A cell-based bioluminescence reporter assay of human Sonic Hedgehog protein autoprocessing to identify inhibitors and activators.

The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value. Here, we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing noninvasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing using endogenous cholesterol to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a WT SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. Additionally, a conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the activation of the cholesterol substrate. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intramolecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel anionic phosphonylated coelenterazine, CLZ-2P, as the nanoluciferase substrate. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.

Metacaspase of Saccharomyces cerevisiae (ScMCA-Ia) presents different catalytic cysteine in a processed and non-processed form.

Metacaspases are cysteine proteases belonging to the CD clan of the C14 family. They possess important characteristics, such as specificity for cleavage after basic residues (Arg/Lys) and dependence on calcium ions to exert their catalytic activity. They are defined by the presence of a large subunit (p20) and a small subunit (p10) and are classified into types I, II, and III. Type I metacaspases have a characteristic pro-domain at the N-terminal of the enzyme, preceding a region rich in glutamine and asparagine. In the yeast Saccharomyces cerevisiae, a type I metacaspase is found. This organism encodes a single metacaspase that participates in the process of programmed cell death by apoptosis. The study focuses on cloning, expressing, and mutating Saccharomyces cerevisiae metacaspase (ScMCA-Ia). Mutations in Cys155 and Cys276 were introduced to investigate autoprocessing mechanisms. Results revealed that Cys155 plays a crucial role in autoprocessing, initiating a conformational change that activates ScMCA-Ia. Comparative analysis with TbMCA-IIa highlighted the significance of the N-terminal region in substrate access to the active site. The study proposes a two-step processing mechanism for type I metacaspases, where an initial processing step generates the active form, followed by a distinct intermolecular processing step. This provides new insights into ScMCA-Ia's activation and function. The findings hold potential implications for understanding cellular processes regulated by metacaspases. Overall, this research significantly advances knowledge in metacaspase biology.

Natural Occurring Polymorphisms in HIV-1 Integrase and RNase H Regulate Viral Release and Autoprocessing.

Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy-naive patients reported that 14 naturally occurring nonsynonymous single-nucleotide polymorphisms (SNPs) in HIV derived from antiretrovirus drug-naive patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing a Met-to-Ile change at codon 50 in integrase [HIV(IN:M50I)] was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P < 0.001). Transmission electron microscopy analysis revealed that abnormal bud accumulation on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins' autoprocessing in the HIV(IN:M50I) particles, although Förster resonance energy transfer (FRET) assay displayed that GagPol containing IN:M50I forms a homodimer with a similar efficiency with GagPol (wild type). The impaired maturation and replication were rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 of integrase and Asn-to-Ser change at codon 79 of RNase H. These data demonstrate that Gag and GagPol assembly, virus release, and autoprocessing are regulated by not only integrase but also RNase H. IMPORTANCE Nascent HIV-1 is a noninfectious viral particle. Cleaving Gag and GagPol polyproteins in the particle by mature HIV protease (PR), the nascent virus becomes an infectious virus. PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by self-cleavage is called autoprocessing. Here, during the evaluation of the roles of naturally emerging nonsynonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase GagPol. Other coexisting SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, recovered this defect, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

Characterization of substrate specificity and novel autoprocessing mechanism of dipeptidase A from Prevotella intermedia.

Prevotella intermedia, a Gram-negative anaerobic rod, is frequently observed in subgingival polymicrobial biofilms from adults with chronic periodontitis. Peptidases in periodontopathic bacteria are considered to function as etiological reagents. Prevotella intermedia OMA14 cells abundantly express an unidentified cysteine peptidase specific for Arg-4-methycoumaryl-7-amide (MCA). BAU17746 (locus tag, PIOMA14_I_1238) and BAU18827 (locus tag, PIOMA14_II_0322) emerged as candidates of this peptidase from the substrate specificity and sequence similarity with C69-family Streptococcus gordonii Arg-aminopeptidase. The recombinant form of the former solely exhibited hydrolyzing activity toward Arg-MCA, and BAU17746 possesses a 26.6% amino acid identity with the C69-family Lactobacillus helveticus dipeptidase A. It was found that BAU17746 as well as L. helveticus dipeptidase A was a P1-position Arg-specific dipeptidase A, although the L. helveticus entity, a representative of the C69 family, had been reported to be specific for Leu and Phe. The full-length form of BAU17746 was intramolecularly processed to a mature form carrying the N-terminus of Cys15. In conclusion, the marked Arg-MCA-hydrolyzing activity in Pre. intermedia was mediated by BAU17746 belonging to the C69-family dipeptidase A, in which the mature form carries an essential cysteine at the N-terminus.

Palmitoylation of Hepatitis C Virus NS2 Regulates Its Subcellular Localization and NS2-NS3 Autocleavage.

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly.IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.

Deletion of a 19-Amino-Acid Region in Clostridioides difficile TcdB2 Results in Spontaneous Autoprocessing and Reduced Cell Binding and Provides a Nontoxic Immunogen for Vaccination.

