Journal: bioRxiv

Article Title: Development of a New N-Terminomic Method to Study the Pathodegradome of the Staphylococcus aureus V8 Protease in Human Neutrophils

doi: 10.1101/2024.11.08.622692

Figure Lengend Snippet: Analysis of the amino acid sequence surrounding the site of proteolysis provided immediate validation of V8 induced cleavage of our 346 neutrophil targets by showcasing the known precision of this enzyme to cleave primarily at the carboxyl side of glutamic acid (E) residues. Demonstrating this, ∼90% of residues upstream of the cleavage site accounted for glutamic acid (E) residues ( A ). We performed an additional dimension of analysis using ICELOGO whereby we looked at the percentage difference of significantly increased and decreased residues at the five positions (P5-P5’) surrounding the sessile bond. Here we found that only glutamic acid was significantly increased at P1 or upstream of the cleavage site ( B ).

Article Snippet: Neutrophil proteomes were standardized to 1mg/ml and incubated ±200ng of V8 protease (MilliporeSigma), at 37°C for 16h.

Techniques: Sequencing, Biomarker Discovery

Journal: bioRxiv

Article Title: Development of a New N-Terminomic Method to Study the Pathodegradome of the Staphylococcus aureus V8 Protease in Human Neutrophils

doi: 10.1101/2024.11.08.622692

Figure Lengend Snippet: Green molecules are highlighted as key neutrophil proteins cleaved by V8. Displayed adjacent to these are the corresponding neutrophil processes they play a role in. These include: ( A ) Leukocyte Extravasation, ( B ) Integrin Interactions, ( C ) Neutrophil Degranulation, ( D ) ROS Production, ( E ) Apoptosis, and ( F )Phagocytosis.

Article Snippet: Neutrophil proteomes were standardized to 1mg/ml and incubated ±200ng of V8 protease (MilliporeSigma), at 37°C for 16h.

Techniques:

Journal: bioRxiv

Article Title: Development of a New N-Terminomic Method to Study the Pathodegradome of the Staphylococcus aureus V8 Protease in Human Neutrophils

doi: 10.1101/2024.11.08.622692

Figure Lengend Snippet: Neutrophil proteomes were exposed to 200ng of the V8 protease for 16h at 37°C whilst control conditions remained untreated. Degradation patterns and their corresponding approximate molecular weights (calculated using Bioinformatics.org ) according to the cleavage events captured by our N-terminomic data are outlined in the relevant protein schematic. Also highlighted in the schematics are the identified sites of V8 cleavage. Western blot analysis of neutrophil V8 targets include: ( A ) Integrin alpha-L, ( B ) Kindlin, ( C ) Myeloperoxidase, and ( D ) Pyruvate kinase PKM2. VWFA = von Willebrand A. FERM = Ferm domain. PH = PH domain.

Article Snippet: Neutrophil proteomes were standardized to 1mg/ml and incubated ±200ng of V8 protease (MilliporeSigma), at 37°C for 16h.

Techniques: Control, Western Blot

Journal: bioRxiv

Article Title: Development of a New N-Terminomic Method to Study the Pathodegradome of the Staphylococcus aureus V8 Protease in Human Neutrophils

doi: 10.1101/2024.11.08.622692

Figure Lengend Snippet: Recombinant proteins of ( A ) Human beta actin and ( B ) BAK1 were treated with varying concentrations of the V8 protease and compared to untreated controls. Purified V8 was also included as a control. A schematic of the corresponding protein is presented adjacent to the Coomassie Blue stained gel. Green coloring indicates the corresponding portion of the protein that was purchased. Cleavage sites detected by TAGS-CR are highlighted with the asterisk and the surrounding residues, as well as the resulting degradation products and their corresponding molecular weights. The blue arrow indicates bands that were excised for in-gel digest for ( B ) BAK1.

Article Snippet: Neutrophil proteomes were standardized to 1mg/ml and incubated ±200ng of V8 protease (MilliporeSigma), at 37°C for 16h.

Techniques: Recombinant, Purification, Control, Staining

Journal: bioRxiv

Article Title: Development of a New N-Terminomic Method to Study the Pathodegradome of the Staphylococcus aureus V8 Protease in Human Neutrophils

doi: 10.1101/2024.11.08.622692

Figure Lengend Snippet: HL-60 derived human neutrophils were infected with either the S. aureus LAC wild type or the sspA (V8 encoding gene) mutant strain using an MOI of 13.5. CFU/mL was then determined after a 24-hour period and data displayed represents the average of 3 biological replicates for each strain. The significance of relative bacterial burden between strains was determined using an unpaired t-test with unequal variance, **p=<0.01. Error bars are ± SEM.

Article Snippet: Neutrophil proteomes were standardized to 1mg/ml and incubated ±200ng of V8 protease (MilliporeSigma), at 37°C for 16h.

Techniques: Derivative Assay, Infection, Mutagenesis