Search

A distinct boundary exists between the progenitor cells in the basal limbal epithelium and the more differentiated corneal epithelial basal cells. We have shown that reciprocal expression patterns of EphA2 and Ephrin-A1 are likely to contribute to normal limbal-corneal epithelial compartmentalization as well as play a role in response to injury. How this signaling axis is regulated remains unclear. We have demonstrated that microRNAs (miRNAs) play critical roles in corneal epithelial wound healing and several miRNAs (e.g. miR-210) have been predicted to target Ephrins. Previous expression profiling experiments demonstrated that miR-210 is prominently expressed in corneal epithelial cells. RNA-seq data acquired from miR-210-depleted HCECs showed up-regulation of genes involved in cellular migration. In addition, miR-210 is decreased after corneal injury while EphA2 is increased. Moreover, antago-210-treated HCECs markedly enhanced wound closure in a scratch wound assay. Antago-210 treatment resulted in increased EphA2 protein levels as well as pS897-EphA2, the pro-migratory form of EphA2. As expected, Ephrin-A1 levels were reduced, while levels of a well-known target of miR-210, Ephrin-A3, were increased by antago-210 treatment. The increase in migration with antago-210 could be inhibited by Ephrin-A1 overexpression, Ephrin-A1-Fc treatment or siRNA depletion of EphA2. However, depletion of Ephrin-A3 did not have effects on the antago-210-induced increase in migration. In addition, Ephrin-A1 overexpression and siEphA2 dampened EGFR signaling, which is increased by antago-210. Our data clearly demonstrate a link between miR-210 and EphA2/Ephrin-A1 signaling that regulates, in part, corneal epithelial migration. This interaction might potentially control the limbal-corneal epithelial boundary.

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)-catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne-modified TMM analogue (O-AlkTMM-C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM-based reporters bearing alkyne, azide, trans-cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one- or two-step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell-surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole-cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies.

Oligomerization of aggregation-prone intrinsically disordered proteins (IDPs), such as α-synuclein, amyloid β, and tau, has been shown to be associated with the pathogenesis of several neurodegenerative diseases, including Parkinson's and Alzheimer's disease. The proteasome is charged with regulating cellular levels of IDPs, but this degradation pathway can become dysregulated leading to their accumulation and subsequent aggregation. Although the pathogenesis of these neurodegenerative diseases is still under intense investigation, it has been shown that the oligomeric forms of IDPs, including α-synuclein and amyloid β, can impair proteasome function. This leads to additional accumulation of the IDPs, further promoting disease progression. Herein, we report the use of small molecule activators of the 20S subcomplex of the proteasome to restore impaired 20S proteasome activity and prevent IDP accumulation and oligomerization. We found that fluspirilene and its new synthetic analog (16) show strong 20S proteasome enhancement (doubling 20S proteolytic activity at ∼2 μM, with maximum fold enhancement of ∼1000%), overcome impaired proteasome function, and prevent the accumulation of pathogenic IDPs. These findings provide support for the use of 20S enhancers as a possible therapeutic strategy to combat neurodegenerative diseases.

Aggregates or oligomeric forms of many intrinsically disordered proteins (IDPs), including α-synuclein, are hallmarks of neurodegenerative diseases, like Parkinson's and Alzheimer's disease, and key contributors to their pathogenesis. Due to their disordered nature and therefore lack of defined drug-binding pockets, IDPs are difficult targets for traditional small molecule drug design and are often referred to as "undruggable". The 20S proteasome is the main protease that targets IDPs for degradation and therefore small molecule 20S proteasome enhancement presents a novel therapeutic strategy by which these undruggable IDPs could be targeted. The concept of 20S activation is still relatively new, with few potent activators having been identified thus far. Herein, we synthesized and evaluated a library of dihydroquinazoline analogues and discovered several promising new 20S proteasome activators. Further testing of top hits revealed that they can enhance 20S mediated degradation of α-synuclein, the IDP associated with Parkinson's disease.

The surface functionality of amorphous carbons is difficult to directly measure because of a lack of crystallinity and overwhelming signals derived from the bulk material. Biochar, a form of amorphous carbon containing considerable oxygen surface functionality, was probed using metal ions and X-ray photoelectron spectroscopy to simultaneously measure the presence and proximity of functional groups and determine the preferred binding modes of a variety of metal ions. These binding motifs were correlated to the efficiency of metal adsorption as determined using the Langmuir isotherm and stability with respect to leaching. Three binding motifs were apparent: physisorption (Cd2+, Mn2+, and Zn2+), chelation (Cu2+, Ni2+, and Zn2+), and hydrolysis/precipitation (Cu2+, Ni2+, and Pb2+).

Whereas much is known about the genes regulated by ΔNp63α in keratinocytes, how ΔNp63α is regulated is less clear. During studies with the hydroxylase, factor inhibiting hypoxia-inducible factor 1 (FIH-1), we observed increases in epidermal ΔNp63α expression along with proliferative capacity in a conditional FIH-1 transgenic mouse. Conversely, loss of FIH-1 in vivo and in vitro attenuated ΔNp63α expression. To elucidate the FIH-1/p63 relationship, BioID proteomics assays identified FIH-1 binding partners that had the potential to regulate p63 expression. FIH-1 interacts with two previously unknown partners, Plectin1 and signal transducer and activator of transcription 1 (STAT1) leading to the regulation of ΔNp63α expression. Two known interactors of FIH-1, apoptosis-stimulating of P53 protein 2 (ASPP2) and histone deacetylase 1 (HDAC1), were also identified. Knockdown of ASPP2 upregulated ΔNp63α and reversed the decrease in ΔNp63α by FIH-1 depletion. Additionally, FIH-1 regulates growth arrest and DNA damage-45 alpha (GADD45α), a negative regulator of ΔNp63α by interacting with HDAC1. GADD45α knockdown rescued reduction in ΔNp63α by FIH-1 depletion. Collectively, our data reveal that FIH-1 positively regulates ΔNp63α in keratinocytes via variety of signaling partners: (a) Plectin1/STAT1, (b) ASPP2, and (c) HDAC1/GADD45α signaling pathways.

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.

Trehalosamine (2-amino-2-deoxy-α,α-d-trehalose) is an aminoglycoside with antimicrobial activity against Mycobacterium tuberculosis, and it is also a versatile synthetic intermediate used to access imaging probes for mycobacteria. To overcome inefficient chemical synthesis approaches, we report a two-step chemoenzymatic synthesis of trehalosamine that features trehalose synthase (TreT)-catalyzed glycosylation as the key transformation. Soluble and recyclable immobilized forms of TreT were successfully employed. We demonstrate that chemoenzymatically synthesized trehalosamine can be elaborated to two complementary imaging probes, which label mycobacteria via distinct pathways.

Protein O-mycoloylation is a unique post-translational lipidation that was recently discovered in Corynebacterium. We describe an alkyne-modified trehalose monomycolate chemical reporter that can metabolically tag O-mycoloylated proteins in C. glutamicum, enabling their detection and identification through click chemistry.