OC 04.3 - Prospective FVIII intracellular trafficking routes involving GABARAP

Saturday, June 24, 2023

13:30 – 13:45 ET

Room: 520

Session Available As: On-Demand

Presenting Author(s)

Salime El Hazzouri

PhD candidate
University Clinic Bonn, Institute of experimental Haematology and Transfusion medicine
Bonn, Nordrhein-Westfalen, Germany

Co-Author(s)

NN

Nicole Nüsgen

Medizinisch-technische Assistentin
Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
bonn, Nordrhein-Westfalen, Germany
Rawya Al Rifai

PhD candidat
Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
Bonn, Nordrhein-Westfalen, Germany
HS

Heike Singer

Dr. rer. nat
University Clinic Bonn, Institue of experimental Haematology and Transfusion medicine
Bonn, Nordrhein-Westfalen, Germany
Nasim Shahidi Hamedani, PharmD PhD (she/her/hers)

Scientific Staff Member
University Hospital Bonn; Institute of Experimental Haematologie and Transfusion Medicine
Bonn, Nordrhein-Westfalen, Germany
Johannes Oldenburg, MD (he/him/his)

Professor, Chairman and Director
University Clinic Bonn AöR, Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany
Bonn, Nordrhein-Westfalen, Germany
Osman EL-Maarri, Priv Doz, PhD (he/him/his)

Priv.-Doz. Dr.
Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
bonn, Nordrhein-Westfalen, Germany

Background: We previously revealed the effect of GABARAP protein on trafficking and secretion of FVIII. Our previous results hinted to the existence of alternative routes for FVIII along with the known conventional secretory pathway. Downstream studies on the generated CRISPR/Cas9-KOs: Calnexin, Calreticulin, LMAN1, MCFD2, GABARAP, GABARAPL1 and Atg7 in FVIII-expressing HEK293 cells (HEK28), showed that both the autophagy machinery and the endosomal system appear to be potential alternative routes for FVIII cellular processing.

Aims: To determine more precisely the mode of involvement and the intracellular location of GABARAP-FVIII interaction

Methods: In this study, we used three experimental approaches. First: immunofluorescent staining and co-localization analysis to determine the degree of correlation of GABARAP to 25 intracellular marker. Second: BLItz assay to determine the binding affinity between candidate interacting proteins (GABARAP, Full length and B-domain deleted FVIII, and Calreticulin). Third: chemical treatment with Chloroquine, Glucose-deficient medium, BrefeldinA and Rab7 inhibitor, to affect specific cellular pathways and subcellular compartments in both wild type HEK28 cells and GABARAP knockout.

Results: GABARAP was observed to co-localize with proteins of classical secretory pathway (LMAN1 PCC=0.17, COPI/ COPII PCC=0.15, and Cis-Golgi PCC=0.16), higher with endolysosomal system (Rab7 PCC=0.16, VAMP8 PCC=0.41) and to an even higher extent with ubiquitin/proteasome (PCC=0.34/0.45). Additionally, absence of GABARAP showed decreased FVIII co-localization with VAMP8 (pvalue < 0.05). Based on BLItz assay, presence of Calreticulin appeared to enhance/stabilize GABARAP-FVIII complex (KD = 445.2 µM to KD = 276.2 µM). When autophagy was inhibited; FVIII decreased in the media of GABARAP-KO (40% residual) and disappeared completely in LMAN1-KO media. A non-unsimilar effect of glucose-starvation was seen, where FVIII secretion decreased in GABARAP-KO (50-70% residual) and slightly changed in LMAN1-KO with residual FVIII activity.

Conclusion(s): It appears that GABARAP influences the stability of FVIII’s cellular interactions in different cellular compartments, in favor of the homeostasis/equilibrium of FVIII intracellular life.

Learning Objectives:

FVIII basic research involving verification of known and investigation of novel biosynthesis, trafficking , and secretion pathways as well as interacting partners and new compartments involved in the cellular machinery of the FVIII protein.
Investigation of the specific relation between the FVIII protein and GABARAP, an Atg8 (autophagy) family member, that also represents a versatile role including functions in conventional and unconventional protein secretion and trafficking pathways.
Presenting tools of basic cellular and molecular research of FVIII such as Immunofluorescent staining, binding/interaction assays and data analysis ….etc in order to better generate perspectives on future steps for deeper and further investigations on FVIII.