Roberto Giambruno

JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia.

The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor–mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.

Germline RBBP6 mutations in familial myeloproliferative neoplasms

To the editor: Myeloproliferative neoplasms (MPNs) comprise a heterogeneous group of hematologic disorders characterized by clonal overproduction of differentiated myeloid cells, propensity to thrombosis, hemorrhage, and increased risk of leukemia. Three MPN subtypes, polycythemia vera (PV),

A Modular Synthesis of Modified Phosphoanhydrides

Phosphoanhydrides (P-anhydrides) are ubiquitously occurring modifications in nature. Nucleotides and their conjugates, for example, are among the most important building blocks and signaling molecules in cell biology. To study and manipulate their biological functions, a diverse range of analogues have been developed. Phosphate-modified analogues have been successfully applied to study proteins that depend on these abundant cellular building blocks, but very often both the preparation and purification of these molecules are challenging. This study discloses a general access to P-anhydrides, including different nucleotide probes, that greatly facilitates their preparation and isolation. The convenient and scalable synthesis of, for example, (18) O labeled nucleoside triphosphates holds promise for future applications in phosphoproteomics.

Affinity purification strategies for proteomic analysis of transcription factor complexes.

Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein–protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities.

Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry.

Mass spectrometry-based in vitro kinase screens play an essential role in the discovery of kinase substrates, however, many suffer from biological and technical noise or necessitate genetically-altered enzyme-cofactor systems. We describe a method that combines stable γ-[18O2]-ATP with classical in vitro kinase assays within a contemporary quantitative proteomic workflow. Our approach improved detection of known substrates of the non-receptor tyrosine kinase ABL1; and identified potential, new in vitro substrates.