Mechanism-based traps enable protease and hydrolase substrate discovery.

Hydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-especially those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes.

Structural Basis of Protein Arginine Methyltransferase Activation by a Catalytically Dead Homolog (Prozyme).

Prozymes are pseudoenzymes that stimulate the function of weakly active enzymes through complex formation. The major Trypanosoma brucei protein arginine methyltransferase, TbPRMT1 enzyme (ENZ), requires TbPRMT1 prozyme (PRO) to form an active heterotetrameric complex. Here, we present the X-ray crystal structure of the TbPRMT1 ENZ-Δ52PRO tetrameric complex with the cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.4 Å resolution. The individual ENZ and PRO units adopt the highly-conserved PRMT domain architecture and form an antiparallel heterodimer that corresponds to the canonical homodimer observed in all previously reported PRMTs. In turn, two such heterodimers assemble into a tetramer both in the crystal and in solution with twofold rotational symmetry. ENZ is unstable in absence of PRO and incapable of forming a homodimer due to a steric clash of an ENZ-specific tyrosine within the dimerization arm, rationalizing why PRO is required to complement ENZ to form a PRMT dimer that is necessary, but not sufficient for PRMT activity. The PRO structure deviates from other, active PRMTs in that it lacks the conserved η2 310-helix within the Rossmann fold, abolishing cofactor binding. In addition to its chaperone function for ENZ, PRO substantially contributes to substrate binding. Heterotetramerization is required for catalysis, as heterodimeric ENZ-PRO mutants lack binding affinity and methyltransferase activity toward the substrate protein TbRGG1. Together, we provide a structural basis for TbPRMT1 ENZ activation by PRO heterotetramer formation, which is conserved across all kinetoplastids, and describe a chaperone function of the TbPRMT1 prozyme, which represents a novel mode of PRMT regulation.

Trypanosoma brucei PRMT1 Is a Nucleic Acid Binding Protein with a Role in Energy Metabolism and the Starvation Stress Response.

In Trypanosoma brucei and related kinetoplastid parasites, transcription of protein coding genes is largely unregulated. Rather, mRNA binding proteins, which impact processes such as transcript stability and translation efficiency, are the predominant regulators of gene expression. Arginine methylation is a posttranslational modification that preferentially targets RNA binding proteins and is, therefore, likely to have a substantial impact on T. brucei biology. The data presented here demonstrate that cells depleted of T. brucei PRMT1 (TbPRMT1), a major type I protein arginine methyltransferase, exhibit decreased virulence in an animal model. To understand the basis of this phenotype, quantitative global proteomics was employed to measure protein steady-state levels in cells lacking TbPRMT1. The approach revealed striking changes in proteins involved in energy metabolism. Most prominent were a decrease in glycolytic enzyme abundance and an increase in proline degradation pathway components, changes that resemble the metabolic remodeling that occurs during T. brucei life cycle progression. The work describes several RNA binding proteins whose association with mRNA was altered in TbPRMT1-depleted cells, and a large number of TbPRMT1-interacting proteins, thereby highlighting potential TbPRMT1 substrates. Many proteins involved in the T. brucei starvation stress response were found to interact with TbPRMT1, prompting analysis of the response of TbPRMT1-depleted cells to nutrient deprivation. Indeed, depletion of TbPRMT1 strongly hinders the ability of T. brucei to form cytoplasmic mRNA granules under starvation conditions. Finally, this work shows that TbPRMT1 itself binds nucleic acids in vitro and in vivo, a feature completely novel to protein arginine methyltransferases.IMPORTANCETrypanosoma brucei infection causes human African trypanosomiasis, also known as sleeping sickness, a disease with a nearly 100% fatality rate when untreated. Current drugs are expensive, toxic, and highly impractical to administer, prompting the community to explore various unique aspects of T. brucei biology in search of better treatments. In this study, we identified the protein arginine methyltransferase (PRMT), TbPRMT1, as a factor that modulates numerous aspects of T. brucei biology. These include glycolysis and life cycle progression signaling, both of which are being intensely researched toward identification of potential drug targets. Our data will aid research in those fields. Furthermore, we demonstrate for the first time a direct association of a PRMT with nucleic acids, a finding we believe could translate to other organisms, including humans, thereby impacting research in fields as distant as human cancer biology and immune response modulation.

The Major Protein Arginine Methyltransferase in Trypanosoma brucei Functions as an Enzyme-Prozyme Complex.

Prozymes are catalytically inactive enzyme paralogs that dramatically stimulate the function of weakly active enzymes through complex formation. The two prozymes described to date reside in the polyamine biosynthesis pathway of the human parasite Trypanosoma brucei, an early branching eukaryote that lacks transcriptional regulation and regulates its proteome through posttranscriptional and posttranslational means. Arginine methylation is a common posttranslational modification in eukaryotes catalyzed by protein arginine methyltransferases (PRMTs) that are typically thought to function as homodimers. We demonstrate that a major T. brucei PRMT, TbPRMT1, functions as a heterotetrameric enzyme-prozyme pair. The inactive PRMT paralog, TbPRMT1PRO, is essential for catalytic activity of the TbPRMT1ENZ subunit. Mutational analysis definitively demonstrates that TbPRMT1ENZ is the cofactor-binding subunit and carries all catalytic activity of the complex. Our results are the first demonstration of an obligate heteromeric PRMT, and they suggest that enzyme-prozyme organization is expanded in trypanosomes as a posttranslational means of enzyme regulation.

