Journal: Biochemical Journal

Article Title: Cyclophilin D reduces Ca 2+ sequestration by complement 1q binding protein

doi: 10.1042/BCJ20253361

Figure Lengend Snippet: (A) Sequence of the CypD, with mutations R55K and R82K highlighted in red, used in this study (upper row) aligned with the full-length protein UniProt P30405 (lower row) ( (B) Sequence of C1qBP used in this study (upper row) aligned with the full-length protein UniProt Q07021 (lower row). The numbering system for the truncated proteins used in the text and NMR spectra are shown

Article Snippet: Detection was performed using a rabbit anti-C1qBP antibody (Proteintech, U.S.A., Cat. No. 24474–1-AP).

Techniques: Sequencing

Journal: Biochemical Journal

Article Title: Cyclophilin D reduces Ca 2+ sequestration by complement 1q binding protein

doi: 10.1042/BCJ20253361

Figure Lengend Snippet: ( A ) Coomassie-stained SDS–PAGE demonstrating the interaction between CypD and His-C1qBP in a pull-down assay. Lanes contain 10 µl of loaded eluate from each reaction condition. ( B ) Coomassie-stained SDS–PAGE of isolated and equimolar mixture of CypD and C1qBP over a 144-hour period at 37°C. ( C-D ) ImageJ densitometry measurements quantifying the relative percentage of protein abundance of both ( C ) C1qBP and ( D ) CypD in isolation and in combination (‘*’ denotes P <0.05 when compared with the control group, ‘a’ denotes P <0.05 when compared with C1qBP in mix). ( E-G ) Molecular mass analysis by SEC-MALS showing the fitted traces of ( E ) C1qBP at approximately 86 KDa, ( F ) CypD at approximately 16 KDa and ( G ) CypD:C1qBP complex at approximately 101 KDa. ( H ) Coomassie-stained SDS–PAGE of CypD, C1qBP and their SEC eluted complex (~10 µg loaded), confirming their presence and interaction. ( I-J ) DLS measurements of C1qBP and CypD in isolation and in complex, showing the ( I ) cumulant radius and ( J ) static light scattering (SLS). Bar charts display mean values ( n =3) with corresponding standard error of the mean (SEM) error bars. Data were analysed using two-way Analysis of Variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. An asterisk (*) indicates a statistically significant difference ( P <0.05) compared with the 0-hour time point, while ‘a’ denotes P <0.05 compared with the mixed treatment within each group.

Article Snippet: Detection was performed using a rabbit anti-C1qBP antibody (Proteintech, U.S.A., Cat. No. 24474–1-AP).

Techniques: Staining, SDS Page, Pull Down Assay, Isolation, Quantitative Proteomics, Control

Journal: Biochemical Journal

Article Title: Cyclophilin D reduces Ca 2+ sequestration by complement 1q binding protein

doi: 10.1042/BCJ20253361

Figure Lengend Snippet: Two-dimensional 15 N- 1 H HSQC spectrum, 20 mM Na₂HPO₄, 20 mM NaCl, pH 6.5 298K of ( A ) 15 N-uniformly labelled CypD alone, ( B ) 15 N-uniformly labelled CypD in the presence of unlabelled C1qBP (CypD:trimeric C1qBP 4:1 molar ratio). ( C ) Spectrum of 15 N-CypD:C1qBP complex following the addition of CsA (CypD:CsA 1:1 molar ratio). Assignments of these spectra were obtained in-house using standard triple resonance 1 H, 13 C, 15 N experiments and compared with deposited assignments in the Biological Magnetic Resonance Databank.

Article Snippet: Detection was performed using a rabbit anti-C1qBP antibody (Proteintech, U.S.A., Cat. No. 24474–1-AP).

