Karine Berthelot

Isopentenyl diphosphate isomerase: A checkpoint to isoprenoid biosynthesis

Even if the isopentenyl diphosphate (IPP) isomerases have been discovered in the 50s, it is only in the last decade that the genetical, enzymatical, structural richness and cellular importance of this large family of crucial enzymes has been uncovered. Present in all living kingdoms, they can be classified in two subfamilies: type 1 and type 2 IPP isomerases, which show clearly distinct characteristics. They all perform the regulatory isomerization of isopentenyl diphosphate into dimethylallyl diphosphate, a key rate-limiting step of the terpenoid biosynthesis, via a protonation/deprotonation mechanism. Due to their importance in the isoprenoid metabolism and the increasing interest of industry devoted to terpenoid production, it is foreseen that the biotechnological development of such enzymes should be under intense scrutiny in the near future.

Rubber Elongation Factor (REF), a Major Allergen Component in Hevea brasiliensis Latex Has Amyloid Properties

REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom.

Hevea brasiliensis REF (Hev b 1) and SRPP (Hev b 3): An overview on rubber particle proteins

This review article aims to gather all the knowledge on two important proteins associated with Hevea brasiliensis rubber particles: namely the rubber elongation factor (REF) and the small rubber particle protein (SRPP). It covers more then three decades of research on these two proteins and their homologues in plants, and particularly emphasizes on the different possible properties or functions of these various proteins found in plants.

Homologous Hevea brasiliensis REF (Hevb1) and SRPP (Hevb3) present different auto-assembling

HbREF and HbSRPP are two Hevea brasiliensis proteins present on rubber particles, and probably involved in the coagulation of latex. Their function is unclear, but we previously discovered that REF had amyloid properties, which could be of particular interest during the coagulation process. First, we confirmed that REF and SRPP, homologous and principal proteins in hevea latex, are not glycoproteins. In this work, we investigated various aspects of protein interactions: aggregation, auto-assembling, yeast and erythrocyte agglutination, co-interactions by various biochemical (PAGE, spectroscopy, microscopy), biophysical (DLS, ellipsometry) and structural (TEM, ATR-FTIR, PM-IRRAS) approaches. We demonstrated that both proteins are auto-assembling into different aggregative states: REF polymerizes as an amyloid rich in β-sheets and forms quickly large aggregates (>μm), whereas SRPP auto-assembles in solution into stable nanomultimers of a more globular nature. Both proteins are however able to interact together, and SRPP may inhibit the amyloidogenesis of REF. REF is also able to interact with the membranes of yeasts and erythrocytes, leading to their agglutination. In addition, we also showed that both REF and SRPP did not have antimicrobial activity, whereas their activity on membranes has been clearly evidenced. We may suspect that these aggregative properties, even though they are clearly different, may occur during coagulation, when the membrane is destabilized. The interaction of proteins with membranes could help in the colloidal stability of latex, whereas the protein-protein interactions would contribute to the coagulation process, by bringing rubber particles together or eventually disrupting the particle monomembranes.

A Yeast Toxic Mutant of HET-s(218-289) Prion Displays Alternative Intermediates of Amyloidogenesis

Amyloids are thought to be involved in various types of neurodegenerative disorders. Several kinds of intermediates, differing in morphology, size, and toxicity, have been identified in the multistep amyloidogenesis process. However, the mechanisms explaining amyloid toxicity remain unclear. We previously generated a toxic mutant of the nontoxic HET-s((218-289)) amyloid in yeast. Here we report that toxic and nontoxic amyloids differ not only in their structures but also in their assembling process. We used multiple and complementary methods to investigate the intermediates formed by these two amyloids. With the methods used, no intermediates were observed for the nontoxic amyloid; however, under the same experimental conditions, the toxic mutant displayed visible oligomeric and fibrillar intermediates.

Highlights on Hevea brasiliensis (pro)hevein proteins.

