The role of post-translational modifications and protein turnover during neutrophil differentiation and maturation

Thesis

Neutrophils are frontline effectors of the innate immune system, rapidly mobilised in response to both inflammation and microbial invasion. Their development from hematopoietic progenitors is a tightly regulated process involving lineage commitment, functional maturation, and priming for rapid immune responses. Although key transcriptional and signalling pathways involved in neutrophil differentiation have been described, the contribution of post-translational modifications (PTMs) – critical modulators of protein activity, stability, and interactions – remains largely unexplored to date.

This work presents the first comprehensive, unbiased characterisation of global proteomic and PTM dynamics during neutrophil differentiation, using state-of-the-art mass spectrometry-based proteomics. By applying parallel enrichment strategies for ubiquitinated (di-Gly-modified) and phosphorylated peptides, over ~6,000 proteins, ~30,000 ubiquitination sites, and ~14,000 phosphorylation events across defined stages of neutrophil development were quantified. Moreover, potential regulators of ubiquitination changes during neutrophil differentiation were interrogated using CRISPR-Cas9 technology to target the deubiquitinases (DUBs) in a phenotypic screen.

These datasets reveal stage-specific patterns of PTM regulation, implicating a coordinated remodelling of signalling networks and effector functions during differentiation. Integrative bioinformatic analyses identified potential candidate master regulators, including previously unrecognised E3 ligases, and deubiquitinases (DUBs), whose activity or modification state strongly correlates with distinct differentiation phases. The targeted CRISPR-Cas9 phenotypic screen identified candidate DUBs that may participate in the regulation of neutrophil differentiation. These included YOD1, BAP1, COPS6, VCPIP1, USP7, USP47, OTUB2, and USP44. Several of these hits are currently under experimental validation to assess their mechanistic role in neutrophil lineage specification and function. Moreover, increasing levels of NLRP3 inflammasome components were detected during neutrophil differentiation, suggesting that NOD-like receptor signalling pathways may contribute to neutrophil development.

These findings provide a high-resolution dynamic molecular PTM landscape of neutrophil differentiation, highlighting the underappreciated role of protein phosphorylation and ubiquitination in shaping innate immune cell fate. This work opens new avenues towards better understanding neutrophil biology and developing strategies to therapeutically modulate immune responses in inflammatory and infectious diseases.

Authors: Akbor, MA

• DOI: 10.5287/ora-exax7ddyd
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: mass spectrometry; phosphoproteomics; bioinformatics; CRISPR screen; differentiation; transcriptomics; epigenomics; integrative omics; HoxB8; ubiquitomics; proteomics; protein post-translational modifications; HL60; neutrophils
• Subjects: Life sciences; Proteomics; Post-translational modification; Neutrophils--Immunology