Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay.

A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics.

Leveraging a physiologically-based quantitative translational modeling platform for designing B cell maturation antigen-targeting bispecific T cell engagers for treatment of multiple myeloma.

Bispecific T cell engagers (TCEs) are an emerging anti-cancer modality that redirects cytotoxic T cells to tumor cells expressing tumor-associated antigens (TAAs), thereby forming immune synapses to exert anti-tumor effects. Designing pharmacokinetically acceptable TCEs and optimizing their size presents a considerable protein engineering challenge, particularly given the complexity of intercellular bridging between T cells and tumor cells. Therefore, a physiologically-relevant and clinically-verified computational modeling framework is of crucial importance to understand the protein engineering trade-offs. In this study, we developed a quantitative, physiologically-based computational framework to predict immune synapse formation for a variety of molecular formats of TCEs in tumor tissues. Our model incorporates a molecular size-dependent biodistribution using the two-pore theory, extravasation of T cells and hematologic cancer cells, mechanistic bispecific intercellular binding of TCEs, and competitive inhibitory interactions by shed targets. The biodistribution of TCEs was verified by positron emission tomography imaging of [89Zr]AMG211 (a carcinoembryonic antigen-targeting TCE) in patients. Parameter sensitivity analyses indicated that immune synapse formation was highly sensitive to TAA expression, degree of target shedding, and binding selectivity to tumor cell surface TAAs over shed targets. Notably, the model suggested a "sweet spot" for TCEs' CD3 binding affinity, which balanced the trapping of TCEs in T-cell-rich organs. The final model simulations indicated that the number of immune synapses is similar (~55/tumor cell) between two distinct clinical stage B cell maturation antigen (BCMA)-targeting TCEs, PF-06863135 in an IgG format and AMG420 in a BiTE format, at their respective efficacious doses in multiple myeloma patients. This result demonstrates the applicability of the developed computational modeling framework to molecular design optimization and clinical benchmarking for TCEs, thus suggesting that this framework can be applied to other targets to provide a quantitative means to facilitate model-informed best-in-class TCE discovery and development.

An Automated Multicycle Immunoaffinity Enrichment Approach Developed for Sensitive Mouse IgG1 Antibody Drug Analysis in Mouse Plasma Using LC/MS/MS.

In the immuno-oncology field, surrogate mouse monoclonal antibodies are often preferred in establishing proper PK/PD/efficacy correlations as well as supporting anticipated mouse to human translation. Thus, a highly sensitive and specific bioanalytical method is needed in quantifying those surrogate mouse antibodies after dosing in mice. Unfortunately, when specific reagents, such as recombinant target antigen and anti-idiotypic antibody, are not available, measuring mouse surrogate antibody drugs in mice is very challenging for ligand binding assay (LBA) due to the severe cross reactivity potential. Different from LBA, if at least one unique surrogate peptide can be identified from the surrogate antibody sequence, the immunoaffinity enrichment based LC/MS/MS assay may be able to differentiate the analyte response from the high endogenous immunoglobulin background and provide adequate sensitivity. Herein, a new automated multicycle immunoaffinity enrichment method was recently developed to extract a surrogate mouse IgG1 (mIgG1) antibody drug from mouse plasma using a commercially available antimouse IgG1 secondary antibody. In the assay, reuse of the capture antibody up to six times mostly resolved the binding capacity issue caused by the abundant endogenous mIgG1 and made the immunoaffinity enrichment step more cost-effective. Combined with a unique surrogate peptide identified from the antibody, the LC/MS/MS assay achieved a limit of quantitation of 5 ng/mL with satisfactory assay precision, accuracy, and dynamic range. The successful implementation of this novel approach in discovery pharmacokinetic (PK) studies eliminates the dependence on specially generated immunoaffinity capturing reagents.

The Arg/N-Degron Pathway-A Potential Running Back in Fine-Tuning the Inflammatory Response?

