What is ASC oligomerization?

ASC oligomerization is a direct evidence for inflammasome activation and its detection allows a read-out independent of caspase-1 and IL-1β. This protocol describes how to detect the oligomerization of ASC by Western blot. Keywords: Auto-inflammation; Biochemistry; Caspase-1; Inflammasome; Pyroptosis.

How is inflammasome activation measured?

Inflammasome activation is typically measured by Western Blot or ELISA. While these are standard methods, both are cumbersome and time consuming and may require making lysates or using serum-free supernatant. Other drawbacks include variable caspase-1 antibody quality and the limited dynamic range of ELISAs.

What is speck formation?

Speck formation is rapid causing a concomitant drop in cytosolic ASC concentration, approximately 200-fold, to submicromolar concentrations within 100 s (2), and almost all available NLRP3 and ASC oligomerize within minutes (11). Speck assembly is microtubule-dependent (12–14).

Where is the inflammasome found?

Inflammasomes are expressed primarily by myeloid cells and are located within the cell. The macromolecular inflammasome structure can be visualized by cryo-electron microscopy. This complex has been found to play a role in a variety of disease models in mice and several have been genetically linked to human diseases.

What are ASC specks?

A hallmark of inflammasome activation is the ASC speck, a micrometre-sized structure formed by the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which consists of a pyrin domain (PYD) and a caspase recruitment domain (CARD).

What does the Inflammasome do?

The inflammasomes are innate immune system receptors/sensors that regulate the activation of caspase-1 and induce inflammation in response to infectious microbes and molecules derived from host proteins. It has been implicated in a host of inflammatory disorders.

How is Pyroptosis measured?

Subsequent pyroptotic cell lysis is assessed by measuring release of the cytoplasmic enzyme LDH. This technique can be used with many different caspase-1 activating stimuli and provides a rapid (30 min incubation), population-based quantification of cell lysis.

What is ASC inflammasome?

The inflammasome is a multi-protein complex that recruits pro-caspase-1 via ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) and then proceeds to cleave the cytokine precursors pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18.

What are the 3 main components of an inflammasome?

An inflammasome complex comprises a sensor, an adaptor, and a zymogen procaspase-1. The functional output of inflammasome activation includes secretion of cytokines, IL-1β and IL-18, and induction of an inflammatory form of cell death called pyroptosis.

What is inflammasome composed of?

Inflammasomes are typically composed of a sensor, the adaptor molecule ASC [apoptosis-associated speck-like protein containing a caspase-activation and recruitment domain (CARD); also known as PYCARD] and the cysteine protease procaspase-1.

What does the inflammasome do?

Is an inflammasome a PRR?

The activation of inflammasomes, a group of intracellular multimeric protein complexes that activate inflammatory caspase-1, is a major inflammatory pathway [2]. An inflammasome is defined by its sensor protein (a PRR), which oligomerizes to form a pro-caspase-1 activating platform in response to DAMPs or PAMPs.