Absolute proteomic quantification reveals design principles of sperm flagellar chemosensation

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Troetschel, C., Hamzeh, H., Alvarez, L., Pascal, R., Lavryk, F., Boenigk, W., Koerschen, H. G., Mueller, A., Poetsch, A., Rennhack, A., Gui, L., Nicastro, D., Struenker, T., Seifert, R., & Kaupp, U. B. Absolute proteomic quantification reveals design principles of sperm flagellar chemosensation. Embo Journal, 39(4), (2020): e102723, doi:10.15252/embj.2019102723.Cilia serve as cellular antennae that translate sensory information into physiological responses. In the sperm flagellum, a single chemoattractant molecule can trigger a Ca2+ rise that controls motility. The mechanisms underlying such ultra‐sensitivity are ill‐defined. Here, we determine by mass spectrometry the copy number of nineteen chemosensory signaling proteins in sperm flagella from the sea urchin Arbacia punctulata. Proteins are up to 1,000‐fold more abundant than the free cellular messengers cAMP, cGMP, H+, and Ca2+. Opto‐chemical techniques show that high protein concentrations kinetically compartmentalize the flagellum: Within milliseconds, cGMP is relayed from the receptor guanylate cyclase to a cGMP‐gated channel that serves as a perfect chemo‐electrical transducer. cGMP is rapidly hydrolyzed, possibly via “substrate channeling” from the channel to the phosphodiesterase PDE5. The channel/PDE5 tandem encodes cGMP turnover rates rather than concentrations. The rate‐detection mechanism allows continuous stimulus sampling over a wide dynamic range. The textbook notion of signal amplification—few enzyme molecules process many messenger molecules—does not hold for sperm flagella. Instead, high protein concentrations ascertain messenger detection. Similar mechanisms may occur in other small compartments like primary cilia or dendritic spines.We thank Heike Krause for preparing the manuscript. Financial support by the Deutsche Forschungsgemeinschaft (DFG) via the priority program SPP 1726 “Microswimmers” and the Cluster of Excellence 1023 “ImmunoSensation” is gratefully acknowledged. We thank D. Stoddard for management of the UTSW cryo‐electron microscope facility, which is funded in part by a Cancer Prevention and Research Institute of Texas (CPRIT) Core Facility Award (RP170644). This study was supported by HHS|National Institutes of Health (NIH) grant R01 GM083122 and by CPRIT grant RR140082 to D. Nicastro

Presenilin Is the Molecular Target of Acidic γ-Secretase Modulators in Living Cells

The intramembrane-cleaving protease γ-secretase catalyzes the last step in the generation of toxic amyloid-β (Aβ) peptides and is a principal therapeutic target in Alzheimer's disease. Both preclinical and clinical studies have demonstrated that inhibition of γ-secretase is associated with prohibitive side effects due to suppression of Notch processing and signaling. Potentially safer are γ-secretase modulators (GSMs), which are small molecules that selectively lower generation of the highly amyloidogenic Aβ42 peptides but spare Notch processing. GSMs with nanomolar potency and favorable pharmacological properties have been described, but the molecular mechanism of GSMs remains uncertain and both the substrate amyloid precursor protein (APP) and subunits of the γ-secretase complex have been proposed as the molecular target of GSMs. We have generated a potent photo-probe based on an acidic GSM that lowers Aβ42 generation with an IC50 of 290 nM in cellular assays. By combining in vivo photo-crosslinking with affinity purification, we demonstrated that this probe binds the N-terminal fragment of presenilin (PSEN), the catalytic subunit of the γ-secretase complex, in living cells. Labeling was not observed for APP or any of the other γ-secretase subunits. Binding was readily competed by structurally divergent acidic and non-acidic GSMs suggesting a shared mode of action. These findings indicate that potent acidic GSMs target presenilin to modulate the enzymatic activity of the γ-secretase complex

Synthesis of a potent photoreactive acidic gamma-secretase modulator for target identification in cells

Supramolecular self-assembly of amyloidogenic peptides is closely associated with numerous pathological conditions. For instance, Alzheimers disease (AD) is characterized by abundant amyloid plaques originating from the proteolytic cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases. Compounds named gamma-secretase modulators (GSMs) can shift the substrate cleavage specificity of gamma-secretase toward the production of non-amyloidogenic, shorter A beta fragments. Herein, we describe the synthesis of highly potent acidic GSMs, equipped with a photoreactive diazirine moiety for photoaffinity labeling. The probes labeled the N-terminal fragment of presenilin (the catalytic subunit of gamma-secretase), supporting a mode of action involving binding to gamma-secretase. This fundamental step toward the elucidation of the molecular mechanism governing the GSM-induced shift in gamma-secretase proteolytic specificity should pave the way for the development of improved drugs against AD. (C) 2012 Elsevier Ltd. All rights reserved

Synthesis and bioactivity of the γ-secretase modulator photo-probe AR243.

<p>(<b>A</b>) The γ-secretase modulator (GSM) photo-probe AR243 was synthesized by a copper(I)-catalyzed three-component coupling between piperidine-derivative <b>1</b>, diazirinyl-benzaldehyde <b>2</b>, and biotin-alkyne <b>3</b>. The intermediate was then de-protected to yield the free acidic photo-probe. i: CuBr, molecular sieves, Tol. 60°C, 2 d. ii: TBAF, THF, r.t., 2 h. (<b>B</b>) CHO cells with stable co-expression of wild type APP and wild type PSEN1 were treated with increasing concentrations of the photo-probe AR243 or DMSO vehicle, and Aβ levels in conditioned media were quantified by sandwich immunoassay. AR243 caused a dose-dependent decrease in Aβ42 levels with a concomitant increase in Aβ38 levels, confirming its bioactivity as a potent GSM with an IC₅₀ for Aβ42 reduction of 290 nM.</p

The γ-secretase modulator photo-probe AR243 targets endogenous PSEN1 in human HEK293T cells.

<p>Photo-affinity labeling studies were performed with membrane preparations from human HEK293T cells as described in Fig. 1. Western blotting of purified target proteins demonstrated labeling of endogenous PSEN1-NFT but not PSEN1-CTF by AR243. Co-incubation with an excess of parent compound BB25 (100 µM) caused displacement of the photo-probe, demonstrating specificity of the binding to PSEN1-NTF. Input represents 0.02% of the total membrane material.</p