Mechanism-Based Post-Translational Modification and Inactivation in Terpene Synthases

Terpenes are ubiquitous natural chemicals with diverse biological functions spanning all three domains of life. In specialized metabolism, the active sites of terpene synthases (TPSs) evolve in shape and reactivity to direct the biosynthesis of a myriad of chemotypes for organismal fitness. As most terpene biosynthesis mechanistically involves highly reactive carbocationic intermediates, the protein surfaces catalyzing these cascade reactions possess reactive regions possibly prone to premature carbocation capture and potentially enzyme inactivation. Here, we show using proteomic and X-ray crystallographic analyses that cationic intermediates undergo capture by conserved active site residues leading to inhibitory self-alkylation. Moreover, the level of cation-mediated inactivation increases with mutation of the active site, upon changes in the size and structure of isoprenoid diphosphate substrates, and alongside increases in reaction temperatures. TPSs that individually synthesize multiple products are less prone to self-alkylation then TPSs possessing relatively high product specificity. In total, the results presented suggest that mechanism-based alkylation represents an overlooked mechanistic pressure during the evolution of cation-derived terpene biosynthesis

Identification of Microprotein–Protein Interactions via APEX Tagging

Microproteins are peptides and small proteins encoded by small open reading frames (smORFs). Newer technologies have led to the recent discovery of hundreds to thousands of new microproteins. The biological functions of a few microproteins have been elucidated, and these microproteins have fundamental roles in biology ranging from limb development to muscle function, highlighting the value of characterizing these molecules. The identification of microprotein–protein interactions (MPIs) has proven to be a successful approach to the functional characterization of these genes; however, traditional immunoprecipitation methods result in the enrichment of nonspecific interactions for microproteins. Here, we test and apply an in situ proximity tagging method that relies on an engineered ascorbate peroxidase 2 (APEX) to elucidate MPIs. The results demonstrate that APEX tagging is superior to traditional immunoprecipitation methods for microproteins. Furthermore, the application of APEX tagging to an uncharacterized microprotein called C11orf98 revealed that this microprotein interacts with nucleolar proteins nucleophosmin and nucleolin, demonstrating the ability of this approach to identify novel hypothesis-generating MPIs

Improved Identification and Analysis of Small Open Reading Frame Encoded Polypeptides

Computational, genomic, and proteomic approaches have been used to discover nonannotated protein-coding small open reading frames (smORFs). Some novel smORFs have crucial biological roles in cells and organisms, which motivates the search for additional smORFs. Proteomic smORF discovery methods are advantageous because they detect smORF-encoded polypeptides (SEPs) to validate smORF translation and SEP stability. Because SEPs are shorter and less abundant than average proteins, SEP detection using proteomics faces unique challenges. Here, we optimize several steps in the SEP discovery workflow to improve SEP isolation and identification. These changes have led to the detection of several new human SEPs (novel human genes), improved confidence in the SEP assignments, and enabled quantification of SEPs under different cellular conditions. These improvements will allow faster detection and characterization of new SEPs and smORFs

SHMT2 and the BRCC36/BRISC deubiquitinase regulate HIV-1 Tat K63-ubiquitylation and destruction by autophagy

<div><p>HIV-1 Tat is a key regulator of viral transcription, however little is known about the mechanisms that control its turnover in T cells. Here we use a novel proteomics technique, called DiffPOP, to identify the molecular target of JIB-04, a small molecule compound that potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines. Mass-spectrometry analysis of whole-cell extracts from 2D10 Jurkat T cells revealed that JIB-04 targets Serine Hydroxymethyltransferase 2 (SHMT2), a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63Ub-specific deubiquitinase in the BRISC complex. Importantly, knockdown of SHMT1,2 or BRCC36, or exposure of cells to JIB-04, strongly increased Tat K63Ub-dependent destruction via autophagy. Moreover, point mutation of multiple lysines in Tat, or knockdown of BRCC36 or SHMT1,2, was sufficient to prevent destruction of Tat by JIB-04. We conclude that HIV-1 Tat levels are regulated through K63Ub-selective autophagy mediated through SHMT1,2 and the BRCC36 deubiquitinase.</p></div

SHMT2 and the BRCC36/BRISC deubiquitinase controls Tat K63Ub and destruction by autophagy.

