PROTEASOME'S PROTEASES ARE ALTERED DURING PRIMING, INFLAMMATION AND TOLERANCE BY LPS
Authors: Qureshi Nilofer , Silswal Neerupma 
Published: November 2023

Introduction

The role of cellular proteasomes in LPS‐induced inflammation is not fully‐understood. This study is important for developing new strategies for therapeutic intervention of diseases based on inflammation. Proteasomes are cytoplasmic organelles present in all cells and degrade regulatory proteins, thus affecting >90% genes modulated by LPS. The proteasome has numerous functions, besides being involved in antigen presentation as shown by us and others. There are three active proteases within proteasome; X, chymotrypsin‐like (CT‐like, cleaves after non‐polar residues), Y, post‐acidic (PA cleaves after acidic residues), and Z, trypsin‐like (T‐like, cleaves after basic residues). The CT‐like activity greatly affects transcription/degradation of signaling proteins, cytokines, induction of nitric oxide (NO), cell differentiation factors, and expression of several hundreds of genes are modulated in response to LPS.

Objectives

To evaluate the role of proteasome's proteases in inflammation and sepsis. The molecular basis underlying an inflammatory response to LPS and correlation of proteasome's proteases is not well‐defined. We hypothesized that protease subunits present in proteasomes of cells direct the inflammatory response due to LPS/agonist treatment. The LPS‐induced inflammatory response is regulated by proteases of proteasome at gene‐transcription level and degradation of crucial regulatory proteins.

Methods

To test our hypothesis we determined proteasome's protease subunits that are present in several select control human and septic monocytes, murine cell lines, murine macrophage, B‐cells, and T‐cells. The proteasome's subunits present in monocytes/macrophages were analyzed by immunoblotting, qRT‐PCR, confocal microscopy; and these data were correlated with function. Degradation of ubiquitinated proteins following LPS treatment was also studied in macrophages.

Results

Mouse RAW 264.7 macrophages, THP1 cells, CD14+ human monocytes, Jurkat T cells, and B cells differed in their proteasome subunits. We found that RAW 264.7 cells (contain XYZ subunits), mouse peritoneal resident, thioglycollate‐elicited macrophages (XYZ and LMP subunits) and human monocytes, B cells and Jurkat T cells (LMP subunits) expressed different types of proteasome subunits/activities and had different functions. Cells containing predominantly either LMP subunits (such as THP‐1 and human monocytes), or only X, Y, Z subunits (RAW 264.7 cells, unprimed; XYZ and these cells could be primed to proteasomes containing LMP subunits), showed some functions, while cells containing all 6 subunits of proteasome's proteases induced several mediators in response to LPS, and were very active in inducing cytokines and nitric oxide (NO). Priming of cells with LPS caused a change in subunits of proteasomes (XYZ to LMP), while distinct states of inflammation and tolerance (refractoriness to LPS) strongly correlated with an upregulation or downregulation of proteasome's subunits (proteases), respectively, in vitro experiments. The proteasome subunits are ubiquitinated and degraded after treatment and robust downregulation of these proteases causes cell death.

Conclusions

Proteasome's proteases play a vital role during priming, inflammation, tolerance, and death in mouse and human cells. These studies will lead to novel therapeutic strategies for all diseases based on inflammation.

Support or Funding Information

This study was supported in part by grants R01‐GM 50870 (NQ), R01‐GM102631 (NQ), and R01‐GM102631S1 (NQ) from National Institutes of Health, NIGMS.