1. ERp44 Exerts Redox-Dependent Control of Blood Pressure at the ER
Chihiro Hisatsune, Etsuko Ebisui, Masaya Usui, Naoko Ogawa, Akio Suzuki, Nobuko Mataga, Hiromi Takahashi-Iwanaga, Katsuhiko Mikoshiba 
Molecular Cell 
Volume 58, Issue 6, Pages (June 2015)
DOI: /j.molcel
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2015 58, 1015-1027DOI: (10.1016/j.molcel.2015.04.008)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 Erp44−/− Mice Exhibit Hypotension
(A) C57BL/6 Erp44−/− mice have reduced urine volume at birth. White arrows indicate bladder. Lower panel shows the diameter of the bladder. The n for each genotype is indicated on the corresponding bar of the graph. Mean ± SD. ∗∗p < 0.01.
(B) Kidney morphology of C57BL/6 Erp44−/− mice at birth. Arrows indicate enlarged renal tubules. R, renal pelvis. U, ureter. Scale bar, 600 μm. Lower panels show magnified images of kidney cortex. Asterisks show enlarged renal tubules. Scale bar, 250 μm. Erp44+/+ (n = 6), Erp44−/− (n = 6), Erp44+/− (n = 5, data not shown).
(C) Systolic blood pressure (SBP) of FVB/NJ × C57BL/6 Erp44−/− mice and their age-matched littermates (2–12 months old). Upper panel, male; lower panel, female. The n for each genotype is indicated on the corresponding bar of the graph. Mean ± SD. ∗∗p < 0.01.
(D) Representative mass chromatograms of angiotensin II–IV in mouse plasma (upper panel) and internal standards with a stable isotope (angiotensin II+–IV+, lower panel). Multiple reaction monitoring (MRM) run was simultaneously performed using four transitions (precursor ion/product ion) of each angiotensin. Ordinate of mass chromatograms indicates a total ion current (TIC) as the summation of four product ions. 2- to 12-month-old Erp44−/− mice and their littermates were used for the analysis.
(E) Absolute concentration of angiotensin II–IV in FVB/NJ × C57BL/6 Erp44+/+, Erp44+/−, and Erp44−/− mouse plasma. The n for each genotype is indicated on the corresponding bar of the graph. Mean ± SD. ∗p < 0.05.
(F) Ratio between angiotensin III+IV and II in Erp44+/+, Erp44+/−, and Erp44−/− mouse plasma. Mean ± SD. ∗∗∗p <
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

4. Figure 2 Reduced Angiotensin II Stability in Erp44−/− Mouse Plasma
(A) Representative HPLC chromatograms of angiotensin II digestion by mouse plasma.
(B) Quantification of peak amplitudes of angiotensin II and byproducts, III, IV, and YIHPF peptide, after various incubation times. Eight-week-old FVB/NJ × C57BL6 Erp44−/− and their littermate control mouse plasma were used. Mean ± SD (n = 5).
(C) LAP, GOT, albumin, UA, BUN, and glucose levels in serum of 8-week-old adult female FVB/NJ × C57BL6 mice. The n for each genotype is indicated on the corresponding bar of the graph. Mean ± SD. ∗∗∗p <
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

5. Figure 3 Redox-Dependent Interaction of ERp44 with ERAP1
(A) Identification of ERAP1 as an ERp44-interacting protein. The immunoprecipitates with anti-ERp44 antibody from liver lysates of C57BL/6 mice were separated by SDS-PAGE and stained with silver staining. The asterisk marks ERAP1 band.
(B) ERp44 interacts with ERAP1 in HeLa cells in the presence of NEM (10 mM).
(C) DTT treatment decreased ERp44-ERAP1 interaction. Cells overexpressing ERp44 and ERAP1 were treated with 10 mM DTT for 10 min, and the ERAP1 bound to ERp44 was detected.
(D) Quantification of the band intensity of the immunoprecipitated ERAP1 with ERp44 in (B). Mean ± SD n = 5. ∗∗∗p <
(E) Quantification of the band intensity of immunoprecipitated ERAP1 with ERp44 in (C). Mean ± SD n = 4. ∗p < 0.05.
(F) ERp44-ERAP1 complex detection on SDS-PAGE under non-reducing conditions. HeLa cells were transfected with HA-ERp44 and ERAP1-flag, and the cell lysates and immunoprecipitates by anti-ERp44 antibody were separated by SDS-PAGE under reducing and non-reducing conditions, and probed with indicated antibodies. The bands marked by an asterisk are the antibody used for immunoprecipitation. Note that the clear band indicated by an arrow on the left panel that migrates at roughly the same size around the upper-asterisked band is ERAP1. Experiments were performed three times, and the representative data are shown.
In (B), (C), and (F), 1%–2% of lysates used for immunoprecipitation were loaded on the gel. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

