Generic Micropatterned Pull-Down Assays for Quantifying Interaction Dynamics and Stoichiometry of Protein-Protein Interactions
Creators: name: Wedeking, Tim
Publisher: name: Universität Osnabrück
Year: 2024
Subjects: subject: Pull-Down
subject: Protein-Protein Interaction
subject: Interaction Dynamics
subject: Stoichiometry
subject: Quantitative Analysis
subject: 570 - Biowissenschaften, Biologie

description: Many cellular functions are governed by an extensive network of protein-protein interactions (PPI) that exhibit regulatory mechanisms through core features such as affinity, avidity and competition. While fulfillment of essential functions such as cell growth, programmed cell death and immune response require a precisely tuned level of activity, dysregulation of such a tightly controlled network leads to serios consequences such as cancerous cell growth, pathogen susceptibility, or autoimmune diseases. Fundamental biological research at the protein level is therefore of vital interest, enabling breakthroughs in medicine while promoting the development of ever more advanced methods to help characterize the many unresolved PPIs. One of the key factors of cellular signaling is the family of cytokines that regulate hematopoiesis and immunological homeostasis. Serving as the model system of this work, the type I interferon (IFN) signaling pathway plays a prominent role in the response against viral and intracellular bacterial infections. Type I IFN signaling employs a heterodimeric class II cytokine receptor (IFNAR1, IFNAR2), as well as cytosolic associated Janus family tyrosine kinases (JAK) and signal transducers and activators of transcription (STAT) as effector proteins. In general, the JAK-STAT pathway is the hallmark of the class I and II cytokine receptor families. Unique to type I IFN, however, are the negative feedback regulators USP18 and ISG15, which serve to regulate the long-term cellular response by desensitizing IFN signaling. While many insights have been gained in recent years at the receptor and JAK level, better mechanistic understanding of the interplay of cytosolic effector proteins and negative feedback regulators is required. This critically depends on quantifying dynamics and stoichiometry of their molecular interactions. On the one hand, bioanalytical characterization was traditionally tackled by biochemical methods and pull-down assays, but such in vitro techniques investigate PPIs far from their biological context and quantitative approaches are very demanding in terms of protein production and purification. In vivo techniques, on the other hand, are still challenging as it is not possible to change or control the protein concentrations in living cells. Moreover, many of these methods require target-specific optimization and leave much to be desired in terms of their reliability and simplicity of their overall strategy. Overcoming these fundamental challenges, this thesis aimed to develop micropatterned nanobody-based pull-down assays to quantify the interaction dynamics and stoichiometry of cytosolic PPIs. These methods were used to characterize the PPIs of the type I IFN signaling pathway, but we also aspired to develop generically applicable strategies that can be used for a broad range of target proteins. Spatially-resolved nanobody immobilization was achieved by employing a robust poly-L-lysine grafted poly(ethylene glycol) (PLL-PEG) surface architecture, which can be readily generated via microcontact printing and provide surface passivation, biocompatibility and a functional micropatterning in a single step. The pull-down assays employ an anti-GFP nanobody directed against the widely used green fluorescent protein (GFP), providing a generalized targeting strategy instead of targeting a specific protein of interest. To this end, we have developed two methods: The first is nanobody live cell micropatterning, which enables spatiotemporal reorganization of cytosolic GFP-fused proteins inside cells to quantitatively investigate their interaction partners and dynamics. The second is in situ single cell pull-down (SiCPull), which directly captures GFP-fused proteins and interacting proteins from whole-cell lysates on the micropatterned chip, enabling background-free quantitative interaction dynamics analysis, and even stoichiometric analysis of individual protein complexes on the single molecule level (SM-SiCPull). Using the nanobody live cell micropatterning, we could demonstrate that ISG15 acts as a double-edged sword. On the one side, ISG15 canonically rescues USP18 from proteolytic degradation, but on the other side interferes with USP18 binding to STAT2, thus partially obstructing USP18’s recruitment to IFNAR2 via STAT2. Furthermore, investigation on the protein domains revealed steric hindrance, as truncation of the N-terminal ISG15 domain abolished the competition. Moreover, SiCPull demonstrated a robust analysis of interaction dynamics of unstimulated STATs, ranging from stable interactions with a lifetime of half an hour, to transient interactions of only mere seconds. Using SM-SiCPull, we could analyze the stoichiometry of individual protein complexes, revealing that both unstimulated and IFN-induced phosphorylated STAT1 featured complex stoichiometries ranging from monomers and dimers to even tetramers. The here presented methods have the potential for broad application in protein interaction analysis, as they are very robust and well compatible with modern cell biology, requiring only relatively simple equipment for microcontact printing, while employing nanobodies for a generalized pull-down strategy.