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. 2015 Nov;35(21):3684-700.
doi: 10.1128/MCB.00764-15. Epub 2015 Aug 17.

Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder

WooJin Kim  1 , Erin Zekas  1 , Robert Lodge  2 , Delia Susan-Resiga  1 , Edwidge Marcinkiewicz  1 , Rachid Essalmani  1 , Koichiro Mihara  3 , Rithwik Ramachandran  3 , Eugene Asahchop  4 , Benjamin Gelman  5 , Éric A Cohen  2 , Christopher Power  4 , Morley D Hollenberg  6 , Nabil G Seidah  7
Affiliations

Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder

WooJin Kim et al. Mol Cell Biol. 2015 Nov.

Abstract

The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg41↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg41XXXXArg46↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg46↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND.

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Figures

FIG 1
FIG 1
Expression of PCs in human HIV-positive brain and cells undergoing inflammation. (A) mRNA levels of PCs were measured in brains from patients with HIV/AIDS (HIV+) (group B, without neurocognitive impairment; group C, with neurocognitive impairment; group D, with neurocognitive impairment and encephalitis) using quantitative PCR analysis and compared to brains from of HIV-uninfected patients (HIV−) (group A, other disease controls; n = 10 per group). (B) PC expression was also measured in HeLa cells after 24 h of exposure to several cytokines (TNF-α, IL-1β, and IFN-γ). TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β; IFN-γ, gamma interferon (n = 3 per condition). *, P < 0.05; **, P < 0.005. Bars represent the means ± SEM, and P was determined by a Student t test (two-tailed). Casp4, caspase 4.
FIG 2
FIG 2
Increased PAR1 expression in astrocytes in HIVE. (A and B) GFAP immunoreactivity was infrequently detected in astrocytes in HIV− brain compared to results in brain from HIVE, which showed numerous hypertrophied GFAP-positive astrocytes. (C and D) Major histocompatibility antigen class II (MHC class II) immunoreactivity, a marker for activated microglia, was also increased in brain from HIVE compared to that in HIV− brain. (E) PAR1 immunoreactivity (purple) was minimally detected in GFAP-positive astrocytes (brown) in non-HIV brain. (F) PAR1 immunoreactivity (blue) was evident in GFAP-positive astrocytes in HIVE (purple results from blue plus brown), which was localized on the cell body and processes (inset). HIVE, HIV with encephalitis. Original magnification, ×20; inset magnification, ×40.
FIG 3
FIG 3
Localization of PAR1 and PCs in adult mouse brain. In situ hybridization histochemistry was performed in cryosections of mouse brain using 35S-labeled cRNAs. Cb, cerebellum; Cx, cerebral cortex; DG, dentate gyrus; Hi, hippocampus; Hy, hypothalamus; Po, pons; OL, olfactory lobe; St, striatum.
FIG 4
FIG 4
In vitro cleavage of PAR1 by PACE4. A synthetic peptide (19-mer) mimicking the tethered ligand and putative PC cleavage site of human PAR1 was used to assess in vitro cleavage by PCs. Comparative RP-HPLC analyses of PAR1 19-mer digestion with purified soluble PCs or trypsin (which cleaves preferentially at Arg41↓ and at Arg46↓) revealed that PACE4 cleaves human PAR1 at the expected Arg46↓. The products were separated by RP-HPLC on a Varian C18 column, and peptide bonds were detected at 214 nm. The percent cleavage was calculated as the ratio of the normalized peak area (peak area/number of peptide bonds) of C-terminal fragment NPNDKYE to that of the intact 19-mer peptide (at time zero).
FIG 5
FIG 5
Schematic representation of human PAR1 constructs. Human PAR1 construct (wild-type and single-site mutant at Arg46) containing mCherry at its N terminus and eYFP at its C terminus was overexpressed with/without PCs in HEK293 cells to confirm the proteolytic cleavage of PAR1 by PCs. While noncleaved PAR1 would contain both mCherry (red) and eYFP (yellow) fluorescence, cleaved-PAR1 would lose mCherry fluorescence.
FIG 6
FIG 6
Ex vivo cleavage of PAR1 by soluble PC5A and PACE4. A human PAR1 construct containing mCherry at its N terminus and eYFP at its C terminus was overexpressed with PCs in HEK293. Thrombin cleaves PAR1 at Arg41↓, and its activity is inhibited by anti-thrombin III. Soluble PC5A and PACE4 cleave PAR1 at Arg46↓. Scale bars, 20 μm.
FIG 7
FIG 7
PAR1 colocalizes with the cell surface low-density lipoprotein receptor (LDLR). A human PAR1 construct containing mCherry at its N terminus and eYFP at its C terminus overexpressed with LDLR-V5 (green; detected with an Alexa Fluor 647-conjugated secondary donkey anti-mouse antibody) in HEK293 demonstrates the colocalization of PAR1 and LDLR at the cell surface. Scale bar, 20 μm.
FIG 8
FIG 8
Cleavage of PAR1 at Arg46 by PC5A and PACE4. A single-amino acid variant of human PAR1 at Arg46 was coexpressed with PC5A and PACE4. The mutant prevented the cleavage, indicating that PC5A and PACE4 cleave at Arg46. Scale bars, 20 μm.
FIG 9
FIG 9
