A Clickable Aminooxy Probe for Monitoring Cellular ADP-Ribosylation

ADP-ribosylation is essential for cell function, yet there is a dearth of methods for detecting this post-translational modification in cells. Here, we describe a clickable aminooxy alkyne (AO-alkyne) probe that can detect cellular ADP-ribosylation on acidic amino acids following Cu-catalyzed conjugation to an azide-containing reporter. Using AO-alkyne, we show that PARP10 and PARP11 are auto-ADP-ribosylated in cells. We also demonstrate that AO-alkyne can be used to monitor stimulus-induced ADP-ribosylation in cells. Functional studies using AO-alkyne support a previously unknown mechanism for ADP-ribosylation on acidic amino acids, wherein a glutamate or aspartate at the initial C1′-position of ADP-ribose transfers to the C2′ position. This new mechanism for ADP-ribosylation has important implications for how glutamyl/aspartyl-ADP-ribose is recognized by proteins in cells

Engineering the Substrate Specificity of ADP-Ribosyltransferases for Identifying Direct Protein Targets

Adenosine diphosphate ribosyltransferases (ARTDs; ARTD1–17 in humans) are emerging as critical regulators of cell function in both normal physiology and disease. These enzymes transfer the ADP-ribose moiety from its substrate, nicotinamide adenine dinucleotide (NAD⁺), to amino acids of target proteins. The functional redundancy and overlapping target specificities among the 17 ARTDs in humans make the identification of direct targets of individual ARTD family members in a cellular context a formidable challenge. Here we describe the rational design of orthogonal NAD⁺ analogue-engineered ARTD pairs for the identification of direct protein targets of individual ARTDs. Guided by initial inhibitor studies with nicotinamide analogues containing substituents at the C-5 position, we synthesized an orthogonal NAD⁺ variant and found that it is used as a substrate for several engineered ARTDs (ARTD1, -2, and -6) but not their wild-type counterparts. Comparing the target profiles of ARTD1 (PARP1) and ARTD2 (PARP2) in nuclear extracts highlighted the semi-complementary, yet distinct, protein targeting. Using affinity purification followed by tandem mass spectrometry, we identified 42 direct ARTD1 targets and 301 direct ARTD2 targets. This represents a powerful new technique for identifying direct protein targets of individual ARTD family members, which will facilitate studies delineating the pathway from ARTD activation to a given cellular response

Cell-specific Profiling of Nascent Proteomes Using Orthogonal Enzyme-mediated Puromycin Incorporation

Translation regulation is a fundamental component of gene expression, allowing cells to respond rapidly to a variety of stimuli in the absence of new transcription. The lack of methods for profiling nascent proteomes in distinct cell populations in heterogeneous tissues has precluded an understanding of translational regulation in physiologically relevant contexts. Here, we describe a chemical genetic method that involves orthogonal enzyme-mediated incorporation of a clickable puromycin analogue into nascent polypeptides. Using this method, we show that we can label newly synthesized proteins in a cell-specific manner in cells grown in culture and in heterogeneous tissues. We also show that we can identify the nascent proteome in genetically targeted cell populations using affinity enrichment and tandem mass spectrometry. Our method has the potential to provide unprecedented insights into cell-specific translational regulation in heterogeneous tissues

Role of histone kinases in Tat transactivation.

<p>(A) Chromatin immunoprecipitation analysis of Jurkat T cells containing an integrated HIV promoter in the absence or presence of Tat. Immunoprecipitations were performed with α-phospho-histone H3 antibodies (serine 10) followed by radioactive PCR with primers specific for the HIV LTR, the c-fos, or the β-globin genes. (B) Jurkat 1G5 cells containing an integrated HIV LTR luciferase construct were transiently transfected with Tat/FLAG (25 ng) and kinase-deficient (KD) kinase expression vectors (200 ng). (C) Western blot analysis of cellular lysates from 293 cells cotransfected with the indicated expression plasmids. (D) Transfection of CMV luciferase (25 ng) with the KD RSK2 expression plasmid (200 ng) in Jurkat cells. (E) Transfection of 5xUAS luciferase and Gal4-CDK9 (20 ng) with the KD RSK2 expression plasmid (200 ng) in Jurkat cells. Values are means±SEM of three experiments.</p

Superinduction of Tat activity in CLS fibroblasts.

<p>(A) Western blot analysis of cellular extracts of fibroblasts from a patient with CLS and control human fibroblasts. (B) Nuclear microinjection of CLS fibroblasts with synthetic Tat (amino acids 1–72), the HIV LTR luciferase reporter, a CMV-GFP expression plasmid, and either the empty vector, an RSK2 expression construct, or a plasmid expressing kinase-deficient RSK2. Values are means±SEM of five experiments. (C) Coinjection of the 5xUAS luciferase reporter, a plasmid expressing the Gal4-VP16 transactivator and CMV-GFP with either the RSK2-expressing plasmid or the vector alone. Values are means±SEM of three experiments.</p

Activation of RSK2 by Tat.

<p>(A) Autoradiography of radioactive <i>in vitro</i> synthesized RSK2 proteins before (Input) and after binding to biotinylated synthetic Tat (amino acids 1–72) or to beads alone. Increasing amounts of <i>in vitro</i> translated RSK2 were included in the binding reaction. (B) Kinase assay of endogenous RSK2 immunoprecipitated from Cos7 cells transfected with wild type Tat/FLAG, TatF38A/FLAG, or empty vector. Values are means±SEM of four experiments. (C) Western blotting of nuclear extracts isolated from Cos7 cells cotransfected with RSK2/HA and Tat/FLAG or with RSK2/HA and Tat F38A/FLAG constructs. Densitometric quantification of the phospho-S227-specific bands was performed using the NIH Image software. (D) Chromatin immunoprecipitation analysis of the Jurkat T cell line A2, latently infected with an HIV-based lentiviral vector expressing Tat/FLAG from the HIV LTR after treatment with TNF-α. At indicated time points, cells were harvested and immunoprecipitations were performed in duplicate with α-phospho-S227 antibodies followed by PCR with primers specific for the HIV LTR or the c-fos gene.</p