Paul F. van Swieten

Skin-Depigmenting Agent Monobenzone Induces Potent T-Cell Autoimmunity toward Pigmented Cells by Tyrosinase Haptenation and Melanosome Autophagy

In this study, we report the previously unknown mechanism of inducing robust anti-melanoma immunity by the vitiligo-inducing compound monobenzone. We show monobenzone to increase melanocyte and melanoma cell immunogenicity by forming quinone-haptens to the tyrosinase protein and by inducing the release of tyrosinase- and melanoma antigen recognized by T cells-1 (MART-1)-containing CD63+ exosomes following melanosome oxidative stress induction. Monobenzone further augments the processing and shedding of melanocyte-differentiation antigens by inducing melanosome autophagy and enhanced tyrosinase ubiquitination, ultimately activating dendritic cells, which induced cytotoxic human melanoma-reactive T cells. These T cells effectively eradicate melanoma in vivo, as we have reported previously. Monobenzone thereby represents a promising and readily applicable compound for immunotherapy in melanoma patients.

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

Bioorthogonal organic chemistry in living cells: novel strategies for labeling biomolecules

The chemical labeling of biomolecules continues to be an important tool for the study of their function and cellular fate. Attention is increasingly focused on labeling of biomolecules in living cells, since cell lysis introduces many artefacts. In addition, with the advances in biocompatible synthetic organic chemistry, a whole new field of opportunity has opened up, affording high diversity in the nature of the label as well as a choice of ligation reactions. In recent years, several different two-step labeling strategies have emerged. These rely on the introduction of a bioorthogonal attachment site into a biomolecule, then ligation of a reporter molecule to this site using bioorthogonal organic chemistry. This Perspective focuses on these techniques, their implications and future directions.

Endocytosis targets exogenous material selectively to cathepsin S in live human dendritic cells, while cell‐penetrating peptides mediate nonselective transport to cysteine cathepsins

The way the MHC II‐associated proteolytic system of APC handles exogenous antigen is key to the stimulation of the T cell in infections and immunotherapy settings. Using a cell‐impermeable, activity‐based probe (ABP) for papain cathepsins, the most abundant type of endocytic proteases, we have simulated the encounter between exogenous antigen and endocytic proteases in live human monocyte‐derived dendritic cells (MO‐DC). Although cathepsin S (CatS), ‐B, ‐H, and ‐X were active in DC‐derived endocytic fractions in vitro, the peptide‐size tracer was routed selectively to active CatS after internalization by macropinocytosis. Blocking of the vacuolar adenosine triphosphatase abolished this CatS‐selective targeting, and LPS‐induced maturation of DC resulted in degradation of active CatS. Conjugation of the ABP to a protein facilitated the delivery to endocytic proteases and resulted in labeling of sizable amounts of CatB and CatX, although CatS still remained the major protease reached by this construct. Conjugation of the probe to a cell‐penetrating peptide (CPP) routed the tracer to the entire panel of intracellular cathepsins, independently from endocytosis or LPS stimulation. Thus, different means of internalization result in differential targeting of active cathepsins in live MO‐DC. CPP may serve as vehicles to target antigen more efficiently to protease‐containing endocytic compartments.

N-Azidoacetylmannosamine-mediated chemical tagging of gangliosides

Peracetylated N-α-azidoacetylmannosamine (Ac4ManNAz) is metabolized by cells to CMP-azidosialic acid. It has been demonstrated previously that in this way azidosialic acid-containing glycoproteins are formed that can be labeled on the cell surface by a modified Staudinger ligation. Here, we first demonstrate that the same procedure also results in the formation of azidosialic acid-containing gangliosides. Deoxymannojirimycin, an inhibitor of N-glycan processing in proteins, decreases the total cell surface labeling in Jurkat cells by ∼25%. Inhibition of ganglioside biosynthesis with N-[5-(adamantan-1-yl-methoxy)-pentyl]1-deoxynojirimycin reduces cell surface labeling by ∼75%. In conclusion, exposure of cells to Ac4ManNAz allows in vivo chemical tagging of gangliosides.

Chemical Proteomics Profiling of Proteasome Activity

Proteolysis is a key mechanism for protein homeostasis in living cells. This process is effected by different classes of proteases. The proteasome is one of the most abundant and versatile proteases, bearing three different proteolytic active sites. The proteasome plays an important role in essential biological pathways such as antigen presentation, signal transduction, and cell-cycle control feedback loops. The aim of this work is to design novel chemical strategies for capturing, detection, identification, and quantification--in one word, profiling--the active protease fractions of interest, in cells of different phenotypes. Here, a set of chemistry-based functional proteomics techniques is demonstrated by profiling the multi-catalytic protease activities of the proteasome. Importantly, functional profiling is complementary to expression level profiling and is an indispensable parameter for better understanding of mechanisms underlying biological processes.