<p>(<b>A</b>) Immunoblot analysis of Tat and GFP protein levels in 2D10 cells depleted of SHMT2 or Cyclin T1, as indicated. TAF4 served as loading control. (<b>B</b>) Immunoblot analysis of Tat protein expression in 2D10 cells depleted of BRCC36 or Cyclin T1. NF-κB2 protein levels were monitored to assess any change in T cell signaling. TAF4 served as loading control. (<b>C</b>) Immunoblot analysis of Tat expression in Tet-on-Tat-off HeLa cells depleted of SHMT1, SHMT2 or BRCC36, as indicated above each lane. TAF4 served as loading control. (<b>D</b>) Top, immunoblot analysis of the effects of overexpression of Flag-SHMT1 and FH-BRCC36 proteins on Tat protein levels. TAF4 served as loading control. Bottom, Dual:Luc reporter activity of the HIV-Luc reporter. Plasmids expressing the Flag-vector, Flag-SHMT1 and FH-BRCC36 proteins were tested at 0 ng (blue bar), 20 ng (purple bar), 100 ng (yellow bar) and 500 ng (light blue bar), as indicated. (<b>E</b>) Analysis of FLAG-Tat-101 immunoprecipitates from lysates of BRCC36- or SHMT1- knockdown cells. FLAG-Tat-101 proteins were labelled with HA-tagged ubiquitin, either wild-type Ub (WT), or ubiquitin mutants that selectively support only K63 or K48 ubiquitylation, and Tat proteins were monitored using anti-HA antisera. (<b>F</b>) Immunoblot analysis of the effect of JIB-04 on Tat protein levels in 2D10 cells depleted of SHMT2 or BRCC36. (<b>G</b>) The effect of JIB-04 on wildtype or Tat ΔK (+K41) STREP-tagged Tat proteins was shown by immunoblot. TAF4 served as loading control. (<b>H</b>) Model of the role of SHMT2 and BRCC36 in the release of Tat-K63Ub from destruction through chaperone-mediated autophagy or SQSMT1/p62-dependent macroautophagy.</p

JIB-04 has a limited effect on host cell transcription.

<p>(<b>A</b>) Shown is a graph of normalized RNA-seq reads mapped to HIV-1 and eGFP in 2D10 cells. Cells were pre-treated with DMSO or 3 μM JIB-04 for 16 h, and stimulated by TNFα (10 ng/ml) for 0h or 6h, as indicated. (<b>B</b>) RNA-seq heatmap of 100 genes that showed the largest differences in expression in DMSO versus JIB-04-treated 2D10 cells. The effect of JIB-04 on host cell gene expression was monitored both in the absence (0h) or presence (6h) of TNFα. Data were derived from minimum and the maximum rlog values of duplicate RNA-seq experiments in 2D10 cells. Genes are grouped as JIB-04-repressed, JIB-04-activated and TNFα-inducible groups, as indicated on the right. (<b>C</b>) Analysis of mRNA by qRT-PCR for <i>HIV-Env</i>, <i>eGFP</i> and selected TNFα-inducible (top) and non-inducible (bottom) genes in 2D10 cells in DMSO or JIB-04-treated cells. Significant differences between samples treated by DMSO or 3 μM JIB-04 at 6 h TNFα stimulation were calculated by Student’s T-test (*p<0.05, **p<0.005, ***p<0.0005; ns = non-significant) for each gene.</p

JIB-04 inhibits HIV-1 Tat expression in activated 2D10 T cells.

<p>(<b>A</b>) Immunoblot analysis of HIV-1 Tat expression from the integrated HIV-1 provirus in 2D10 T cells induced with TNFα for 0–24 h, as indicated above each lane. Cells were treated with DMSO (lanes 1–6) or 3 μM JIB-04 (lanes 7–12) overnight. Cyclin T1 served as loading control. (<b>B</b>) As in part A, except that 2D10 cells were activated with PHA (10 μg/mL) and PMA (50 ng/mL) for the times indicated above each lane. (<b>C</b>) Immunoblot analysis of HIV-1 Tat protein levels in 2D10 cells treated with different concentrations of JIB-04 (0.1 μM-50 μM) for 24 h, as indicated. Cyclin T1 and TAF4 (TFIID subunit) served as loading controls. (<b>D</b>) As in part C, except that cells were exposed to the histone demethylase inhibitor GSKJ1, at the concentrations listed above each lane. Cyclin T1 and p65 (NF-κB subunit) served as loading controls. (<b>E</b>) Quantification of Tat and eGFP protein levels in TNFα-stimulated 2D10 cells treated with the JIB-04 at the concentrations indicated in the X-axis. The signals were calculated by Image J and averaged from three independent experiments. (<b>F</b>) qRT-PCR analysis <i>d2EGFP</i> (HIV-1 reporter gene), <i>HIV-1 Env</i>, or host cell <i>CXCL10</i> and <i>CDK9</i> mRNAs extracted from 2D10 cells pre-treated with either DMSO or 3 μM JIB-04 for 16 h and stimulated by TNFα (10 ng/ml), for the times indicated below each graph. Values on the Y-axis for <i>d2EGFP</i>, <i>Env</i>, <i>CXCL10</i> and <i>CDK9</i> mRNAs prior to TNFα stimulation were normalized to 1. Differences between DMSO and 3 μM-JIB-04 treated samples were calculated by Student’s T-test at each time point (*p<0.05, **p<0.005, ***p<0.0005). Shown is a representative result from three independent experiments.</p

JIB-04 increases Tat K63Ub and proteolytic destruction.

