The spatial landscape of cancer hallmarks reveals patterns of tumor ecological dynamics and drug sensitivity.

Tumors are complex ecosystems of interacting cell types. The concept of cancer hallmarks distills this complexity into underlying principles that govern tumor growth. Here, we explore the spatial distribution of cancer hallmarks across 63 primary untreated tumors from 10 cancer types using spatial transcriptomics.

Endocytosis of Ubiquitylation-Deficient EGFR Mutants via Clathrin-Coated Pits is Mediated by Ubiquitylation.

Signaling by epidermal growth factor receptor (EGFR) is controlled by endocytosis. However, mechanisms of EGFR endocytosis remain poorly understood. Here, we found that the EGFR mutant lacking known ubiquitylation, acetylation and clathrin adaptor AP-2-binding sites (21KRΔAP2) was internalized at relatively high rates via the clathrin-dependent pathway in human duodenal adenocarcinoma HuTu-80 cells.

Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis.

Activation of epidermal growth factor (EGF) receptor (EGFR) leads to its interaction with Grb2, a dual-function adapter mediating both signaling through Ras and receptor endocytosis. We used time-lapse three-dimensional imaging by spinning disk confocal microscopy to analyze trafficking of EGFR and Grb2 in living HeLa cells stimulated with low, physiological concentrations of EGFR ligands. Endogenous Grb2 was replaced in these cells by Grb2 fused to yellow fluorescent protein (YFP).

Lysine 63-linked polyubiquitination is required for EGF receptor degradation.

Ubiquitination mediates endocytosis and endosomal sorting of various signaling receptors, transporters, and channels. However, the relative importance of mono- versus polyubiquitination and the role of specific types of polyubiquitin linkages in endocytic trafficking remain controversial. We used mass spectrometry-based targeted proteomics to show that activated epidermal growth factor receptor (EGFR) is ubiquitinated by one to two short (two to three ubiquitins) polyubiquitin chains mainly linked via lysine 63 (K63) or conjugated with a single monoubiquitin.

Structural, thermodynamic, and mechanistical studies in uroporphyrinogen III synthase: molecular basis of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a rare autosomal disease ultimately related to deleterious mutations in uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the biosynthetic route of the heme group. UROIIIS catalyzes the cyclization of the linear tetrapyrrol hydroxymethylbilane (HMB), inverting the configuration in one of the aromatic rings. In the absence of the enzyme (or when ill-functioning), HMB spontaneously degrades to the by-product uroporphyrinogen I, which cannot lead to the heme group and accumulates in the body, producing some of the symptoms observed in CEP patients.

Intracellular rescue of the uroporphyrinogen III synthase activity in enzymes carrying the hotspot mutation C73R.

A single mutation (C73R) in the enzyme uroporphyrinogen III synthase (UROIIIS) is responsible for more than one-third of all of the reported cases of the rare autosomal disease congenital erythropoietic porphyria (CEP). CEP patients carrying this hotspot mutation develop a severe phenotype of the disease, including reduced life expectancy. Here, we have investigated the molecular basis for the functional deficit in the mutant enzyme both in vitro and in cellular systems.

Uroporphyrinogen III synthase mutations related to congenital erythropoietic porphyria identify a key helix for protein stability.

In the present study we have investigated deleterious mutants in the uroporphyrinogen III synthase (UROIIIS) that are related to the congenital erythropoietic porphyria (CEP). The 25 missense mutants found in CEP patients have been cloned, expressed, and purified. Their enzymatic activities have been measured relative to wild-type UROIIIS activity.