Cleavage of PAR1 by PC5A and PACE4 on cell surface. Both PC specific inhibitors, cell-permeable RVKR-cmk and nonpermeable D6R, prevented the cleavage of PAR1, indicating that PC5A and PACE4 cleave PAR1 on cell surface rather than in the TGN. Scale bars, 20 μm.
FIG 10
FIG 10
No effect of membrane-bound PC5B and PC7 on PAR1. A human PAR1 construct containing mCherry at its N terminus and eYFP at its C terminus was overexpressed with PCs in HEK293. Exogenous overnight addition of 1 unit/ml thrombin cleaved PAR1 while membrane-bound PC5B and PC7 had no effect. Scale bars, 20 μm.
FIG 11
FIG 11
PACE4 and PC5A cleavage of PAR1 decreases calcium mobilization in response to thrombin stimulation. HEK293 cells were mock transfected or transiently transfected with cDNAs coding for PACE4 or PC5A, as indicated. The next day the cells were incubated for 30 min with fluo-4-AM no-wash calcium indicator dye and then treated with 20 nM thrombin. Intracellular calcium mobilization was immediately assayed by the fluorescence signal (see Materials and Methods). Representative plots of the nonlinear fits of relative fluorescence measured at 516 nm are shown in panel A, and the peak heights are compared in panel B (n = 3). ⭑, P < 0.05, one-way analysis of variance with Dunnett's post hoc test.
FIG 12
FIG 12
PAR1 retention in the TGN by furin. A human PAR1 construct containing mCherry at its N terminus and eYFP at its C terminus overexpressed with furin in HEK293 results in PAR1 retention in the trans-Golgi network as confirmed by a TGN marker, golgin-97. Scale bars, 20 μm.
FIG 13
FIG 13
PAR1 retention in the TGN by PACS1. A human PAR1 construct containing mCherry at its N terminus and eYFP at its C terminus overexpressed with PACS1, a transporting protein of furin from early endosome to TGN, results in similar PAR1 retention in the TGN. Scale bars, 20 μm.
FIG 14
FIG 14
Inhibition of furin cleavage of HIV glycoprotein gp160 by PAR1. Human PAR1 was transiently overexpressed with PCs in COS-1 cells. (A) PAR1 inhibits furin processing of gp160 to gp41. (B) A PAR1 mutant (R46A) prevents inhibition on furin processing. (C) PAR1 does not block processing of Vpr by soluble PACE4. WB, Western blotting. FP, fusion peptide; M, membrane.
FIG 15
FIG 15
PAR1 inhibition of membrane-bound PC5B and PC7. Human PAR1 was transiently overexpressed with PC5A, PC5B, or PC7 in HEK293 cells. (A) PAR1 inhibits processing of proGDF11 by PC5B, not PC5A. The structures of PC5A and PC5B are illustrated below. The arrows represent the autocatalytic cleavage sites. The positions of the active site Asp, His, and Ser and the oxyanion hole Asn are also shown. The transmembrane domain (gray) of PC5B is before the green cytosolic tail. The Cys-rich domain is represented in pink. (B) PAR1 partially inhibits PC7 processing of human TfR1. FG, Flag; s-hTfR1, soluble human TfR1.
FIG 16
FIG 16
A proximity ligation assay (PLA) demonstrates the interaction of furin and PC7 with PAR1. HEK293 cells were transfected with cDNAs as indicated and incubated with rabbit anti-PAR1 and mouse anti-V5 primary antibodies. PLA probes, anti-rabbit antibody minus sense and anti-mouse antibody plus sense, were utilized to detect the interaction of mCherry-hPAR1-eYFP with V5-tagged furin or PC7. The dots (indicating interactions of PLA probes) per cell were manually counted. The enlarged pictures emphasize transfected cells where PAR1 colocalizes with either furin or PC7, indicated by green dots. Based on the distribution of dots/cell (right panels), we determined that control cells averaged <2 dots/cell whereas those that express furin or PC7 averaged 35 and 23 dots/cell, respectively. Scale bars, 15 μm.
FIG 17
FIG 17
Inhibition of furin processing of gp160 of HIV-1 by PAR1 reduces virus infectivity. (A) HEK293T cells were cotransfected with the indicated amounts of plasmids (μg), and cellular protein expression was analyzed following SDS-PAGE and Western blotting, as described in Materials and Methods. (B) Quantitative analyses of selected intracellular viral proteins. (C) Infection assay for HIV-1 viruses. HEK293T cells were transfected with 5 μg of proviral NL4-3 DNA together with either 0.1 μg of mCherry-hPAR1-eYFP R46A mutant plus 0.9 μg of control EGFP-C1 vector or with 0.1 μg of WT mCherry-hPAR1-eYFP plus 0.9 μg of control EGFP-C1 vector. A dilution curve of viral load confirmed nonsaturating conditions (1 ng of virus per 750,000 TZM-bl cells). Note the ∼70% inhibition of virus infectivity by WT PAR1. ***, P < 0.001 (Student's t test). These data are representative of at least two independent experiments.
FIG 18
FIG 18
Schematic diagram of the effects of PAR1 on PCs. Human PAR1 is processed by thrombin at Arg41↓ and by the soluble PC5A and PACE4 at Arg46↓, resulting in inflammatory and cytoprotective effects, respectively. On the other hand, PAR1 inhibits the membrane-bound furin and PC5B and partially PC7 and is relocalized to the TGN by furin.
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