Erythropoietin signaling regulates heme biosynthesis.

Heme is required for survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for many cellular processes, how it is coupled to cellular differentiation is unknown. Here, using zebrafish, murine, and human models, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, AKAP10, regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane. This integrated pathway culminates with the direct phosphorylation of the crucial heme biosynthetic enzyme, ferrochelatase (FECH) by protein kinase A (PKA). Biochemical, pharmacological, and genetic inhibition of this signaling pathway result in a block in hemoglobin production and concomitant intracellular accumulation of protoporphyrin intermediates. Broadly, our results implicate aberrant PKA signaling in the pathogenesis of hematologic diseases. We propose a unifying model in which the erythroid transcriptional program works in concert with post-translational mechanisms to regulate heme metabolism during normal development.

Covalent modification of the NF-κB essential modulator (NEMO) by a chemical compound can regulate its ubiquitin binding properties in vitro.

Post-translational modification by ubiquitin plays important roles in multiple physiological and pathological processes. Ubiquitin-binding proteins play a critical role in recognizing and relaying polyubiquitin-based signaling. NEMO (NF-κB Essential Modulator) is a central player in canonical NF-κB signaling whose major function is to bind to Lys-63- and/or M1- (or linear) linked polyubiquitin chains generated in response to cell stimulation. Here we show that Withaferin A (WA), a steroidal lactone, causes a change in NEMO's interaction with specific types of polyubiquitin chains in vitro. WA induces full-length recombinant NEMO to bind to long Lys-48-linked polyubiquitin chains but not tetra-ubiquitin species. Significantly, the UBAN (ubiquitin binding in ABIN and NEMO) domain, essential for the ability of NEMO to bind M1/Lys-63-linked polyubiquitin, is dispensable for the WA-induced gain-of-function activity. Mass spectrometric analysis demonstrated that WA covalently modifies NEMO on a cysteine residue within the C-terminal zinc finger (ZF) domain. Point mutations to the ZF can reverse the WA-induced Lys-48-polyubiquitin binding phenotype. Our study demonstrates the feasibility of directly altering the ubiquitin interaction properties of an ubiquitin-binding protein by a chemical compound, thereby shedding light on a novel drug class to potentially alter polyubiquitin-based cellular processes.

The one hour yeast proteome.

We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS(2) acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS(1) and 80,460 MS(2) scans (per run) to produce 43,400 (x) peptide spectral matches and 34,255 (x) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.