Recognition of danger signals by a cell initiates a powerful cascade of events generally leading to inflammation. Inflammatory caspases and several other proteases become activated and subsequently cleave their target proinflammatory mediators. The irreversible nature of this process implies that the newly generated proinflammatory fragments need to be sequestered, inhibited, or degraded in order to cancel the proinflammatory program or prevent chronic inflammation. The Arg/N-degron pathway is a ubiquitin-dependent proteolytic pathway that specifically degrades protein fragments bearing N-degrons, or destabilizing residues, which are recognized by the E3 ligases of the pathway. Here, we report that the Arg/N-degron pathway selectively degrades a number of proinflammatory fragments, including some activated inflammatory caspases, contributing in tuning inflammatory processes. Partial ablation of the Arg/N-degron pathway greatly increases IL-1ß secretion, indicating the importance of this ubiquitous pathway in the initiation and resolution of inflammation. Thus, we propose a model wherein the Arg/N-degron pathway participates in the control of inflammation in two ways: in the generation of inflammatory signals by the degradation of inhibitory anti-inflammatory domains and as an "off switch" for inflammatory responses through the selective degradation of proinflammatory fragments.

Novel Apoptotic Mediators Identified by Conservation of Vertebrate Caspase Targets.

Caspases are proteases conserved throughout Metazoans and responsible for initiating and executing the apoptotic program. Currently, there are over 1800 known apoptotic caspase substrates, many of them known regulators of cell proliferation and death, which makes them attractive therapeutic targets. However, most caspase substrates are by-standers, and identifying novel apoptotic mediators amongst all caspase substrates remains an unmet need. Here, we conducted an in silico search for significant apoptotic caspase targets across different species within the Vertebrata subphylum, using different criteria of conservation combined with structural features of cleavage sites. We observed that P1 aspartate is highly conserved while the cleavage sites are extensively variable and found that cleavage sites are located primarily in coiled regions composed of hydrophilic amino acids. Using the combination of these criteria, we determined the final list of the 107 most relevant caspase substrates including 30 novel targets previously unknown for their role in apoptosis and cancer. These newly identified substrates can be potential regulators of apoptosis and candidates for anti-tumor therapy.

Downregulation of the Arg/N-degron Pathway Sensitizes Cancer Cells to Chemotherapy In Vivo.

The N-degron pathway is an emerging target for anti-tumor therapies, because of its capacity to positively regulate many hallmarks of cancer, including angiogenesis, cell proliferation, motility, and survival. Thus, inhibition of the N-degron pathway offers the potential to be a highly effective anti-cancer treatment. With the use of a small interfering RNA (siRNA)-mediated approach for selective downregulation of the four Arg/N-degron-dependent ubiquitin ligases, UBR1, UBR2, UBR4, and UBR5, we demonstrated decreased cell migration and proliferation and increased spontaneous apoptosis in cancer cells. Chronic treatment with lipid nanoparticles (LNPs) loaded with siRNA in mice efficiently downregulates the expression of UBR-ubiquitin ligases in the liver without any significant toxic effects but engages the immune system and causes inflammation. However, when used in a lower dose, in combination with a chemotherapeutic drug, downregulation of the Arg/N-degron pathway E3 ligases successfully reduced tumor load by decreasing proliferation and increasing apoptosis in a mouse model of hepatocellular carcinoma, while avoiding the inflammatory response. Our study demonstrates that UBR-ubiquitin ligases of the Arg/N-degron pathway are promising targets for the development of improved therapies for many cancer types.

Beta-amyloid induces apoptosis of neuronal cells by inhibition of the Arg/N-end rule pathway proteolytic activity.

