Kidney Week

Abstract: SA-PO098

Nuclear-Localized Complement Factor B (CFB) Is a DNA-Binding Enzyme Regulating Histone Degradation

Session Information

• AKI: Inflammation and Cell Cycle
  October 26, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

• 103 AKI: Mechanisms

Authors

• Tajmul, Md, Immunoregulation Section, Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, United States

Md Tajmul, PhD

• Freiwald, Tilo, III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Tilo Freiwald, MD, DrMed

• Trujillo-Ochoa, Jorge Luis, Immunoregulation Section, Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, United States

Jorge Luis Trujillo-Ochoa, PhD

• Chauss, Daniel, Immunoregulation Section, Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, United States

Daniel Chauss, PhD

• Kumar, Dhaneshwar, Immunoregulation Section, Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, United States

Dhaneshwar Kumar, PhD

• Karim, Baktiar O., Leidos Inc, Reston, Virginia, United States

Baktiar O. Karim, PA

• Gunkel, Maja, III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Maja Gunkel

• Guo, Yubing, III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Yubing Guo, PhD

• Jäger, Julius F.E., III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Julius F.E. Jäger

• Kemper, Claudia, Complement and Inflammation Research Section, National Heart Lung and Blood Institute, NIH, Bethesda, Maryland, United States

Claudia Kemper, PhD

• Huber, Tobias B., III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Tobias B. Huber, MD, FASN

• Wiech, Thorsten, Institute of Pathology, Section for Renal Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Thorsten Wiech, DrMed

• Kolev, Martin, Apellis Pharmaceuticals Inc, Waltham, Massachusetts, United States

Martin Kolev, PhD

• Briggs, Scott D., Department of Biochemistry, Purdue University, West Lafayette, Indiana, United States

Scott D. Briggs, PhD

• Kazemian, Majid, Department of Biochemistry, Purdue University, West Lafayette, Indiana, United States

Majid Kazemian

• Portilla, Didier, Division of Nephrology and Center for Immunity, Inflammation, and Regenerative Medicine, University of Virginia, Charlottesville, Virginia, United States

Didier Portilla, MD

• Afzali, Behdad, Immunoregulation Section, Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, United States

Behdad Afzali, MD, PhD

Background

The complement (C’) system is essential for innate immunity and pivotal to lytic pathogen destruction, recognition of danger signals, and orchestration of inflammatory responses. Although traditionally viewed as an extracellular, serum-based defense mechanism, recent insights have revealed critically important intracellular functions of C’ components, suggesting roles beyond classical immune responses. Despite extensive studies on circulating factor B (CFB), its intracellular functions remain under-explored. We have observed inducible CFB expression in injured kidney epithelial cells, leading us to hypothesize that intracellular CFB also regulates cellular processes.

Methods

We studied mouse proximal tubular epithelial cells using primary cells, TKPTS cell lines, confocal imaging, a small molecule CFB inhibitor, CUT&Tag, electrophoretic mobility shift assay (EMSA), computational predictions with AlphaFold2 and GraphSite and protein assays with wild-type and mutated CFB.

Results

Confocal microscopy revealed significant nuclear accumulation of CFB in kidney epithelial cells. To explore potential DNA-binding by CFB, we employed CUT&Tag, identifying a consensus motif enriched at CFB binding sites within the genome. EMSA confirmed the binding of CFB to this motif, with a Kd of 0.71uM, indicating a meaningful interaction. In silico modeling suggested a DNA-binding pocket with 11 DNA-contacting residues, supported in vitro with CFB mutants showing reduced DNA binding. Considering the serine protease activity of CFB, we proposed that CFB targets and degrades DNA-associated proteins, such as histones, within its vicinity. In vitro experiments, including those with a CFB inhibitor, confirmed this hypothesis, showing the ability of CFB to degrade nucleosomal histones. Ongoing experiments aim to map specific histone cleavage sites to guide future functional studies

Conclusion

These findings expand understanding of intracellular C’ functions and highlight a potential role for CFB in modulating transcriptional outcomes, with potential implications for therapeutic strategies targeting CFB in the intracellular or extracellular space in patients.

Funding

• NIDDK Support – Apellis Pharmaceuticals