6. Figure 4 ERp44-Mediated ERAP1 Retention within the ER in Living Cells
(A) Interaction of ERAP1 with ERp44 mutants in HeLa cells.
(B) Quantification of ERAP1 band intensity in (A). The n is indicated on the corresponding bar of the graph. Mean ± SD. ∗∗p < 0.01.
(C) Schematic illustration of ERp44 deletion mutants (left panel). a, b, and b′: thioredoxin-like domain. Interaction of ERp44 deletion mutants with ERAP1 in HeLa cells (right panel). Note that the b domain is unstable by itself and is difficult to express.
(D) Alignment of ERAP1 and P-LAP from various species.
(E) ERAP1 C487A, but not C475A, greatly decreased binding with ERp44.
(F) The quantification of the ERAP1 band intensity in 4E. The n is indicated on the corresponding bar of the graph. Mean ± SD. ∗∗p < 0.01.
(G) Immunostaining of HeLa cells overexpressing both ERAP1 and RFP-ERp44. Scale bar, 20 μm. The cells were stained with rabbit anti-ERAP1 (green) and rat anti-ERp44 (red) antibodies.
(H) Endogenous ERAP1 in RFP-ERp44-transfected (arrows) and nontransfected (asterisks) cells. Cells were stained with rabbit anti-ERAP1 (green) and rat anti-ERp44 (red) antibodies. Upper and lower pictures are from the same field of view but with different exposure times. Scale bar, 20 μm.
(I) LAP activity in the culture medium of HeLa cells overexpressing wild-type (WT) and various mutant forms of ERp44 and ERAP1. Mean ± SD; n = 3.
(J) LAP activity of the culture medium of HeLa cells overexpressing WT and mutant forms of ERAP1 and ERp44. ERAP1 C487 is critical for ERp44-mediated ERAP1 retention within the ER. Mean ± SD; n = 3.
In (A), (C), and (E), 1%–2% of lysates used for immunoprecipitation were loaded on the gel. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

