Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis.

Heparan sulfate proteoglycans interact with many extracellular matrix constituents, growth factors and enzymes. Degradation of heparan sulfate by endoglycosidic heparanase cleavage affects a variety of biological processes. We have purified a 50-kDa heparanase from human hepatoma and placenta, and now report cloning of the cDNA and gene encoding this enzyme. Expression of the cloned cDNA in insect and mammalian cells yielded 65-kDa and 50-kDa recombinant heparanase proteins. The 50-kDa enzyme represents an N-terminally processed enzyme, at least 100-fold more active than the 65-kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and specimens of human breast, colon and liver carcinomas. Low metastatic murine T-lymphoma and melanoma cells transfected with the heparanase cDNA acquired a highly metastatic phenotype in vivo, reflected by a massive liver and lung colonization. This represents the first cloned mammalian heparanase, to our knowledge, and provides direct evidence for its role in tumor metastasis. Cloning of the heparanase gene enables the development of specific molecular probes for early detection and treatment of cancer metastasis and autoimmune disorders.     

Heparanase Promotes Engraftment and Prevents Graft versus Host Disease in Stem Cell Transplantation

Background

Heparanase, endoglycosidase that cleaves heparan sulfate side chains of heparan sulfate proteoglycans, plays important roles in cancer metastasis, angiogenesis and inflammation.

Design and Methods

Applying a mouse model of bone marrow transplantation and transgenic mice over-expressing heparanase, we evaluated the effect of heparanase on the engraftment process and the development of graft-versus-host disease.

Results

Analysis of F1 mice undergoing allogeneic bone marrow transplantation from C57BL/6 mice demonstrated a better and faster engraftment in mice receiving cells from donors that were pretreated with heparanase. Moreover, heparanase treated recipient F1 mice showed only a mild appearance of graft-versus-host disease and died 27 days post transplantation while control mice rapidly developed signs of graft-versus-host disease (i.e., weight loss, hair loss, diarrhea) and died after 12 days, indicating a protective effect of heparanase against graft-versus-host disease. Similarly, we applied transgenic mice over-expressing heparanase in most tissues as the recipients of BMT from C57BL/6 mice. Monitoring clinical parameters of graft-versus-host disease, the transgenic mice showed 100% survival on day 40 post transplantation, compared to only 50% survival on day 14, in the control group. In vitro and in vivo studies revealed that heparanase inhibited T cell function and activation through modulation of their cytokine repertoire, indicated by a marked increase in the levels of Interleukin-4, Interleukin-6 and Interleukin-10, and a parallel decrease in Interleukin-12, tumor necrosis factor-alfa and interferon-gamma. Using point mutated inactive enzyme, we found that the shift in cytokine profile was independent of heparanase enzymatic activity.

Conclusions

Our results indicate a significant role of heparanase in bone marrow transplantation biology, facilitating engraftment and suppressing graft-versus-host disease, apparently through an effect on T cell activation and cytokine production pattern.     

Aβ(39-42) modulates Aβ oligomerization but not fibril formation

Recently, certain C-terminal fragments (CTFs) of Aβ42 have been shown to be effective inhibitors of Aβ42 toxicity. Here, we examine the interactions between the shortest CTF in the original series, Aβ(39-42), and full-length Aβ. Mass spectrometry results indicate that Aβ(39-42) binds directly to Aβ monomers and to the n = 2, 4, and 6 oligomers. The Aβ42:Aβ(39-42) complex is further probed using molecular dynamics simulations. Although the CTF was expected to bind to the hydrophobic C-terminus of Aβ42, the simulations show that Aβ(39-42) binds at several locations on Aβ42, including the C-terminus, other hydrophobic regions, and preferentially in the N-terminus. Ion mobility-mass spectrometry (IM-MS) and electron microscopy experiments indicate that Aβ(39-42) disrupts the early assembly of full-length Aβ. Specifically, the ion-mobility results show that Aβ(39-42) prevents the formation of large decamer/dodecamer Aβ42 species and, moreover, can remove these structures from solution. At the same time, thioflavin T fluorescence and electron microscopy results show that the CTF does not inhibit fibril formation, lending strong support to the hypothesis that oligomers and not amyloid fibrils are the Aβ form responsible for toxicity. The results emphasize the role of small, soluble assemblies in Aβ-induced toxicity and suggest that Aβ(39-42) inhibits Aβ-induced toxicity by a unique mechanism, modulating early assembly into nontoxic hetero-oligomers, without preventing fibril formation.     

Zn2+-Aβ40 complexes form metastable quasi-spherical oligomers that are cytotoxic to cultured hippocampal neurons

The roles of metal ions in promoting amyloid β-protein (Aβ) oligomerization associated with Alzheimer disease are increasingly recognized. However, the detailed structures dictating toxicity remain elusive for Aβ oligomers stabilized by metal ions. Here, we show that small Zn(2+)-bound Aβ1-40 (Zn(2+)-Aβ40) oligomers formed in cell culture medium exhibit quasi-spherical structures similar to native amylospheroids isolated recently from Alzheimer disease patients. These quasi-spherical Zn(2+)-Aβ40 oligomers irreversibly inhibit spontaneous neuronal activity and cause massive cell death in primary hippocampal neurons. Spectroscopic and x-ray diffraction structural analyses indicate that despite their non-fibrillar morphology, the metastable Zn(2+)-Aβ40 oligomers are rich in β-sheet and cross-β structures. Thus, Zn(2+) promotes Aβ40 neurotoxicity by structural organization mechanisms mediated by coordination chemistry.     