Hevein, from Hevea brasiliensis (rubber tree), was identified in 1960. It is the most abundant soluble protein (22%) found in latex. Hevein is formed from a larger protein called prohevein. The 187 amino-acid prohevein is cleaved into two fragments: the N-terminal 43 amino-acid hevein, a lectin bearing a chitin-binding motif with antifungal properties, and a C-terminal domain (C-ter), which possesses amyloid properties. Hevein-like proteins are also widely represented in the plant kingdom and belong to a larger family related to stress and pathogenic responses. During the last 55 years, these proteins have attracted the interest of numerous specialists from the fields of plant physiology, genetics, molecular and structural biology, and physico-chemistry to allergology. This review highlights various aspects of hevein, prohevein, and C-ter from the point of view of these various fields, and examines their potential roles in latex as well as their beneficial and negative biological effects (e.g. wound sealing and resistance to pathogens which is mediated by agglutination, antimicrobial activity, and/or allergenicity). It covers results and observations from 1960 up to the most recent research.

Rubber particle proteins REF1 and SRPP1 interact differently with native lipids extracted from Hevea brasiliensis latex

Rubber particle membranes from the Hevea latex contain predominantly two proteins, REF1 and SRPP1 involved in poly(cis-1,4-isoprene) synthesis or rubber quality. The repartition of both proteins on the small or large rubber particles seems to differ, but their role in the irreversible coagulation of the rubber particle is still unknown. In this study we highlighted the different modes of interactions of both recombinant proteins with different classes of lipids extracted from Hevea brasiliensis latex, and defined as phospholipids (PL), glycolipids (GL) and neutral lipids (NL). We combined two biophysical methods, polarization modulated-infrared reflection adsorption spectroscopy (PM-IRRAS) and ellipsometry to elucidate their interactions with monolayers of each class of lipids. REF1 and SRPP1 interactions with native lipids are clearly different; SRPP1 interacts mostly in surface with PL, GL or NL, without modification of its structure. In contrast REF1 inserts deeply in the lipid monolayers with all lipid classes. With NL, REF1 is even able to switch from α-helice conformation to β-sheet structure, as in its aggregated form (amyloid form). Interaction between REF1 and NL may therefore have a specific role in the irreversible coagulation of rubber particles.

Unexpected dimerization of isoprene in a gas chromatography inlet. A study by gas chromatography/mass spectrometry coupling.

During analysis of pure isoprene by gas chromatography/mass spectrometry (GC-MS) using a programmed temperature vaporization (PTV) inlet, the presence of several isoprene dimers was detected in the total ion chromatograms (TICs). This study intends to determine the part of the instrument where dimerization occurs and the relative importance of the dimer amounts under different experimental conditions. The reference thermal dimerization of isoprene gives four six-membered cyclic dimers and two eight-membered ones. In all samples containing different amounts of freshly distilled isoprene, only peaks corresponding to the former appeared in TICs. For the same temperature, their amounts increase as the concentration of injected isoprene increases. The main products are diprene (from 80 to 100%) of the total dimers and dipentene (from 1 to 14%). The sum of the two other dimers is never higher than 6%. In conclusion, isomeric dimers are produced through a dimerization in the inlet. No dimerization of isoprene occurs in the mass spectrometer source. Then care is needed when analyzing terpenic compounds in the presence of isoprene by GC-MS because structures, retention times and mass spectra of diprene and dipentene are close.

Hevea brasiliensis prohevein possesses a conserved C-terminal domain with amyloid-like properties in vitro.

Prohevein is a wound-induced protein and a main allergen from latex of Hevea brasiliensis (rubber tree). This 187 amino-acid protein is cleaved in two fragments: a N-terminal 43 amino-acids called hevein, a lectin bearing a chitin-binding motif with antifungal properties and a C-terminal domain (C-ter) far less characterized. We provide here new insights on the characteristics of prohevein, hevein and C-terminal domain. Using complementary biochemical (ThT/CR/chitin binding, agglutination) and structural (modeling, ATR-FTIR, TEM, WAXS) approaches, we show that this domain clearly displays all the characteristics of an amyloid-like proteins in vitro, that could confer agglutination activity in synergy with its chitin-binding activity. Additionally, this C-ter domain is highly conserved and present in numerous plant prohevein-like proteins or pathogenesis-related (PR and WIN) proteins. This could be the hallmark of the eventual presence of proteins with amyloid properties in plants, that could potentially play a role in defense through aggregation properties.