7. Figure 5 Elevated ERAP1 Level in Erp44−/− Mouse Plasma
(A) ERAP1 levels in total cell lysate and the P2/3 membrane fraction of Erp44+/+ and Erp44−/− MEF cells. Lower panel shows the quantification of band intensity of ERAP1 in Erp44+/+ and Erp44−/− fibroblast cell lysates. Mean ± SD; n = 3. ∗∗∗p < We loaded 75 μg of protein for each lane.
(B) Detection of ERAP1 in the culture medium of Erp44+/+ and Erp44−/− MEF cells. Lower panel shows the quantification of ERAP1 band intensity. Mean ± SD. ∗∗p < 0.01.
(C) LAP activities in the culture medium of Erp44+/+ and Erp44−/− MEF cells. Mean ± SD n = 5.
(D) Overexpression of mRFP-ERp44, but not mRFP, in Erp44−/− MEF cells decreased the LAP activity of the culture medium to the level of that of Erp44+/+ MEF cells. Mean ± SD; n = 6–8.
(E) Depletion of ERAP1 from the culture medium of Erp44−/− MEF cells decreased the LAP activity to the level of that of Erp44+/+ MEF cells. Mean ± SD; n = 6.
(F) The expression level of ERAP1 mRNA in Erp44+/+and Erp44−/− MEF cells. The ERAP1 mRNA level was normalized by GAPDH mRNA. Mean ± SD; n = 6.
(G) ERAP1 protein levels in 8-week-old FVB/NJ × C57BL/6 Erp44+/+(WT), Erp44+/− (Het), and Erp44−/− (KO) mouse plasma. 0.25 μl of plasma was blotted with anti-ERAP1 antibody. Lower panel showed the membrane stained with amido black.
(H) Quantification of ERAP1 level in the mouse plasma. The ERAP1 band intensity was normalized by the immunoglobulin heavy chain. Mean ± SD. ∗∗∗p < The n for each genotype is indicated on the corresponding bar of the graph.
(I) Deglycosylation of ERAP1 in Erp44+/+ MEF cell lysates and in Erp44−/− mouse plasma. Degly, deglycosidase treatment. Experiments were performed three times and the representative data are shown.
(J) Pulse chase assay of ERAP1 in the cell lysate (upper panel) and culture medium (lower panel) of Erp44+/+ and Erp44−/− MEF cells. ERAP1 was pulse labeled with 35S for 30 min and immunoprecipitated with anti-ERAP1 antibody from the cell lysates or culture medium at the indicated time. Arrows indicates the positions of ERAP1 that were differently modified with glycosylation. Experiments were performed three times, and the representative data are shown.
(K) Depletion of ERAP1 from Erp44−/− mouse plasma decreased LAP activity to the level of Erp44+/+ mouse plasma. Mean ± SD; n = 9–10.
(L) Angiotensin II degradation assay with ERAP1-depleted Erp44−/− mouse plasma. Plasma from 8-week-old FVB/NJ × C57BL/6 Erp44−/− mice and their littermates was used for analysis. The experiments were performed three times, and the representative data are shown.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

8. Figure 6
Increased ERp44-ERAP1 Association upon Systemic Inflammation Regulates Blood Pressure
(A) Blood pressure of C57BL/6 Erp44+/+ and Erp44+/− mice during sepsis. Mean ± SEM. ∗p < 0.05; n = 7.
(B) Drop in blood pressure in Erp44+/+ and Erp44+/− mice. Mean ± SEM. ∗p < 0.05; n = 7.
(C) Expression of various proteins in the liver of C57BL/6 WT mice 48 hr post-IFN-γ + LPS injection. Each lane corresponds to an individual mouse sample. We loaded 75 μg of protein from the S1 fraction for each lane.
(D) Quantification of band intensity of ERAP1, ERp44, BIP, and PDI in the liver after 48 hr of IFN-γ + LPS or control PBS injection. Mean ± SD n = 4. ∗p < 0.05, ∗∗∗p < The values are normalized to the average values of control and IFN-γ + LPS-treated samples.
(E) ERp44-ERAP1 association is increased in IFN-γ + LPS-injected mouse liver. Each lane corresponds to an individual 8-week-old C57BL/6 wild-type mouse. Lower panel shows the quantification of band intensity of ERAP1 co-immunoprecipitated with ERp44. Mean ± SD; n = 4. ∗p < The values are normalized to the ERAP1 band intensity of control sample.
(F) ERAP1-ERp44 complex in C57BL/6 wild-type mouse liver lysates on SDS-PAGE under non-reducing and reducing conditions. The membrane was blotted with anti-ERAP1 (upper panel) or anti-ERp44 antibody (lower panel). Right panels show the quantification of band intensity of the ERp44-ERAP1 complex detected with anti-ERAP1 (upper panel) or ERp44 (lower panel) antibody. Mean ± SD; n = 4. ∗p < The values are normalized to the ERAP1 or ERp44 band intensity of the control sample.
(G) LAP and GOT activities in C57BL/6 Erp44+/+ and Erp44+/− mice upon sepsis. Mean ± SEM; n = 6. ∗p < We used C57BL/6 genetic background Erp44+/+ and ERp44+/− mice in Figure 6, because pure-background mice are suitable to observe an effect of small changes in ERp44 expression on blood pressure upon sepsis.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions