Human pregnancy zone protein stabilizes misfolded proteins including preeclampsia- and Alzheimer's-associated amyloid beta peptide.

Protein misfolding underlies the pathology of a large number of human disorders, many of which are age-related. An exception to this is preeclampsia, a leading cause of pregnancy-associated morbidity and mortality in which misfolded proteins accumulate in body fluids and the placenta. We demonstrate that pregnancy zone protein (PZP), which is dramatically elevated in maternal plasma during pregnancy, efficiently inhibits in vitro the aggregation of misfolded proteins, including the amyloid beta peptide (Aß) that is implicated in preeclampsia as well as with Alzheimer's disease. The mechanism by which this inhibition occurs involves the formation of stable complexes between PZP and monomeric Aß or small soluble Aß oligomers formed early in the aggregation pathway. The chaperone activity of PZP is more efficient than that of the closely related protein alpha-2-macroglobulin (α2M), although the chaperone activity of α2M is enhanced by inducing its dissociation into PZP-like dimers. By immunohistochemistry analysis, PZP is found primarily in extravillous trophoblasts in the placenta. In severe preeclampsia, PZP-positive extravillous trophoblasts are adjacent to extracellular plaques containing Aß, but PZP is not abundant within extracellular plaques. Our data support the conclusion that the up-regulation of PZP during pregnancy represents a major maternal adaptation that helps to maintain extracellular proteostasis during gestation in humans. We propose that overwhelming or disrupting the chaperone function of PZP could underlie the accumulation of misfolded proteins in vivo. Attempts to characterize extracellular proteostasis in pregnancy will potentially have broad-reaching significance for understanding disease-related protein misfolding.

Nanobody engineering: computational modelling and design for biomedical and therapeutic applications.

Nanobodies, the smallest functional antibody fragment derived from camelid heavy-chain-only antibodies, have emerged as powerful tools for diverse biomedical applications. In this comprehensive review, we discuss the structural characteristics, functional properties, and computational approaches driving the design and optimisation of synthetic nanobodies. We explore their unique antigen-binding domains, highlighting the critical role of complementarity-determining regions in target recognition and specificity. This review further underscores the advantages of nanobodies over conventional antibodies from a biosynthesis perspective, including their small size, stability, and solubility, which make them ideal candidates for economical antigen capture in diagnostics, therapeutics, and biosensing. We discuss the recent advancements in computational methods for nanobody modelling, epitope prediction, and affinity maturation, shedding light on their intricate antigen-binding mechanisms and conformational dynamics. Finally, we examine a direct example of how computational design strategies were implemented for improving a nanobody-based immunosensor, known as a Quenchbody. Through combining experimental findings and computational insights, this review elucidates the transformative impact of nanobodies in biotechnology and biomedical research, offering a roadmap for future advancements and applications in healthcare and diagnostics.

A Novel Role for Plasminogen Activator Inhibitor Type-2 as a Hypochlorite-Resistant Serine Protease Inhibitor and Holdase Chaperone.

Plasminogen activator inhibitor type-2 (PAI-2), a member of the serpin family, is dramatically upregulated during pregnancy and in response to inflammation. Although PAI-2 exists in glycosylated and non-glycosylated forms in vivo, the majority of in vitro studies of PAI-2 have exclusively involved the intracellular non-glycosylated form. This study shows that exposure to inflammation-associated hypochlorite induces the oligomerisation of PAI-2 via a mechanism involving dityrosine formation. Compared to plasminogen activator inhibitor type-1 (PAI-1), both forms of PAI-2 are more resistant to hypochlorite-induced inactivation of its protease inhibitory activity. Holdase-type extracellular chaperone activity plays a putative non-canonical role for PAI-2. Our data demonstrate that glycosylated PAI-2 more efficiently inhibits the aggregation of Alzheimer's disease and preeclampsia-associated amyloid beta peptide (Aß), compared to non-glycosylated PAI-2 in vitro. However, hypochlorite-induced modification of non-glycosylated PAI-2 dramatically enhances its holdase activity by promoting the formation of very high-molecular-mass chaperone-active PAI-2 oligomers. Both PAI-2 forms protect against Aß-induced cytotoxicity in the SH-SY5Y neuroblastoma cell line in vitro. In the villous placenta, PAI-2 is localised primarily to syncytiotrophoblast with wide interpersonal variation in women with preeclampsia and in gestational-age-matched controls. Although intracellular PAI-2 and Aß staining localised to different placental cell types, some PAI-2 co-localised with Aß in the extracellular plaque-like aggregated deposits abundant in preeclamptic placenta. Thus, PAI-2 potentially contributes to controlling aberrant fibrinolysis and the accumulation of misfolded proteins in states characterised by oxidative and proteostasis stress, such as in Alzheimer's disease and preeclampsia.

Alpha-2-Macroglobulin, a Hypochlorite-Regulated Chaperone and Immune System Modulator.

Alpha-macroglobulins are ancient proteins that include monomeric, dimeric, and tetrameric family members. In humans, and many other mammals, the predominant alpha-macroglobulin is alpha-2-macroglobulin (α 2M), a tetrameric protein that is constitutively abundant in biological fluids (e.g., blood plasma, cerebral spinal fluid, synovial fluid, ocular fluid, and interstitial fluid). α 2M is best known for its remarkable ability to inhibit a broad spectrum of proteases, but the full gamut of its activities affects diverse biological processes. For example, α 2M can stabilise and facilitate the clearance of the Alzheimer's disease-associated amyloid beta (Aß) peptide. Additionally, α 2M can influence the signalling of cytokines and growth factors including neurotrophins. The results of several studies support the idea that the functions of α 2M are uniquely regulated by hypochlorite, an oxidant that is generated during inflammation, which induces the native α 2M tetramer to dissociate into dimers. This review will discuss the evidence for hypochlorite-induced regulation of α 2M and the possible implications of this in neuroinflammation and neurodegeneration.

PZP and PAI-2: Structurally-diverse, functionally similar pregnancy proteins?

Pregnancy zone protein (PZP) and plasminogen activator inhibitor type 2 (PAI-2) are two multifunctional proteins that are elevated in normal pregnancy and numerous other inflammatory states. Both proteins were originally identified as protease inhibitors, but current evidence supports the notion that they may also function as modulators of T-helper cells and/or extracellular chaperones. Exacerbated inflammation, fibrinolytic disturbances and misfolded proteins are all implicated in the pathology of preeclampsia, a leading cause of maternal and foetal mortality and morbidity. Notably, reduced levels of PZP or PAI-2 are associated with preeclampsia and clarification of their diverse functions in normal pregnancy could provide much needed insight regarding the pathogenesis of this disorder. Given that inflammation and protein misfolding underlie the pathology of a very large number of disorders, the contributions of PZP and PAI-2 to extracellular proteostasis and immunoregulation could be broad-reaching.