Alzheimer's disease (AD) is accompanied by the dysfunction of intracellular protein homeostasis systems, in particular the ubiquitin-proteasome system (UPS). Beta-amyloid peptide (Aß), which is involved in the processes of neurodegeneration in AD, is a substrate of this system, however its effect on UPS activity is still poorly explored. Here we found that Aß peptides inhibited the proteolytic activity of the antiapoptotic Arg/N-end rule pathway that is a part of UPS. We identified arginyltransferase Ate1 as a specific component of the Arg/N-end rule pathway targeted by Aßs. Aß bearing the familial English H6R mutation, known to cause early-onset AD, had an even greater inhibitory effect on protein degradation through the Arg/N-end rule pathway than intact Aß. This effect was associated with a significant decrease in Ate1-1 and Ate1-3 catalytic activity. We also found that the loss of Ate1 in neuroblastoma Neuro-2a cells eliminated the apoptosis-inducing effects of Aß peptides. Together, our results show that the apoptotic effect of Aß peptides is linked to their impairment of Ate1 catalytic activity leading to suppression of the Arg/N-end rule pathway proteolytic activity and ultimately cell death.

Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays.

Nα-terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified ("canonical") residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu, and (oxidized) Cys. Over the last decade, several publications have suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only α-amino groups of N-terminal residues but also γ-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of a substrate's canonical (Nt-arginylatable) N-terminal residue, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R-transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about the specificity of some Ate1 isoforms for N-terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1.

Formyl-methionine as a degradation signal at the N-termini of bacterial proteins.

In bacteria, all nascent proteins bear the pretranslationally formed N-terminal formyl-methionine (fMet) residue. The fMet residue is cotranslationally deformylated by a ribosome-associated deformylase. The formylation of N-terminal Met in bacterial proteins is not strictly essential for either translation or cell viability. Moreover, protein synthesis by the cytosolic ribosomes of eukaryotes does not involve the formylation of N-terminal Met. What, then, is the main biological function of this metabolically costly, transient, and not strictly essential modification of N-terminal Met, and why has Met formylation not been eliminated during bacterial evolution? One possibility is that the similarity of the formyl and acetyl groups, their identical locations in N-terminally formylated (Nt-formylated) and Nt-acetylated proteins, and the recently discovered proteolytic function of Nt-acetylation in eukaryotes might also signify a proteolytic role of Nt-formylation in bacteria. We addressed this hypothesis about fMet-based degradation signals, termed fMet/N-degrons, using specific E. coli mutants, pulse-chase degradation assays, and protein reporters whose deformylation was altered, through site-directed mutagenesis, to be either rapid or relatively slow. Our findings strongly suggest that the formylated N-terminal fMet can act as a degradation signal, largely a cotranslational one. One likely function of fMet/N-degrons is the control of protein quality. In bacteria, the rate of polypeptide chain elongation is nearly an order of magnitude higher than in eukaryotes. We suggest that the faster emergence of nascent proteins from bacterial ribosomes is one mechanistic and evolutionary reason for the pretranslational design of bacterial fMet/N-degrons, in contrast to the cotranslational design of analogous Ac/N-degrons in eukaryotes.

Liat1, an arginyltransferase-binding protein whose evolution among primates involved changes in the numbers of its 10-residue repeats.

The arginyltransferase Ate1 is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. At least six isoforms of mouse Ate1 are produced through alternative splicing of Ate1 pre-mRNA. We identified a previously uncharacterized mouse protein, termed Liat1 (ligand of Ate1), that interacts with Ate1 but does not appear to be its arginylation substrate. Liat1 has a higher affinity for the isoforms Ate1(1A7A) and Ate1(1B7A). Liat1 stimulated the in vitro N-terminal arginylation of a model substrate by Ate1. All examined vertebrate and some invertebrate genomes encode proteins sequelogous (similar in sequence) to mouse Liat1. Sequelogs of Liat1 share a highly conserved ∼30-residue region that is shown here to be required for the binding of Liat1 to Ate1. We also identified non-Ate1 proteins that interact with Liat1. In contrast to Liat1 genes of nonprimate mammals, Liat1 genes of primates are subtelomeric, a location that tends to confer evolutionary instability on a gene. Remarkably, Liat1 proteins of some primates, from macaques to humans, contain tandem repeats of a 10-residue sequence, whereas Liat1 proteins of other mammals contain a single copy of this motif. Quantities of these repeats are, in general, different in Liat1 of different primates. For example, there are 1, 4, 13, 13, 17, and 17 repeats in the gibbon, gorilla, orangutan, bonobo, neanderthal, and human Liat1, respectively, suggesting that repeat number changes in this previously uncharacterized protein may contribute to evolution of primates.