Techniques:

Journal: Biochemical Journal

Article Title: Cyclophilin D reduces Ca 2+ sequestration by complement 1q binding protein

doi: 10.1042/BCJ20253361

Figure Lengend Snippet: ( A ) Histogram showing total integrals of observable resonances of 15 N-uniformly labelled CypD under the different sample conditions. Repeats were performed for the 15 NCypD:C1qBP complex spectra, with the data showing similar intensities each time. ( B, C ) Two-dimensional 15 N- 1 H HSQC spectrum, 20 mM Na₂HPO₄, 20 mM NaCl, pH 6.5 298K of ( B ) 15 N-uniformly labelled CypD R55K, ( C ) 15 N-uniformly labelled CypD R82K in the presence of unlabelled C1qBP (pH 6.5) (CypD:trimeric C1qBP 4:1 molar ratio). Assignments of these spectra were obtained in-house using standard triple resonance 1 H, 13 C, 15 N experiments and compared with deposited assignments in the Biological Magnetic Resonance Databank.

Article Snippet: Detection was performed using a rabbit anti-C1qBP antibody (Proteintech, U.S.A., Cat. No. 24474–1-AP).

Techniques:

Journal: Biochemical Journal

Article Title: Cyclophilin D reduces Ca 2+ sequestration by complement 1q binding protein

doi: 10.1042/BCJ20253361

Figure Lengend Snippet: ( A ) Adaptive Poisson–Boltzmann Solver (APBS) electrostatic surface analysis of trimeric C1qBP (PDB: 1P32) and CypD (PDB: 2BIT). Electrostatic potential mapping reveals distinct positive (blue) and negative (red) surface regions for both proteins. The colour bar is shown at the bottom of the panel. ( B ) AlphaFold-predicted interaction between CypD (green) and C1qBP (wheat), with the electrostatic surface overlaid over the cartoon representation. The colour bar is shown at the bottom of the panel. ( C, D ) Two-dimensional 15 N- 1 H HSQC spectra, 20 mM Na₂HPO₄, 20 mM NaCl, 298K of uniformly 15 N-labelled CypD in the presence of unlabelled C1qBP at pH 7.4 ( C ) and pH 8.0 ( D ).

Article Snippet: Detection was performed using a rabbit anti-C1qBP antibody (Proteintech, U.S.A., Cat. No. 24474–1-AP).

Techniques:

Journal: Biochemical Journal

Article Title: Cyclophilin D reduces Ca 2+ sequestration by complement 1q binding protein

doi: 10.1042/BCJ20253361

Figure Lengend Snippet: ( A ). Molecular surface of AlphaFold model of CypD in complex with C1qBP. Residues that show significant attenuation of NMR 15 N- 1 H HSQC signal intensities at pH 8.0 are coloured in yellow, with a transparent cartoon representation of C1qBP (coloured in wheat) in the foreground. ( B ) 180° rotation of A to show the reverse surface of CypD, with C1qBP in the background, highlighting little chemical shift perturbations in the complex 15 N- 1 H HSQC spectrum. ( C ) 90° rotation of A to show the interactions involving C1qBP acidic loop N61–F95 from one of the subunits of the trimer. The structures were created using the program PyMOL (Schrödinger, LLC).

Article Snippet: Detection was performed using a rabbit anti-C1qBP antibody (Proteintech, U.S.A., Cat. No. 24474–1-AP).

Techniques:

Journal: Biochemical Journal

Article Title: Cyclophilin D reduces Ca 2+ sequestration by complement 1q binding protein

doi: 10.1042/BCJ20253361

Figure Lengend Snippet: ( A ) NanoDifferential Scanning Fluorimetry (NanoDSF) thermal unfolding profiles of C1qBP (0.8 mg/ml; 30 µM) without and with Ca 2+ (2.5 mM) showing a 3°C increase in melting temperature (Tm), indicating Ca 2+ -dependent stabilization ( P <0.001). The accompanying bar chart quantifies this shift (bar charts represent mean values ( n =4) with corresponding SEM error bars. Welch’s two-sample t-test was used to compare group means). ( B ) Isothermal titration calorimetry (ITC) of C1qBP binding to Ca 2+ at pH 7.4, revealing biphasic binding kinetics with two distinct stoichiometries. ( C ) ITC overlay comparing Ca 2+ binding to C1qBP alone, in the presence of CypD and with a negative control (PDZ1), demonstrating reduction in the binding capacity of C1qBP for Ca 2+ in the presence of CypD. Thermodynamics values are representative of mean ± SEM, ( n ≥ 2).

Article Snippet: Detection was performed using a rabbit anti-C1qBP antibody (Proteintech, U.S.A., Cat. No. 24474–1-AP).

