Raymond Miquelis

Protein-disulfide isomerase (PDI) in FRTL5 cells. pH-dependent thyroglobulin/PDI interactions determine a novel PDI function in the post-endoplasmic reticulum of thyrocytes.

Thyroglobulin (TG) is secreted by the thyrocytes into the follicular lumen of the thyroid. After maturation and hormone formation, TG is endocytosed and delivered to lysosomes. Quality control mechanisms may occur during this bidirectional traffic since 1) several molecular chaperones are cosecreted with TG in vivo, and 2) lysosomal targeting of immature TG is thought to be prevented via the interaction, in acidic conditions, between the Ser789–Met1172 TG hormonogenic domain (BD) and an unidentified membrane receptor. We investigated the secretion and cell surface expression of PDI and other chaperones in the FRTL5 thyroid cell line, and then studied the characteristics of the interaction between TG and PDI. We demonstrated that PDI, but also other chaperones such as calnexin and KDEL-containing proteins are exposed at the cell surface. We observed on living cells or membrane preparations that PDI specifically binds TG in acidic conditions, and that only BD is involved in binding. Surface plasmon resonance analysis of TG/PDI interactions indicated: 1) that PDI bound TG but only in acidic conditions, and that it preferentially recognized immature molecules, and 2) BD is involved in binding even if cysteine-rich modules are deleted. The notion that PDI acts as an “escort” for immature TG in acidic post-endoplasmic reticulum compartments is discussed.

A protein chip membrane-capture assay for botulinum neurotoxin activity

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC(50)s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC(50) of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.

Synaptic vesicle chips to assay botulinum neurotoxins

BoNTs (botulinum neurotoxins), considered to be the most toxic of all biological substances, inhibit neurotransmission through proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins [VAMP (vesicle-associated membrane protein, or synaptobrevin), SNAP-25 (25 kDa synaptosome-associated protein) or syntaxin]. Expansion in the use of BoNTs as therapeutic and cosmetic agents, and the potential threat they constitute as biological weapons, underlines the need for rapid and sensitive in vitro assays. Here, we present new automatized bioassays to detect VAMP cleavage by BoNT/B and F. Western blotting and SPR (surface plasmon resonance) methods revealed that BoNT/B and F totally cleave their substrate on immunoisolated SVs (synaptic vesicles). Real-time monitoring of the immunocapture of native SVs from crude lysates on SPR sensor chips enabled the detection of picogram amounts of different SV proteins. Pre-incubation of a membrane fraction containing SVs with BoNT specifically inhibited capture by anti-VAMP antibodies, and amounts as low as 0.1 pg of BoNT/B were detected. This automated SPR assay is approx. 200 times more sensitive, and 25 times more rapid, than the in vivo BoNT/B test currently used. Moreover, the method can be performed using a few thousand cultured neurons and constitutes a new screening assay for inhibitors. Our data indicate that native VAMP is an optimal substrate for in vitro BoNT assays that can be monitored by SPR.

Mammalian peptidylglycine alpha-amidating monooxygenase mRNA expression can be modulated by the La autoantigen.

ABSTRACT Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal α-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3′ untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3′ UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3′ UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3′ UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.