Calpain-generated natural protein fragments as short-lived substrates of the N-end rule pathway.

Calpains are Ca(2+)-dependent intracellular proteases. We show here that calpain-generated natural C-terminal fragments of proteins that include G protein-coupled receptors, transmembrane ion channels, transcriptional regulators, apoptosis controllers, kinases, and phosphatases (Phe-GluN2a, Lys-Ica512, Arg-Ankrd2, Tyr-Grm1, Arg-Atp2b2, Glu-Bak, Arg-Igfbp2, Glu-IκBα, and Arg-c-Fos), are short-lived substrates of the Arg/N-end rule pathway, which targets destabilizing N-terminal residues. We also found that the identity of a fragment's N-terminal residue can change during evolution, but the residue's destabilizing activity is virtually always retained, suggesting selection pressures that favor a short half-life of the calpain-generated fragment. It is also shown that a self-cleavage of a calpain can result in an N-end rule substrate. Thus, the autoprocessing of calpains can control them by making active calpains short-lived. These and related results indicate that the Arg/N-end rule pathway mediates the remodeling of oligomeric complexes by eliminating protein fragments that are produced in these complexes through cleavages by calpains or other nonprocessive proteases. We suggest that this capability of the Arg/N-end rule pathway underlies a multitude of its previously known but mechanistically unclear functions.

Ubiquitin reference technique and its use in ubiquitin-lacking prokaryotes.

In a pulse-chase assay, the in vivo degradation of a protein is measured through a brief labeling of cells with, for example, a radioactive amino acid, followed by cessation of labeling and analysis of cell extracts prepared at different times afterward ("chase"), using immunoprecipitation, electrophoresis and autoradiography of a labeled protein of interest. A conventional pulse-chase assay is fraught with sources of data scatter, as the efficacy of labeling and immunoprecipitation can vary, and sample volumes can vary as well. The ubiquitin reference technique (URT), introduced in 1996, addresses these problems. In eukaryotes, a DNA-encoded linear fusion of ubiquitin to another protein is cleaved by deubiquitylases at the ubiquitin-protein junction. A URT assay uses a fusion in which the ubiquitin moiety is located between a downstream polypeptide (test protein) and an upstream polypeptide (a long-lived reference protein). The cotranslational cleavage of a URT fusion by deubiquitylases after the last residue of ubiquitin produces, at the initially equimolar ratio, a test protein with a desired N-terminal residue and a reference protein containing C-terminal ubiquitin moiety. In addition to being more accurate than pulse-chases without a reference, URT makes it possible to detect and measure the degradation of a test protein during the pulse (before the chase). Because prokaryotes, including Gram-negative bacteria such as, for example, Escherichia coli and Vibrio vulnificus, lack the ubiquitin system, the use of URT in such cells requires ectopic expression of a deubiquitylase. We describe designs and applications of plasmid vectors that coexpress, in bacteria, both a URT-type fusion and Ubp1, a deubiquitylase of the yeast Saccharomyces cerevisiae. This single-plasmid approach extends the accuracy-enhancing URT assay to studies of protein degradation in prokaryotes.

Neurodegeneration-associated protein fragments as short-lived substrates of the N-end rule pathway.

