5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.

Protein motions that occur on the µs to ms timescale have been linked to enzymatic rates observed for catalytic turnovers, but not to transition-state barrier crossing. It has been hypothesized that enzyme motions on the fs time-scale of bond vibrations play a role in transition state formation. Here we perturb fs motion by substituting all non-exchangeable carbon, nitrogen, and hydrogen atoms with 13C, 15N, and 2H and observe the catalytic effects in HIV-1 protease. According to the Born-Oppenheimer approximation, isotopic substitution alters vibrational frequency with unchanged electrostatic properties. Using a fluorescent peptide to report on multiple steps in the reaction, we observe significantly reduced rates in the heavy enzyme relative to the light enzyme. A possible interpretation of our results is that there exists a dynamic link between mass-dependent bond vibrations of the enzyme and events in the reaction coordinate.

Expression of the human ␤-amyloid peptide (A␤) in a transgenic Caenorhabditis elegans Alzheimer disease model leads to the induction of HSP-16 proteins, a family of small heat shockinducible proteins homologous to vertebrate ␣B crystallin. These proteins also co-localize and co-immunoprecipitate with A␤ in this model (Fonte, V., Kapulkin, V., Taft Accumulation of the ␤-amyloid (A␤)2 peptide in the brain has been proposed to be causally linked to Alzheimer disease (the "Amyloid Cascade" hypothesis (1)), even though the specific mechanisms by which the A␤ peptide induces AD pathology have not been resolved. Intracellular A␤ accumulation has also been proposed to underlie the muscle pathology observed in inclusion body myositis (2). To investigate A␤ toxicity in a genetically tractable model, we have engineered Caenorhabditis elegans nematodes to express the human A␤-(1-42) peptide in either body wall muscle (3) or neurons (4).In C. elegans transgenic models with muscle expression of A␤, the peptide accumulates in intracellular cytoplasmic deposits (5) despite the inclusion of a signal peptide in the transgene construct. The appropriate removal of the signal peptide and the association of Abeta with hsp-3, an ER chaperone homologous to mammalian GRP78/BiP (6), have led us to propose that Abeta is routed to the secretory pathway in this model but is retrotranslocated out of the ER because it is recognized as an abnormal protein (4). We have also demonstrated a role for autophagosomes and lysosomes in the clearance of Abeta in this model, suggesting that Abeta may also exist in these subcellular compartments (8). Intracellular Abeta is observed in the muscles of IBM patients or in transgenic mouse models of IBM (9, 10), although the subcellular distribution of Abeta has not been determined. Intracellular A␤ has also been observed in human brain neurons (11), and the relevance of intracellular A␤ in Alzheimer disease has been supported by studies with the LaFerla 3ϫ transgenic AD mouse model, where accumulation of intracellular A␤ precedes neurofibrillary tangle formation (12). A number of neurodegenerative diseases (Parkinson, Huntington, amyotrophic lateral sclerosis, etc.) are characterized by intracellular cytoplasmic accumulation of proteins causally associated with theses diseases, and thus the C. elegans transgenic model described in this study may be generally relevant to the proteotoxicity underlying neurodegenerative diseases. In this context, a transgenic C. elegans strain expressing human A␤ has been used recently to investigate the roles of insulin-like signaling and heat shock factor in proteotoxicity (13).A robust finding in these transgenic C. elegans models is the induction of the HSP-16 family of small chaperone proteins by A␤ expression (14, 15). HSP-16 proteins readily co-immunoprecipitate with A␤ in transgenic C. elegans worms and closely associate with intracellular A␤ deposits as observed by immunohistochemistry (16). The HSP-16 family proteins are homologous to ␣B crystallin and have been show...

The highly conserved 70 kDa heat shock proteins (Hsp70) play an integral role in proteostasis such that dysregulation has been implicated in numerous diseases. Elucidating the precise role of Hsp70 family members in the cellular context, however, has been hampered by the redundancy and intricate regulation of the chaperone network, and relatively few selective and potent tools. We have characterized a natural product, novolactone, that targets cytosolic and ER-localized isoforms of Hsp70 through a highly conserved covalent interaction at the interface between the substrate-binding and ATPase domains. Biochemical and structural analyses indicate that novolactone disrupts interdomain communication by allosterically inducing a conformational change in the Hsp70 protein to block ATP-induced substrate release and inhibit refolding activities. Thus, novolactone is a valuable tool for exploring the requirements of Hsp70 chaperones in diverse cellular contexts.

The TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR’s ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.DOI: http://dx.doi.org/10.7554/eLife.13964.001

HIV-1 protease is an important target for the treatment of HIV/ AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease transition state has not been previously resolved using these methods. We have measured primary 14 at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage. A transition structure consistent with the KIE values involves proton transfer from the active site Asp-125 (1.32 Å) with partial hydrogen bond formation to the accepting nitrogen (1.20 Å) and partial bond loss from the carbonyl carbon to the amide leaving group (1.52 Å). The KIEs measured for the native and I84V enzyme indicate nearly identical transition states, implying that a true transition-state analogue should be effective against both enzymes.aspartyl protease | protease mechanism | transition-state structure | drug design

Protein lysine methyltransferases (PKMTs) are key players in epigenetic regulation and have been associated with a variety of diseases, including cancers. The catalytic subunit of Polycomb Repressive Complex 2, EZH2 (EC 2.1.1.43), is a PKMT and a member of a family of SET domain lysine methyltransferases that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to lysine 27 of histone 3 (H3K27). Wild-type (WT) EZH2 primarily catalyzes the mono- and dimethylation of H3K27; however, a clinically relevant active site mutation (Y641F) has been shown to alter the reaction specificity, dominantly catalyzing trimethylation of H3K27, and has been linked to tumor genesis and maintenance. Herein, we explore the chemical mechanism of methyl transfer by EZH2 and its Y641F mutant with pH-rate profiles and solvent kinetic isotope effects (sKIEs) using a short peptide derived from histone H3 [H3(21-44)]. A key component of the chemical reaction is the essential deprotonation of the ε-NH3(+) group of lysine to accommodate subsequent methylation. This deprotonation has been suggested by independent studies (1) to occur prior to binding to the enzyme (by bulk solvent) or (2) to be facilitated within the active site following binding, either (a) by the enzyme itself or (b) by a water molecule with access to the binding pocket. Our pH-rate and sKIE data best support a model in which lysine deprotonation is enzyme-dependent and at least partially rate-limiting. Furthermore, our experimental data are in agreement with prior computational models involving enzyme-dependent solvent deprotonation through a channel providing bulk solvent access to the active site. The mechanism of deprotonation and the rate-limiting catalytic steps appear to be unchanged between the WT and Y641F mutant enzymes, despite their activities being highly dependent on different substrate methylation states, suggesting determinants of substrate and product specificity in EZH2 are independent of catalytic events limiting the steady-state rate.

The transition state for the Trypanosoma cruzi uridine phosphorylase (TcUP) reaction has an expanded SN2 character. We used binding isotope effects (BIE’s) to probe uridine distortion in the complex with TcUP and sulfate to mimic the Michaelis complex. Inverse 1′-3H and 5′-3H BIE’s indicate a constrained bonding environment of these groups in the complex. Quantum chemical modeling identified a uridine conformer whose calculated BIE’s match the experimental values. This conformer differs in sugar pucker and uracil orientation from the unbound conformer and the transition-state structure. These results support ground-state stabilization in the Michaelis complex.