Integrity of the core mitochondrial RNA-binding complex 1 is vital for trypanosome RNA editing.

Trypanosoma brucei is the causative agent of the human and veterinarian diseases African sleeping sickness and nagana. A majority of its mitochondrial-encoded transcripts undergo RNA editing, an essential process of post-transcriptional uridine insertion and deletion to produce translatable mRNA. Besides the well-characterized RNA editing core complex, the mitochondrial RNA-binding 1 (MRB1) complex is one of the key players. It comprises a core complex of about six proteins, guide RNA-associated proteins (GAPs) 1/2, which form a heterotetramer that binds and stabilizes gRNAs, plus MRB5390, MRB3010, and MRB11870, which play roles in initial stages of RNA editing, presumably guided by the first gRNA:mRNA duplex in the case of the latter two proteins. To better understand all functions of the MRB1 complex, we performed a functional analysis of the MRB8620 core subunit, the only one not characterized so far. Here we show that MRB8620 plays a role in RNA editing in both procyclic and bloodstream stages of T. brucei, which reside in the tsetse fly vector and mammalian circulatory system, respectively. While RNAi silencing of MRB8620 does not affect procyclic T. brucei fitness when grown in glucose-containing media, it is somewhat compromised in cells grown in the absence of this carbon source. MRB8620 is crucial for integrity of the MRB1 core, such as its association with GAP1/2, which presumably acts to deliver gRNAs to this complex. In contrast, GAP1/2 is not required for the fabrication of the MRB1 core. Disruption of the MRB1 core assembly is followed by the accumulation of mRNAs associated with GAP1/2.

A core MRB1 complex component is indispensable for RNA editing in insect and human infective stages of Trypanosoma brucei.

Uridine insertion/deletion RNA editing is a unique and vital process in kinetoplastids, required for creation of translatable open reading frames in most mitochondrially-encoded RNAs. Emerging as a key player in this process is the mitochondrial RNA binding 1 (MRB1) complex. MRB1 comprises an RNA-independent core complex of at least six proteins, including the GAP1/2 guide RNA (gRNA) binding proteins. The core interacts in an RNA-enhanced or -dependent manner with imprecisely defined TbRGG2 subcomplexes, Armadillo protein MRB10130, and additional factors that comprise the dynamic MRB1 complex. Towards understanding MRB1 complex function in RNA editing, we present here functional characterization of the pentein domain-containing MRB1 core protein, MRB11870. Inducible RNAi studies demonstrate that MRB11870 is essential for proliferation of both insect vector and human infective stage T. brucei. MRB11870 ablation causes a massive defect in RNA editing, affecting both pan-edited and minimally edited mRNAs, but does not substantially affect mitochondrial RNA stability or processing of precursor transcripts. The editing defect in MRB1-depleted cells occurs at the initiation stage of editing, as pre-edited mRNAs accumulate. However, the gRNAs that direct editing remain abundant in the knockdown cells. To examine the contribution of MRB11870 to MRB1 macromolecular interactions, we tagged core complexes and analyzed their composition and associated proteins in the presence and absence of MRB11870. These studies demonstrated that MRB11870 is essential for association of GAP1/2 with the core, as well as for interaction of the core with other proteins and subcomplexes. Together, these data support a model in which the MRB1 core mediates functional interaction of gRNAs with the editing machinery, having GAP1/2 as its gRNA binding constituents. MRB11870 is a critical component of the core, essential for its structure and function.

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei.

A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.

Architecture of the trypanosome RNA editing accessory complex, MRB1.

Trypanosoma brucei undergoes an essential process of mitochondrial uridine insertion and deletion RNA editing catalyzed by a 20S editosome. The multiprotein mitochondrial RNA-binding complex 1 (MRB1) is emerging as an equally essential component of the trypanosome RNA editing machinery, with additional functions in gRNA and mRNA stabilization. The distinct and overlapping protein compositions of reported MRB1 complexes and diverse MRB1 functions suggest that the complex is composed of subcomplexes with RNA-dependent and independent interactions. To determine the architecture of the MRB1 complex, we performed a comprehensive yeast two-hybrid analysis of 31 reported MRB1 proteins. We also used in vivo analyses of tagged MRB1 components to confirm direct and RNA-mediated interactions. Here, we show that MRB1 contains a core complex comprised of six proteins and maintained by numerous direct interactions. The MRB1 core associates with multiple subcomplexes and proteins through RNA-enhanced or RNA-dependent interactions. These findings provide a framework for interpretation of previous functional studies and suggest that MRB1 is a dynamic complex that coordinates various aspects of mitochondrial gene regulation.