Protein aggregates are a common feature of neurodegenerative syndromes. Specific protein fragments were found to be aggregated in disorders including Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. Here, we show that the natural C-terminal fragments of Tau, TDP43, and α-synuclein are short-lived substrates of the Arg/N-end rule pathway, a processive proteolytic system that targets proteins bearing "destabilizing" N-terminal residues. Furthermore, a natural TDP43 fragment is shown to be metabolically stabilized in Ate1(-/-) fibroblasts that lack the arginylation branch of the Arg/N-end rule pathway, leading to accumulation and aggregation of this fragment. We also found that a fraction of Aß42, the Alzheimer's disease-associated fragment of APP, is N-terminally arginylated in the brains of 5xFAD mice and is degraded by the Arg/N-end rule pathway. The discovery that neurodegeneration-associated natural fragments of TDP43, Tau, α-synuclein, and APP can be selectively destroyed by the Arg/N-end rule pathway suggests that this pathway counteracts neurodegeneration.

The auto-generated fragment of the Usp1 deubiquitylase is a physiological substrate of the N-end rule pathway.

Deamidation of N-terminal Gln by the Ntaq1 Nt(Q)-amidase is a part of the Arg/N-end rule pathway, a ubiquitin-dependent proteolytic system. Here we identify Gln-Usp1(Ct), the C-terminal fragment of the autocleaved Usp1 deubiquitylase, as the first physiological Arg/N-end rule substrate that is targeted for degradation through deamidation of N-terminal Gln. Usp1 regulates genomic stability, in part through the deubiquitylation of monoubiquitylated PCNA, a DNA polymerase processivity factor. The autocleaved Usp1 remains a deubiquitylase because its fragments remain associated with Uaf1, an enhancer of Usp1 activity, until the Gln-Usp1(Ct) fragment is selectively destroyed by the Arg/N-end rule pathway. We also show that metabolic stabilization of Gln-Usp1(Ct) results in a decreased monoubiquitylation of PCNA and in a hypersensitivity of cells to ultraviolet irradiation. Thus, in addition to its other functions in DNA repair and chromosome segregation, the Arg/N-end rule pathway regulates genomic stability through the degradation-mediated control of the autocleaved Usp1 deubiquitylase.

The N-end rule pathway counteracts cell death by destroying proapoptotic protein fragments.

In the course of apoptosis, activated caspases cleave ∼500 to ∼1,000 different proteins in a mammalian cell. The dynamics of apoptosis involve a number of previously identified, caspase-generated proapoptotic protein fragments, defined as those that increase the probability of apoptosis. In contrast to activated caspases, which can be counteracted by inhibitor of apoptosis proteins, there is little understanding of antiapoptotic responses to proapoptotic protein fragments. One possibility is the regulation of proapoptotic fragments through their selective degradation. The previously identified proapoptotic fragments Cys-RIPK1, Cys-TRAF1, Asp-BRCA1, Leu-LIMK1, Tyr-NEDD9, Arg-BID, Asp-BCL(XL), Arg-BIM(EL), Asp-EPHA4, and Tyr-MET bear destabilizing N-terminal residues. Tellingly, the destabilizing nature (but not necessarily the actual identity) of N-terminal residues of proapoptotic fragments was invariably conserved in evolution. Here, we show that these proapoptotic fragments are short-lived substrates of the Arg/N-end rule pathway. Metabolic stabilization of at least one such fragment, Cys-RIPK1, greatly augmented the activation of the apoptosis-inducing effector caspase-3. In agreement with this understanding, even a partial ablation of the Arg/N-end rule pathway in two specific N-end rule mutants is shown to sensitize cells to apoptosis. We also found that caspases can inactivate components of the Arg/N-end rule pathway, suggesting a mutual suppression between this pathway and proapoptotic signaling. Together, these results identify a mechanistically specific and functionally broad antiapoptotic role of the Arg/N-end rule pathway. In conjunction with other apoptosis-suppressing circuits, the Arg/N-end rule pathway contributes to thresholds that prevent a transient or otherwise weak proapoptotic signal from reaching the point of commitment to apoptosis.

Glutamine-specific N-terminal amidase, a component of the N-end rule pathway.