Techniques: Nano Differential Scanning Fluorimetry, Isothermal Titration Calorimetry, Binding Assay, Negative Control

Journal: Biochemical Journal

Article Title: Cyclophilin D reduces Ca 2+ sequestration by complement 1q binding protein

doi: 10.1042/BCJ20253361

Figure Lengend Snippet: (A) Static light scattering (SLS) analysis of C1qBP and the CypD:C1qBP complex in the absence and presence of calcium. Bar graphs represent mean values ( n =3) with error bars indicating the standard error of the mean (SEM). (B) 1D 1 H NMR spectra of CypD (upper) in the absence (green) and presence (red) of 15 mM Ca 2+ , and of C1qBP (with substoichiometric amounts of CypD) (lower) in the absence (black) and presence (red) of 15 mM Ca 2+ . The intensities of the peaks are normalised to proton resonances from buffer additives. (C) AlphaFold-predicted model of hexameric C1qBP showing sequestration of calcium ions within the inter-trimeric space. The two trimeric C1qBP units are depicted in wheat and light blue, respectively, while Ca 2+ is depicted in red.

Article Snippet: Detection was performed using a rabbit anti-C1qBP antibody (Proteintech, U.S.A., Cat. No. 24474–1-AP).

Techniques:

Journal: Biochemical Journal

Article Title: Cyclophilin D reduces Ca 2+ sequestration by complement 1q binding protein

doi: 10.1042/BCJ20253361

Figure Lengend Snippet: ( A ) In the presence of CypD, the ability of C1qBP to sequester Ca 2+ is impaired, leading to excessive mitochondrial matrix calcium accumulation, which subsequently triggers mPTP activation. ( B ) CsA binding to CypD dissociates the CypD:C1qBP complex, restoring the capacity of C1qBP to sequester and store Ca 2+ , thereby lowering matrix Ca 2+ levels and preventing mPTP activation, which would otherwise require excessive Ca 2+ following C1qBP saturation for reactivation. This model highlights the dynamic regulation of mPTP by CypD–C1qBP interactions and Ca 2+ availability. The figure was created in BioRender.com.

Article Snippet: Detection was performed using a rabbit anti-C1qBP antibody (Proteintech, U.S.A., Cat. No. 24474–1-AP).

Techniques: Activation Assay, Binding Assay

Journal: Military Medical Research

Article Title: Trace element-dictated exosome modules and self-adaptive dual-network hydrogel orchestrate diabetic foot regeneration through complement-mitochondria-autophagy circuitry

doi: 10.1186/s40779-025-00658-4

Figure Lengend Snippet: The 3D-TE-Exo with high activity were constructed by Fe-Mg-Zn-Mn-Se multi-TE synergy and 3D dynamic culture. 3D-TE-Exo mitigates inflammation and promotes angiogenesis through a triple mechanism involving C1QBP-mediated regulation of the complement pathway, restoration of mitochondrial membrane potential, and remodeling of autophagic flux, thereby disrupting the pathological cycle. HA hyaluronic acid, CS chitosan, EDC ethyldimethylaminopropyl carbodiimide, NHS N-hydroxysuccinimide, 3D three-dimensional, 3D-TE-Exo exosome derived from trace element-supplemented medium, MDA malondialdehyde, SOD superoxide dismutase, GSH glutathione, TNF-α tumour necrosis factor-alpha, IL-1β interleukin-1β, IL-6 interleukin-6, IL-10 interleukin-10, SIRT1 silent information regulator 1, p62 sequestosome 1, LC3 microtubule-associated protein light chain 3, ATP adenosine triphosphate, ROS reactive oxygen species, HUVECs human umbilical vein endothelial cells, HaCaT human immortal keratinocyte line, H 2 O 2 hydrogen peroxide, ΔΨm mitochondrial membrane potential, UV ultraviolet, LPS lipopolysaccharide, C1QBP complement 1q binding protein, hUC-MSCs human umbilical cord mesenchymal stem cells, DC dendritic cells

Article Snippet: Silent information regulator 1 (SIRT1; 13161–1-AP) and complement 1q binding protein (C1QBP) monoclonal antibody (1F9B1) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Activity Assay, Construct, Membrane, Derivative Assay, Binding Assay