<p>(<b>A</b>) Left, immunoblot analysis of HIV-1 Tat expression in 2D10 cells exposed to DMSO, JIB-04, MG132 or JIB-04+MG132. Cells were pre-treated with or without TNFα (10 ng/ml) for 16 h. The COP9 signalosome complex subunit 3 (Csn3) served as loading control. Right, qRT-PCR analysis of <i>Tat</i>, <i>GFP</i> and <i>Env</i> mRNA levels in these cells. Values shown in the Y-axis were normalized to mRNAs from 2D10 cells without TNFα-stimulation. (<b>B</b>) Dual-Luc (HIV-LTR-Luc/SV40-Renilla-Luc) reporter gene analysis in FLAG-Tat101 transfected HeLa P4 cells. Left, immunoblot analysis of FLAG-Tat101 protein levels in cells treated with DMSO or 2 μM JIB-04. Right panels show dual-luc reporter gene activity in these cells. Significant differences between HIV-Luc activity treated by DMSO or 2 μM JIB-04 were calculated by Student’s T-test (*p<0.05, **p<0.005, ***p<0.0005). (<b>C</b>) Dual-Luc reporter gene analysis, as in part B, in Tet-on-Tat-off HeLa cells. Left, immunoblot analysis of HA-Tat86 protein levels in 2D10 cells treated by DMSO or 2.5 μM JIB-04. Right, dual-Luc reporter gene activity in these cells. Significant differences between HIV-Luc activity treated by DMSO or 2.5 μM JIB-04 were calculated by Student’s T-test (*p<0.05, **p<0.005, ***p<0.0005). (<b>D</b>) Analysis of the effect of JIB-04 on endogenous Tat K63Ub levels in HeLa cells. HIV-1 Tat was immunoprecipitated from lysates of HeLa cells exposed to DMSO or JIB-04 (1 μM and 3 μM), and endogenous ubiquitylation was monitored using the antisera indicated to the left of each panel (HC = antibody heavy chain). (<b>E</b>) Immunoprecipitation of FLAG-Tat-101 from lysates of HeLa cells treated with DMSO or JIB-04 (1μM and 3μM). Ubiquitination of FLAG-Tat-101 in the presence of ectopically expressed HA-ubiquitin-WT, HA-ubiquitin-K63-only or HA-ubiquitin-K48-only was assessed using anti-HA antisera.</p

ChIP analysis of the effect of JIB-04 on the binding of transcription factors to the HIV-1 genome.

<p>ChIP analysis comparison of transcription factor binding to the single integrated HIV-1 genome in 2D10 T cells treated either with DMSO (A,C) or with 3 μM JIB-04 (B,D). Cells were treated with TNFα (10 ng/ml) for 0 h (blue line), 0.5 h (pink line), 2 h (yellow line), or 6 h (light blue line). The ChIP values in the Y-axis are expressed as percentage input. The ChIP primers (A-G) that were used are indicated on the X-axis, and their relative location on the HIV-1 genome are shown in the schematic at the bottom left. Antisera used for ChIP antibodies are NF-κB (p65 subunit), Cyclin T1, CDK9, NELF-A, RNAPII CTD, and phosphorylated RNAPII CTD-Ser2 (Ser2P), CTD-Ser5 (Ser5P) and CTD-Ser7 (Ser7P), as indicated above each panel.</p

JIB-04 inhibits HIV replication in HeLa P4.R5 MAGI. indicator cells.

<p>(<b>A</b>) Images of two representative fields from single-cycle infectivity imaging assays for HeLa P4.R5 MAGI cells subjected to HIV-1 infection and treated with either DMSO or JIB-04. The numbers above each panel refer to the percentage of HIV-infected blue cells exposed to DMSO, 1 μM JIB-04, or 5 μM JIB-04. Shown are two representative photos taken from each of three replicate plates, with a 10X10 amplification. (<b>B</b>) Graph plotting the average numbers of total (pink bar), blue (blue bar) or white (white bar) cells from at least three representative photos in the presence of DMSO or different concentrations of JIB-04 (1–5 μM). The percentage HIV-infected (blue) cells is indicated above the blue bars. Significant differences between cell numbers treated by DMSO or different concentrations of JIB-04 for total, blue and white cells were calculated by Student’s T-test (*p<0.05, **p<0.005, ***p<0.0005; ns = non-significant), respectively.</p