En route to early diagnosis of Alzheimer's disease--are we there yet?

As therapeutics for Alzheimer's disease (AD) become available, it will become increasingly important to develop accurate and reliable tools for its early diagnosis. A recent paper by Georganopoulou et al. suggests that nanobiotechnology could help to overcome the limitations with the current diagnostic methods. The paper introduces a nanoparticle-based assay using antibodies that are specific for amyloid beta-protein (Abeta) oligomers with sub-femtomolar sensitivity. This assay appears to be more specific for AD than previous ELISA-based work and, if specificity of the assay for Abeta oligomers can be established, the method developed might provide a sensitive, reliable diagnostic tool for AD.     

Synthesis of a bicyclic BPTI mimetic containing 4-thioproline replacing Cys38

The synthesis of a backbone bicyclic nonapeptide that mimics the binding site of bovine pancreatic trypsin inhibitor (BPTI) is described. The BPTI mimetic, which contains cis-thioproline replacing Cys³⁸ of the protein, inhibits trypsin with a K_i of 76 M.     

New backbone cyclic substance P analogs

Summary Structure-activity relationships of cyclic substance P (SP) analogs were extensively studied in our laboratories utilizing the new concept of backbone cyclization. Employing the C-terminal hexapeptide SP_6–11, we examined the influence of chemical changes in peptides containing backbone-to-amino-end cyclization. These changes in the ring have significant influence on activity, and should be carefully designed in order to optimize pharmacological feature.     

The molecular tweezer CLR01 reduces aggregated,pathologic, and seeding-competent α-synuclein in experimental multiple system atrophy

Multiple system atrophy (MSA) is a fatal, adult-onset neurodegenerative disorder that has no cure and very limited treatment options. MSA is characterized by deposition of fibrillar α-synuclein (α-syn) in glial cytoplasmic inclusions in oligodendrocytes. Similar to other synucleinopathies, α-syn self-assembly is thought to be a key pathologic event and a prominent target for disease modification in MSA. Molecular tweezers are broad-spectrum nanochaperones that prevent formation of toxic protein assemblies and enhance their clearance. The current lead compound, CLR01, has been shown to inhibit α-syn aggregation but has not yet been tested in the context of MSA. To fill this gap, here, we conducted a proof-of-concept study to assess the efficacy of CLR01 in remodeling MSA-like α-syn pathology in the PLP-α-syn mouse model of MSA. Six-month-old mice received intracerebroventricular CLR01 (0.3 or 1 mg/kg per day) or vehicle for 32 days. Open-field test revealed a significant, dose-dependent amelioration of an anxiety-like phenotype. Subsequently, immunohistochemical and biochemical analyses showed dose-dependent reduction of pathological and seeding-competent forms of α-syn, which correlated with the behavioral phenotype. CLR01 treatment also promoted dopaminergic neuron survival in the substantia nigra. To our knowledge, this is the first demonstration of an agent that reduces formation of putative high-molecular-weight oligomers and seeding-competent α-syn in a mouse model of MSA, supporting the view that these species are key to the neurodegenerative process and its cell-to-cell progression in MSA. Our study suggests that CLR01 is an attractive therapeutic candidate for disease modification in MSA and related synucleinopathies, supporting further preclinical development.     

Wind as a negative factor in human comfort and its implications for planning

In all comfort formulae the wind appears as a positive factor with low velocities not exceeding 10 m/s. To date most researchers have ignored the negative effect which strong winds can have on man's comfort. Although they are not destructive, such winds blowing continuously for many hours of the day, over long periods of the year may have a negative effect on human comfort. Therefore, until a formula is developed which incorporates high wind velocities as a possible negative factor, the many comfort indices will not fulfill their role in objectively ascertaining man's comfort. Examples are cited from Israel. The most dominant region is the Jordan Rift Valley and its southern extension to the Gulf of Elat. This region is now undergoing intensive development and the strong winds are a factor which must be considered in regional planning, in settlement location and in their detailed planning.     

Selection of Aptamers for Amyloid β-Protein,the Causative Agent of Alzheimer's Disease

Alzheimer''s disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult¹. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies^2,3.Amyloid β-protein (Aβ) is central to AD pathogenesis. Soluble, oligomeric Aβ assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD^4,5. Various forms of soluble Aβ assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood⁶. Specific molecular recognition tools may unravel the relationships amongst Aβ assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies^7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)^9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).Despite emergence of aptamers as tools in modern biotechnology and medicine¹¹, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)^12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-β¹⁷, a soluble, oligomeric, β-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils¹⁸. Aptamers generated using monomeric and several forms of fibrillar β₂-microglobulin (β₂m) were found to bind fibrils of certain other amyloidogenic proteins besides β₂m fibrils¹⁹. Ylera et al. described RNA aptamers selected against immobilized monomeric Aβ40²⁰. Unexpectedly, these aptamers bound fibrillar Aβ40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric Aβ40²¹ generated using photo-induced cross-linking of unmodified proteins (PICUP)^22,23. Similar to previous findings^17,19,20, these aptamers reacted with fibrils of Aβ and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope²¹. Here, we present the SELEX methodology used in production of these aptamers²¹.Download video file.^{(175M, mp4)}