Robust Colonic Epithelial Regeneration and Amelioration of Colitis via FZD-Specific Activation of Wnt Signaling.

Background And Aims: Current management of inflammatory bowel disease leaves a clear unmet need to treat the severe epithelial damage. Modulation of Wnt signaling might present an opportunity to achieve histological remission and mucosal healing when treating IBD. Exogenous R-spondin, which amplifies Wnt signals by maintaining cell surface expression of Frizzled (Fzd) and low-density lipoprotein receptor-related protein receptors, not only helps repair intestine epithelial damage, but also induces hyperplasia of normal epithelium.

Differential toxicity to murine small and large intestinal epithelium induced by oncology drugs.

Gastrointestinal toxicity is a major concern in the development of drugs. Here, we establish the ability to use murine small and large intestine-derived monolayers to screen drugs for toxicity. As a proof-of-concept, we applied this system to assess gastrointestinal toxicity of ~50 clinically used oncology drugs, encompassing diverse mechanisms of action.

Transit-Amplifying Cells Coordinate Changes in Intestinal Epithelial Cell-Type Composition.

Renewing tissues have the remarkable ability to continually produce both proliferative progenitor and specialized differentiated cell types. How are complex milieus of microenvironmental signals interpreted to coordinate tissue-cell-type composition? Here, we investigate the responses of intestinal epithelium to individual and paired perturbations across eight epithelial signaling pathways. Using a high-throughput approach that combines enteroid monolayers and quantitative imaging, we identified conditions that enrich for specific cell types as well as interactions between pathways.

Generation and Quantitative Imaging of Enteroid Monolayers.

The intestinal epithelium is a single layer of cells that plays a critical role in digestion, absorbs nutrients from food, and coordinates the delicate interplay between microbes in the gut lumen and the immune system. Epithelial homeostasis is crucial for maintaining health; disruption of homeostasis results in disorders including inflammatory bowel disease and cancer. The advent of 3D intestinal epithelial organoids has greatly advanced our understanding of the molecular underpinnings of epithelial homeostasis and disease.

Catalytic linkage between caspase activity and proteostasis in Archaea.

The model haloarchaeon, Haloferax volcanii possess an extremely high, and highly specific, basal caspase activity in exponentially growing cells that closely resembles caspase-4. This activity is specifically inhibited by the pan-caspase inhibitor, z-VAD-FMK, and has no cross-reactivity with other known protease families. Although it is one of the dominant cellular proteolytic activities in exponentially growing H.

TGF-ß Regulates Cathepsin Activation during Normal and Pathogenic Development.

Cysteine cathepsins play roles during development and disease beyond their function in lysosomal protein turnover. Here, we leverage a fluorescent activity-based probe (ABP), BMV109, to track cysteine cathepsins in normal and diseased zebrafish embryos. Using this probe in a model of mucolipidosis II, we show that loss of carbohydrate-dependent lysosomal sorting alters the activity of several cathepsin proteases.

Enteroid Monolayers Reveal an Autonomous WNT and BMP Circuit Controlling Intestinal Epithelial Growth and Organization.

The intestinal epithelium maintains a remarkable balance between proliferation and differentiation despite rapid cellular turnover. A central challenge is to elucidate mechanisms required for robust control of tissue renewal. Opposing WNT and BMP signaling is essential in establishing epithelial homeostasis.

Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation.

Reversible detyrosination of α-tubulin is crucial to microtubule dynamics and functions, and defects have been implicated in cancer, brain disorganization, and cardiomyopathies. The identity of the tubulin tyrosine carboxypeptidase (TCP) responsible for detyrosination has remained unclear. We used chemical proteomics with a potent irreversible inhibitor to show that the major brain TCP is a complex of vasohibin-1 (VASH1) with the small vasohibin binding protein (SVBP).

Bifunctional Probes of Cathepsin Protease Activity and pH Reveal Alterations in Endolysosomal pH during Bacterial Infection.

Cysteine cathepsins are lysosomal proteases involved in regulation of both normal cellular processes and disease. Biochemical studies with peptide substrates indicate that cathepsins have optimal activity at acidic pH and highly attenuated activity at neutral pH. In contrast, there is mounting evidence that cathepsins have biological roles in environments that have non-acidic pH.

Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death.

When innate immune cells such as macrophages are challenged with environmental stresses or infection by pathogens, they trigger the rapid assembly of multi-protein complexes called inflammasomes that are responsible for initiating pro-inflammatory responses and a form of cell death termed pyroptosis. We describe here the identification of an intracellular trigger of NLRP3-mediated inflammatory signaling, IL-1β production and pyroptosis in primed murine bone marrow-derived macrophages that is mediated by the disruption of glycolytic flux. This signal results from a drop of NADH levels and induction of mitochondrial ROS production and can be rescued by addition of products that restore NADH production.

Non-invasive Imaging of Idiopathic Pulmonary Fibrosis Using Cathepsin Protease Probes.

Idiopathic pulmonary fibrosis (IPF) is a lethal, chronic, progressive disease characterized by formation of scar tissue within the lungs. Because it is a disease of unknown etiology, it is difficult to diagnose, to predict disease course and to devise treatment strategies. Recent evidence suggests that activated macrophages play key roles in the pathology of IPF.

Probes to monitor activity of the paracaspase MALT1.

The human paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) plays a central role in nuclear factor-κB (NF-κB) signaling as both a protease and scaffolding protein. Knocking out MALT1 leads to impaired NF-κB signaling and failure to mount an effective immune response. However, it is unclear to which degree it is the scaffolding function versus the proteolytic activity of MALT1 that is essential.

A biocompatible "split luciferin" reaction and its application for non-invasive bioluminescent imaging of protease activity in living animals.

The great complexity of many human pathologies, such as cancer, diabetes, and neurodegenerative diseases, requires new tools for studies of biological processes on the whole organism level. The discovery of novel biocompatible reactions has tremendously advanced our understanding of basic biology; however, no efficient tools exist for real-time non-invasive imaging of many human proteases that play very important roles in multiple human disorders. We recently reported that the "split luciferin" biocompatible reaction represents a valuable tool for evaluation of protease activity directly in living animals using bioluminescence imaging (BLI).

Activity-based profiling of proteases.

Proteolytic enzymes are key signaling molecules in both normal physiological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA and protein often are not good indicators of total protease activity.

Improved quenched fluorescent probe for imaging of cysteine cathepsin activity.

The cysteine cathepsins are a family of proteases that play important roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is upregulated and can be exploited for tumor imaging. Here we present the design and synthesis of a new class of quenched fluorescent activity-based probes (qABPs) containing a phenoxymethyl ketone (PMK) electrophile.

A biocompatible in vivo ligation reaction and its application for noninvasive bioluminescent imaging of protease activity in living mice.

The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase.

Kinetic analysis of the bypass of a bulky DNA lesion catalyzed by human Y-family DNA polymerases.

1-Nitropyrene (1-NP), a mutagen and potential carcinogen, is the most abundant nitro polyaromatic hydrocarbon in diesel exhaust, which reacts with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)). If not repaired, this DNA lesion is presumably bypassed in vivo by any of human Y-family DNA polymerases kappa (hPolκ), iota (hPolι), eta (hPolη), and Rev1 (hRev1). Our running start assays demonstrated that each of these enzymes was indeed capable of traversing a site-specifically placed dG(AP) on a synthetic DNA template but that hRev1 was stopped after lesion bypass.

Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis.

The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals.