Haiteng Deng

Labeling Substrates of Protein Arginine Methyltransferase with Engineered Enzymes and Matched S-Adenosyl-L-methionine Analogues

Elucidating physiological and pathogenic functions of protein methyltransferases (PMTs) relies on knowing their substrate profiles. S-adenosyl-L-methionine (SAM) is the sole methyl-donor cofactor of PMTs. Recently, SAM analogues have emerged as novel small-molecule tools to efficiently label PMT substrates. Here we reported the development of a clickable SAM analogue cofactor, 4-propargyloxy-but-2-enyl SAM, and its implementation to label substrates of human protein arginine methyltransferase 1 (PRMT1). In the system, the SAM analogue cofactor, coupled with matched PRMT1 mutants rather than native PRMT1, was shown to label PRMT1 substrates. The transferable 4-propargyloxy-but-2-enyl moiety of the SAM analogue further allowed corresponding modified substrates to be characterized through a subsequent click chemical ligation with an azido-based probe. The SAM analogue, in combination with a rational protein-engineering approach, thus shows potential to label and identify PMT targets in the context of a complex cellular mixture.

Expanding cofactor repertoire of protein lysine methyltransferase for substrate labeling.

Protein lysine methyltransferases (PKMTs) play crucial roles in normal physiology and disease processes. Profiling PKMT targets is an important but challenging task. With cancer-relevant G9a as a target, we have demonstrated success in developing S-adenosyl-L-methionine (SAM) analogues, particularly (E)-hex-2-en-5-ynyl SAM (Hey-SAM), as cofactors for engineered G9a. Hey-SAM analogue in combination with G9a Y1154A mutant modifies the same set of substrates as their native counterparts with remarkable efficiency. (E)-Hex-2-en-5-ynylated substrates undergo smooth click reaction with an azide-based probe. This approach is thus suitable for substrate characterization of G9a and expected to further serve as a starting point to evolve other PKMTs to utilize a similar set of cofactors.

The CREB coactivator CRTC2 controls hepatic lipid metabolism by regulating SREBP1.

Abnormal accumulation of triglycerides in the liver, caused in part by increased de novo lipogenesis, results in non-alcoholic fatty liver disease and insulin resistance. Sterol regulatory element-binding protein 1 (SREBP1), an important transcriptional regulator of lipogenesis, is synthesized as an inactive precursor that binds to the endoplasmic reticulum (ER). In response to insulin signalling, SREBP1 is transported from the ER to the Golgi in a COPII-dependent manner, processed by proteases in the Golgi, and then shuttled to the nucleus to induce lipogenic gene expression; however, the mechanisms underlying enhanced SREBP1 activity in insulin-resistant obesity and diabetes remain unclear. Here we show in mice that CREB regulated transcription coactivator 2 (CRTC2) functions as a mediator of mTOR signalling to modulate COPII-dependent SREBP1 processing. CRTC2 competes with Sec23A, a subunit of the COPII complex, to interact with Sec31A, another COPII subunit, thus disrupting SREBP1 transport. During feeding, mTOR phosphorylates CRTC2 and attenuates its inhibitory effect on COPII-dependent SREBP1 maturation. As hepatic overexpression of an mTOR-defective CRTC2 mutant in obese mice improved the lipogenic program and insulin sensitivity, these results demonstrate how the transcriptional coactivator CRTC2 regulates mTOR-mediated lipid homeostasis in the fed state and in obesity.

Bioorthogonal profiling of protein methylation using azido derivative of S-adenosyl-L-methionine.

Protein methyltransferases (PMTs) play critical roles in multiple biological processes. Because PMTs often function in vivo through forming multimeric protein complexes, dissecting their activities in the native contexts is challenging but relevant. To address such a need, we envisioned a Bioorthogonal Profiling of Protein Methylation (BPPM) technology, in which a SAM analogue cofactor can be utilized by multiple rationally engineered PMTs to label substrates of the corresponding native PMTs. Here, 4-azidobut-2-enyl derivative of S-adenosyl-L-methionine (Ab-SAM) was reported as a suitable BPPM cofactor. The resultant cofactor-enzyme pairs were implemented to label specifically the substrates of closely related PMTs (e.g., EuHMT1 and EuHMT2) in a complex cellular mixture. The BPPM approach, coupled with mass spectrometric analysis, enables the identification of the nonhistone targets of EuHMT1/2. Comparison of EuHMT1/2s methylomes indicates that the two human PMTs, although similar in terms of their primary sequences, can act on the distinct sets of nonhistone targets. Given the conserved active sites of PMTs, Ab-SAM and its use in BPPM are expected to be transferable to other PMTs for target identification.

Profiling Genome-Wide Chromatin Methylation with Engineered Posttranslation Apparatus within Living Cells

Protein methyltransferases (PMTs) have emerged as important epigenetic regulators in myriad biological processes in both normal physiology and disease conditions. However, elucidating PMT-regulated epigenetic processes has been hampered by ambiguous knowledge about in vivo activities of individual PMTs particularly because of their overlapping but nonredundant functions. To address limitations of conventional approaches in mapping chromatin modification of specific PMTs, we have engineered the chromatin-modifying apparatus and formulated a novel technology, termed clickable chromatin enrichment with parallel DNA sequencing (CliEn-seq), to probe genome-wide chromatin modification within living cells. The three-step approach of CliEn-seq involves in vivo synthesis of S-adenosyl-L-methionine (SAM) analogues from cell-permeable methionine analogues by engineered SAM synthetase (methionine adenosyltransferase or MAT), in situ chromatin modification by engineered PMTs, subsequent enrichment and sequencing of the uniquely modified chromatins. Given critical roles of the chromatin-modifying enzymes in epigenetics and structural similarity among many PMTs, we envision that the CliEn-seq technology is generally applicable in deciphering chromatin methylation events of individual PMTs in diverse biological settings.

Se-adenosyl-L-selenomethionine cofactor analogue as a reporter of protein methylation.

Posttranslational methylation by S-adenosyl-L-methionine(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases. However, broad application of these compounds has been limited by their general incompatibility with native enzymes. Here we report a SAM surrogate, ProSeAM (propargylic Se-adenosyl-l-selenomethionine), as a reporter of methyltransferases. ProSeAM can be processed by multiple protein methyltransferases for substrate labeling. In contrast, sulfur-based propargylic SAM undergoes rapid decomposition at physiological pH, likely via an allene intermediate. In conjunction with fluorescent/affinity-based azide probes, copper-catalyzed azide-alkyne cycloaddition chemistry, in-gel fluorescence visualization and proteomic analysis, we further demonstrated ProSeAMs utility to profile substrates of endogenous methyltransferases in diverse cellular contexts. These results thus feature ProSeAM as a convenient probe to study the activities of endogenous protein methyltransferases.

Defining efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation

Significance Many proteins undergo various posttranslational modifications for proper functions. One such modification is methylation carried out by enzyme–cofactor pairs of protein methyltransferases (PMTs) and S-adenosyl-L-methionine (SAM). Identification of methylated proteins is quite challenging because of the small size and chemical inertness of the methyl group. To address this challenge, we have synthesized SAM surrogates by replacing SAM’s methyl group with bulky, chemically active functionalities and demonstrated their utility as alternative cofactors of engineered PMTs for substrate labeling. Proteins modified with such chemical moieties are amenable to bioorthogonal reactions for subsequent enrichment and identification. An engineered enzyme–cofactor pair has been successfully used to reveal numerous methylated proteins. Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. With two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme–cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.

Molecular Mechanism for Inhibition of a Critical Component in the Arabidopsis thaliana Abscisic Acid Signal Transduction Pathways, SnRK2.6, by Protein Phosphatase ABI1

