Presenilin/gamma-secretase cleaves CD46 in response to Neisseria infection - PubMed (original) (raw)

Presenilin/gamma-secretase cleaves CD46 in response to Neisseria infection

Nathan J Weyand et al. J Immunol. 2010.

Abstract

CD46 is a type I transmembrane protein with complement and T cell regulatory functions in human cells. CD46 has signaling and receptor properties in immune and nonimmune cells, many of which are dependent on the expression of cytoplasmic tail (cyt) isoforms cyt1 or cyt2. Little is known about how cyt1 and cyt2 mediate cellular responses. We show that CD46-cyt1 and CD46-cyt2 are substrates for presenilin/gamma-secretase (PS/gammaS), an endogenous protease complex that regulates many important signaling proteins through proteolytic processing. PS/gammaS processing of CD46 releases immunoprecipitable cyt1 and cyt2 tail peptides into the cell, is blocked by chemical inhibitors, and is prevented in dominant negative presenilin mutant cell lines. Two human pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, stimulate PS/gammaS processing of CD46-cyt1 and CD46-cyt2. This stimulation requires type IV pili and PilT, the type IV pilus retraction motor, implying that mechanotransduction plays a role in this event. We present a model for PS/gammaS processing of CD46 that provides a mechanism by which signals are transduced via the cyt1 and cyt2 tails to regulate CD46-dependent cellular responses. Our findings have broad implications for understanding the full range of CD46 functions in infection and noninfection situations.

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Figures

FIGURE 1

Low m.w. CD46 tail fragments accumulate in CHO cells stably expressing CD46 isoforms. A, Illustration of CD46 structure: the ectodomain consists of four complement control protein repeats (–4) an alternatively spliced STP-rich region, and a small segment of unknown function (U). The transmembrane segment (TM) is followed by one of two cytoplasmic tails (cyt1 or cyt2). Known and predicted tail phosphorylation sites are in black boxes; putative nuclear localization sequences are underlined. B and C, CHO cell lysates were resolved by SDS-PAGE and immunoblotted with a mAb specific to the cyt1 or cyt2 tail (29). D, immunoprecipitation (IP) of CHO cell lysates using the cyt2-specific mAb, followed by immunoblotting of the precipitates using the cyt2 mAb. E, As a loading control, GAPDH levels in lysates used for immunoblotting (IB) (B and C) and IP and IB (D) were determined with anti-GAPDH mAb. R: Parent CHO cell line; C1, C2, BC1, and BC2: CHO lines expressing the different CD46 isoforms. IB, immunoblotting; IP, immunoprecipitation; STP, serine, threonine, and proline; TM, transmembrane; U, unknown function.

FIGURE 2

N. gonorrhoeae infection of endocervical cells induces cleavage of CD46-cyt1 and CD46-cyt2 isoforms. End1 human endocervical epithelial cells were infected with strain MS11 at various MOI for 4 h, or mock infected with medium, and cyt1 or cyt2 tail peptides were immunoprecipitated with the cyt1 (A) or cyt2 (B) mAb and immunoblotted using the same mAb. Middle panels: High contrast image of the same immunoblot to illustrate the lower Mr bands. Bottom panels: Loading controls; input lysates were immunoblotted with anti–β-tubulin mAb.

FIGURE 3

N. gonorrhoeae stimulates CD46 processing in a Tfp- and pilT_-dependent manner. End1 cells were mock infected with medium or infected with wt MS11, MS11_pilT, MS11_pilTi_, MS11_pilTi_ + IPTG (4 mM), or MS11_pilE_ for 4 h at an MOI of 500. Cell lysates were immunoprecipitated with the cyt1 (A) or cyt2 (B) mAb and immunoblotted using the same mAb. High and low contrast images of each immunoblot are presented. Bottom panels: Loading controls; input lysates were immunoblotted with anti–β-tubulin mAb.

FIGURE 4

MMP inhibitor GM6001 prevents release and accumulation of CD46 tail peptides in _Neisseria_-infected cells. End1 cells were infected with N. gonorrhoeae strain MS11 in the presence or absence of MMP inhibitor GM6001 (20 μM), GM6001 negative analog (GM negative, 20 μM), or vehicle. Cyt1 and cyt2 tail peptides were immunoprecipitated from the lysates then immunoblotted with the appropriate mAb. Upper panels: cyt1 immunoblots and loading control. Bottom panels: cyt2 immunoblots and loading control. Loading controls: input lysates immunoblotted with the anti–β-tubulin mAb.

FIGURE 5

PS/γS inhibitors prevent release and accumulation of CD46 tail peptides in _Neisseria_-infected cells. 16HBE14o- cells were infected with N. gonorrhoeae strain MS11 (A) or N. meningitidis strain 8013 (B) (MOI of 500, 4 h) in the presence or absence of PS/γS inhibitor DAPT (10 μM), Compound E (200 nM), or L-685-458 (1 μM). Cyt1 and cyt2 tail peptides were immunoprecipitated from the lysates then immunoblotted with the appropriate mAb. Upper panels: cyt1 immunoblots and loading control. Bottom panels: cyt2 immunoblots and loading control. Loading controls: input lysates immunoblotted with the anti–β-tubulin mAb.

FIGURE 6

DN presenilin mutations block release and accumulation of CD46 tail peptides. A, A 16HBE14o- cell line stably expressing empty vector controls and two 16HBE14o- DN PS1/PS2 cell lines (DN.1 and DN.2) were transiently transfected with the Notch ΔE-6MT (Notch ΔE) PS/γS activity reporter. Notch ΔE and Notch ICD were detected by immunoblotting total cell lysates with an Ab to the myc tag at the C-terminal end of the fusion protein. Notch reporter transfections were performed in triplicate for each cell line. B, 16HBE14o- DN PS1/PS2 cell lines(DN.1 and DN.2) and control cells stably expressing empty vectors (V) were mock infected with medium or infected with N. gonorrhoeae MS11 or N. meningitidis 8013 (MOI of 500, 4 h). Cyt1 tail peptide was immunoprecipitated from cell lysates then immunoblotted using the cyt1 mAb. Bottom panels: Loading control; input lysates immunoblotted with the anti–β-tubulin mAb.

FIGURE 7

Model for PS/γ-S processing of CD46-cyt1 and CD46-cyt2. 1: MMPs cleave the cyt1 (left cartoon, gray tail) and cyt2 (right cartoon, white tail) juxtamembrane regions of CD46, generating soluble ectodomains and a ~9 kDa membrane-spanning cyt1 C-terminal fragment (CTF) and a ~12 kDa cyt2 CTF. 2: PS/γS cleaves the CTF TM segments, releasing a ~6 kDa cyt1 ICD and a ~8 kDa cyt2 ICD from the cell membrane. Based on what is known about other PS/γS substrates, we predict that the small peptides generated by PS/γS cleavage of the CTFs remain in the extracellular milieu. Numbered octagons: Complement control protein repeats. Octagons B and C: STP-rich segments. White rectangle: juxtamembrane 12 aa segment. Black rectangle: transmembrane segment. Figures are not drawn to scale. N, N-linked oligosaccharides. O, _O_-linked oligosaccharides.

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Presenilin/gamma-secretase cleaves CD46 in response to Neisseria infection - PubMed (original) (raw)