Deamidation of N-terminal Gln by Nt(Q)-amidase, an N-terminal amidohydrolase, is a part of the N-end rule pathway of protein degradation. We detected the activity of Nt(Q)-amidase, termed Ntaq1, in mouse tissues, purified Ntaq1 from bovine brains, identified its gene, and began analyzing this enzyme. Ntaq1 is highly conserved among animals, plants, and some fungi, but its sequence is dissimilar to sequences of other amidases. An earlier mutant in the Drosophila Cg8253 gene that we show here to encode Nt(Q)-amidase has defective long-term memory. Other studies identified protein ligands of the uncharacterized human C8orf32 protein that we show here to be the Ntaq1 Nt(Q)-amidase. Remarkably, "high-throughput" studies have recently solved the crystal structure of C8orf32 (Ntaq1). Our site-directed mutagenesis of Ntaq1 and its crystal structure indicate that the active site and catalytic mechanism of Nt(Q)-amidase are similar to those of transglutaminases.

Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway.

Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway's ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, and His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, and Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first approximately 700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated approximately 70-residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with a K(d) of approximately 1 microm, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of cognate dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1 type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.

A size filtration approach to purify low affinity complexes for crystallization.

Low affinity protein complexes are difficult to isolate and handle in crystallization experiments. Size-exclusion chromatography often does not allow purification of the homogeneous complex. Here we used a size-filtration approach for the purification and concentration of the 19 microM affinity complex of yeast Rab-GTPase and its guanine nucleotide disassociation inhibitor (GDI). The homogeneous protein complex solution was crystallized and the structure was solved using the molecular replacement method. The resulting model of the low affinity unprenylated Rab-GDI complex should reflect a transient Rab-GDI complex when GDI is bound to the membrane-anchored Rab protein and is poised to extract Rab to cytosol.

Aminoacyl-transferases and the N-end rule pathway of prokaryotic/eukaryotic specificity in a human pathogen.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Primary destabilizing N-terminal residues (Nd(p)) are recognized directly by the targeting machinery. The recognition of secondary destabilizing N-terminal residues (Nd(s)) is preceded by conjugation of an Nd(p) residue to Nd(s) of a polypeptide substrate. In eukaryotes, ATE1-encoded arginyl-transferases (R(D,E,C*)-transferases) conjugate Arg (R), an Nd(p) residue, to Nd(s) residues Asp (D), Glu (E), or oxidized Cys residue (C*). Ubiquitin ligases recognize the N-terminal Arg of a substrate and target the (ubiquitylated) substrate to the proteasome. In prokaryotes such as Escherichia coli, Nd(p) residues Leu (L) or Phe (F) are conjugated, by the aat-encoded Leu/Phe-transferase (L/F(K,R)-transferase), to N-terminal Arg or Lys, which are Nd(s) in prokaryotes but Nd(p) in eukaryotes. In prokaryotes, substrates bearing the Nd(p) residues Leu, Phe, Trp, or Tyr are degraded by the proteasome-like ClpAP protease. Despite enzymological similarities between eukaryotic R(D,E,C*)-transferases and prokaryotic L/F(K,R)-transferases, there is no significant sequelogy (sequence similarity) between them. We identified an aminoacyl-transferase, termed Bpt, in the human pathogen Vibrio vulnificus. Although it is a sequelog of eukaryotic R(D,E,C*)-transferases, this prokaryotic transferase exhibits a "hybrid" specificity, conjugating Nd(p) Leu to Nd(s) Asp or Glu. Another aminoacyl-transferase, termed ATEL1, of the eukaryotic pathogen Plasmodium falciparum, is a sequelog of prokaryotic L/F(K,R)-transferases (Aat), but has the specificity of eukaryotic R(D,E,C*)-transferases (ATE1). Phylogenetic analysis suggests that the substrate specificity of R-transferases arose by two distinct routes during the evolution of eukaryotes.