggered to form cross‐β sheets following denaturation, indicating that endostatin is a protein fragment with an inherent propensity to form amyloid deposits. Like β‐amyloid, found in the brains of patients with Alzheimers disease, amyloid endostatin binds to and is toxic to neuronal cells, whereas soluble endostatin has no effect on cell viability. Our results demonstrate a previously unrecognized functional difference between soluble and insoluble endostatin, only the latter acting as a cytotoxic amyloid substance.

No grip, no growth: the conceptual basis of excessive proteolysis in the treatment of cancer

The formation of new bloodvessels, called angiogenesis, is critical for a tumour to grow beyond a few mm(3) in size. A provisional matrix promotes endothelial cell adhesion, migration, proliferation and survival. Synthesis and degradation of this matrix closely resemble processes that occur during coagulation and fibrinolysis. Degradation of the matrix and fibrinolysis are tightly controlled and balanced by stimulators and inhibitors of the plasminogen activation system. Here we give an overview of these processes during tumour progression. We postulate a novel way to inhibit angiogenesis by removal of the matrix through specific and localised overstimulation of the plasminogen activation system.