monitoring-neutrophil-elastase-cathepsin-g-activity-human-sputum

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical ...

German Center for Lung Research (DZL)

University of Heidelberg

Oregon Health and Science University

We present the latest advances in lipid tool development for studying cellular membrane trafficking and metabolism. We focus on chemical modifications that are introduced to natural lipid structures. The new functionalities are used to follow and interfere with lipid dynamics in intact cells.

Novel lipid tools and probes for biological investigations.

Following stimulation, pancreatic β-cells must orchestrate a plethora of signalling events to ensure the appropriate release of insulin and maintenance of normal glucose homeostasis. Failure at any point in this cascade leads to impaired insulin secretion, elevated blood levels of glucose and eventually type 2 diabetes mellitus. Likewise, β-cell replacement or regeneration strategies for the treatment of both type 1 and type 2 diabetes mellitus might fail if the correct cell signalling phenotype cannot be faithfully recreated. However, current understanding of β-cell function is complicated because of the highly dynamic nature of their intracellular and intercellular signalling as well as insulin release itself. β-Cells must precisely integrate multiple signals stemming from multiple cues, often with differing intensities, frequencies and cellular and subcellular localizations, before converging these signals onto insulin exocytosis. In this respect, optical approaches with high resolution in space and time are extremely useful for properly deciphering the complexity of β-cell signalling. An increased understanding of β-cell signalling might identify new mechanisms underlying insulin release, with relevance for future drug therapy and de novo stem cell engineering of functional islets.

Optical tools for understanding the complexity of β-cell signalling and insulin release.

Among many cellular functions, inositol pyrophosphates (PP-InsPs) are metabolic messengers involved in the regulation of glucose uptake, insulin sensitivity, and weight gain. However, their mechanisms of action are still poorly understood. So far, the influence of PP-InsPs on cellular metabolism has been studied by overexpression or knockout/inhibition of relevant metabolizing kinases (IP6Ks, PPIP5Ks). These approaches are, , limited by time-resolution and potential compensation mechanisms. Here, we describe the synthesis of cell-permeant caged PP-InsPs as tools to rapidly modulate intracellular levels of defined isomers of PP-InsPs in a genetically non-perturbed cellular environment. We show that caged prometabolites readily enter live cells where they are enzymatically converted into still inactive, metabolically stable, photocaged PP-InsPs. Upon light-triggered release of 5-PP-InsP, the major cellular inositol pyrophosphate, oscillations of intracellular Ca levels in MIN6 cells were transiently reduced to spontaneously recover again. In contrast, uncaging of 1-PP-InsP, a minor cellular isomer, was without effect. These results provide evidence that PP-InsPs play an active role in regulating [Ca] oscillations, a key element in triggering exocytosis and secretion in β-cells.

Photolysis of cell-permeant caged inositol pyrophosphates controls oscillations of cytosolic calcium in a β-cell line.

The specific non-invasive control of intracellular signaling events requires advanced tools that enter cells by diffusion and are controllable by light. Here, we detail the synthesis and application of membrane-permeant caged inositol pyrophosphates with respect to cell entry and cell distribution. We recently published the synthesis of these tools as well as their effect on PH-domain localization in HeLa cells and oscillations of the intracellular calcium concentration in β-cells, which are known to drive insulin secretion. In this chapter, we discuss the possibilities and limitations when using cell-penetrating inositol pyrophosphates. We provide a detailed protocol for the application in live mouse β-cells and we discuss the image analysis needed for following effects on calcium signaling.

Photo-releasable derivatives of inositol pyrophosphates.

The islets of Langerhans represent one of the many complex endocrine organs in mammals. Traditionally, islet function is studied by a mixture of physiological, cell biological, and molecular biological methods. Recently, novel techniques stemming from the ever-increasing toolbox provided by chemical laboratories have been added to the repertoire. Many emerging techniques will soon be available to manipulate and monitor islet function at the single-cell level and potentially in intact model animals, as well as in isolated human islets. Here, we review the most current small-molecule-based and genetically encoded molecular tool sets available to study islet function. We provide an outlook regarding future tool developments that will impact islet research, with a special focus on the interplay between different islet cell types.

Chemical Biology Toolbox for Studying Pancreatic Islet Function - A Perspective.

Pharmacological treatment of pancreatic β cells targeting cannabinoid receptors 1 and 2 (CB1 and CB2) has been shown to result in significant effects on insulin release, possibly by modulating intracellular calcium levels ([Ca]). It is unclear how the interplay of CB1 and CB2 affects insulin secretion. Here, we demonstrate by the use of highly specific receptor antagonists and the recently developed photo-releasable endocannabinoid 2-arachidonoylglycerol that both receptors have counteracting effects on cytosolic calcium oscillations. We further show that both receptors are juxtaposed in a way that increases [Ca] oscillations in silent β cells but dampens them in active ones. This study highlights a functional role of CB1 and CB2 acting in concert as a compensator/attenuator switch for regulating β cell excitability.

Article	Year
Novel lipid tools and probes for biological investigations. Current opinion in cell biology\| PubMed ID: 30015291	2018
Optical tools for understanding the complexity of β-cell signalling and insulin release. Nature reviews. Endocrinology\| PubMed ID: 30356209	2018
Photolysis of cell-permeant caged inositol pyrophosphates controls oscillations of cytosolic calcium in a β-cell line. Chemical science\| PubMed ID: 30996985	2019
Photo-releasable derivatives of inositol pyrophosphates. Methods in enzymology\| PubMed ID: 32713537	2020
Chemical Biology Toolbox for Studying Pancreatic Islet Function - A Perspective. Cell chemical biology\| PubMed ID: 32822616	2020
Regulation of Calcium Oscillations in β-Cells by Co-activated Cannabinoid Receptors. Cell chemical biology\| PubMed ID: 33147441	2021

Carsten Schultz

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