Background: The antagonistic complex of SnRK2.6 and ABI1 regulates abscisic acid (ABA) signaling in plants. Results: Presented here are the structure of SnRK2.6 kinase domain, and biochemical and computational characterizations of the ABI1-SnRK2.6 complex. Conclusion: Our studies revealed the molecular basis for ABI1-mediated inhibition of SnRK2.6. Significance: The studies advanced our understanding of the downstream signal transduction of ABA through SnRK2s and protein phosphatase type 2Cs. Subclass III SnRK2s (SnRK2.6/2.3/2.2) are the key positive regulators of abscisic acid (ABA) signal transduction in Arabidopsis thaliana. The kinases, activated by ABA or osmotic stress, phosphorylate stress-related transcription factors and ion channels, which ultimately leads to the protection of plants from dehydration or high salinity. In the absence of stressors, SnRK2s are subject to negative regulation by group A protein phosphatase type 2Cs (PP2C), whereas the underlying molecular mechanism remains to be elucidated. Here we report the crystal structure of the kinase domain of SnRK2.6 at 2.6-Å resolution. Structure-guided biochemical analyses identified two distinct interfaces between SnRK2.6 and ABI1, a member of group A PP2Cs. Structural modeling suggested that the two interfaces lock SnRK2.6 and ABI1 in an orientation such that the activation loop of SnRK2.6 is posited to the catalytic site of ABI1 for dephosphorylation. These studies revealed the molecular basis for PP2Cs-mediated inhibition of SnRK2s and provided important insights into the downstream signal transduction of ABA.

Profiling Substrates of Protein Arginine N-Methyltransferase 3 with S-Adenosyl-l-methionine Analogues

Protein arginine N-methyltransferase 3 (PRMT3) belongs to the family of type I PRMTs and harbors the activity to use S-adenosyl-l-methionine (SAM) as a methyl-donor cofactor for protein arginine labeling. However, PRMT3s functions remain elusive with the lacked knowledge of its target scope in cellular settings. Inspired by the emerging Bioorthogonal Profiling of Protein Methylation (BPPM) using engineered methyltransferases and SAM analogues for target identification, the current work documents the endeavor to systematically explore the SAM-binding pocket of PRMT3 and identify suitable PRMT3 variants for BPPM. The M233G single point mutation transforms PRMT3 into a promiscuous alkyltransferase using sp(2)-β-sulfonium-containing SAM analogues as cofactor surrogates. Here the conserved methionine was defined as a hot spot that can be engineered alone or in combination with nearby residues to render cofactor promiscuity of multiple type I PRMTs. With this promiscuous variant and the matched 4-propargyloxy-but-2-enyl (Pob)-SAM analogue as the BPPM reagents, more than 80 novel proteins were readily uncovered as potential targets of PRMT3 in the cellular context. Subsequent target validation and functional analysis correlated the PRMT3 methylation to several biological processes such as cytoskeleton dynamics, whose roles might be compensated by other PRMTs. These BPPM-revealed substrates are primarily localized but not restricted in cytoplasm, the preferred site of PRMT3. The broad localization pattern may implicate the diverse roles of PRMT3 in the cellular setting. The revelation of PRMT3 targets and the transformative character of BPPM for other PRMTs present unprecedented pathways toward elucidating physiological and pathological roles of diverse PRMTs.

Overexpression of CD38 Decreases Cellular NAD Levels and Alters the Expression of Proteins Involved in Energy Metabolism and Antioxidant Defense

Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells and mediates multiple cellular signaling pathways. In the present study, a 35% decrease of cellular NAD level is achieved by stable expression of the N-terminal truncated CD38, a NAD hydrolase. CD38-expressing (CD38(+)) cells have the lower growth rate and are more susceptive to oxidative stress than the wild type cells and empty vector-transfected (CD38(-)) cells. Quantitative proteomic analysis shows that 178 proteins are down-regulated in CD38(+) cells, which involve in diverse cellular processes including glycolysis, RNA processing and protein synthesis, antioxidant, and DNA repair. Down regulation of six selected proteins is confirmed by Western blotting. However, down-regulation of mRNA expressions of genes associated with glycolysis, antioxidant, and DNA repair is less significant than the corresponding change in protein expression, suggesting the low NAD level impairs the protein translational machinery in CD38(+) cells. Down-regulation of antioxidant protein and DNA-repair protein expression contributes to the susceptibility of CD38(+) cells to oxidative stress. Taken together, these results demonstrate that CD38(+) cells are a useful model to study effects of the cellular NAD levels on cellular processes and establish a new linker between cellular NAD levels and oxidative stress.