Clostridioides difficile toxin B (TcdB) is an intracellular toxin responsible for many of the pathologies of C. difficile infection. The two variant forms of TcdB (TcdB1 and TcdB2) share 92% sequence identity but have reported differences in rates of cell entry, autoprocessing, and overall toxicity. This 2,366-amino-acid, multidomain bacterial toxin glucosylates and inactivates small GTPases in the cytosol of target cells, ultimately leading to cell death. Successful cell entry and intoxication by TcdB are known to involve various conformational changes in the protein, including a proteolytic autoprocessing event. Previous studies found that amino acids 1753 to 1852 influence the conformational states of the proximal carboxy-terminal domain of TcdB and could contribute to differences between TcdB1 and TcdB2. In the current study, a combination of approaches was used to identify sequences within the region from amino acids 1753 to 1852 that influence the conformational integrity and cytotoxicity of TcdB2. Four deletion mutants with reduced cytotoxicity were identified, while one mutant, TcdB2Δ1769-1787, exhibited no detectable cytotoxicity. TcdB2Δ1769-1787 underwent spontaneous autoprocessing and was unable to interact with CHO-K1 or HeLa cells, suggesting a potential change in the conformation of the mutant protein. Despite the putative alteration in structural stability, vaccination with TcdB2Δ1769-1787 induced a TcdB2-neutralizing antibody response and protected against C. difficile disease in a mouse model. These findings indicate that the 19-amino-acid region spanning residues 1769 to 1787 in TcdB2 is crucial to cytotoxicity and the structural regulation of autoprocessing and that TcdB2Δ1769-1787 is a promising candidate for vaccination.

Expression, purification and characterisation of Trypanosoma congolense metacaspase 5 (TcoMCA5) - a potential drug target for animal African trypanosomiasis.

The metacaspases (MCAs) are attractive drug targets for the treatment of African trypanosomiasis as they are not found in the metazoan kingdom and their action has been implicated in cell cycle and cell death pathways in kinetoplastid parasites. Here we report the biochemical characterisation of MCA5 from T. congolense. Upon recombinant expression in E. coli, autoprocessing is evident, and MCA5 further autoprocesses when purified using nickel affinity chromatography, which we term nickel-induced over autoprocessing. When both the catalytic His and Cys residues were mutated (TcoMCA5H147A/C202G), no nickel-induced over autoprocessing was observed and was enzymatically active, suggesting the existence of a secondary catalytic Cys residue, Cys81. Immunoaffinity purification of native TcoMCA5 from the total parasite proteins was achieved using chicken anti-TcoMCA5 IgY antibodies. The full length native TcoMCA5 and the autoprocessed products of recombinant TcoMCA5H147A/C202G were shown to possess gelatinolytic activity, the first report for that of a MCA. Both the native and recombinant enzyme were calcium independent, had a preference for Arg over Lys at the P1 site and were active over a pH range between 6.5 and 9. Partial inhibition (23%) of enzymatic activity was only achieved with leupeptin and antipain. These findings are the first step in the biochemical characterisation of the single copy MCAs from animal infective trypanosomes towards the design of novel trypanocides.

In vitro functional characterization of the novel DHH mutations p.(Asn337Lysfs*24) and p.(Glu212Lys) associated with gonadal dysgenesis.

In humans, mutations of Desert Hedgehog gene (DHH) have been described in patients with 46,XY gonadal dysgenesis (GD), associated or not with polyneuropathy. In this study, we describe two patients diagnosed with GD, both harboring novel DHH compound heterozygous mutations p.[Tyr176*];[Asn337Lysfs*24] and p.[Tyr176*];[Glu212Lys]. To investigate the functional consequences of p.(Asn337Lysfs*24) and p.(Glu212Lys) mutations, located within the C-terminal part of DHh on auto-processing, we performed in vitro cleavage assays of these proteins in comparison with Drosophila melanogaster Hedgehog (Hh). We found that p.(Glu212Lys) mutation retained 50% of its activity and led to a partially abolished DHh auto-processing. In contrast, p.(Asn337Lysfs*24) mutation resulted in a complete absence of auto-proteolysis. Furthermore, we found a different auto-processing profile between Drosophila Hh and human DHh, which suggests differences in the processing mechanism between the two species. Review of the literature shows that proven polyneuropathy and GD is associated with complete disruption of DHh-N, whereas disruption of the DHh auto-processing is only described with GD. We propose a model that may explain the differences between Schwann and Leydig cell development by autocrine versus paracrine DHh signaling. To our knowledge, this is the first study investigating the effect of DHH mutations on DHh in vitro auto-processing.

The maturation mechanism of γ-glutamyl transpeptidases: Insights from the crystal structure of a precursor mimic of the enzyme from Bacillus licheniformis and from site-directed mutagenesis studies.

γ-Glutamyl transpeptidases (γ-GTs) are members of N-terminal nucleophile hydrolase superfamily. They are synthetized as single-chain precursors, which are then cleaved to form mature enzymes. Basic aspects of autocatalytic processing of these pro-enzymes are still unknown. Here we describe the X-ray structure of the precursor mimic of Bacillus licheniformis γ-GT (BlGT), obtained by mutating catalytically important threonine to alanine (T399A-BlGT), and report results of autoprocessing of mutants of His401, Thr415, Thr417, Glu419 and Arg571. Data suggest that Thr417 is in a competent position to activate the catalytic threonine (Thr399) for nucleophilic attack of the scissile peptide bond and that Thr415 plays a major role in assisting the process. On the basis of these new structural results, a possible mechanism of autoprocessing is proposed. This mechanism, which guesses the existence of a six-membered transition state involving one carbonyl and two hydroxyl groups, is in agreement with all the available experimental data collected on γ-GTs from different species and with our new Ala-scanning mutagenesis data.

Zinc inhibits Hedgehog autoprocessing: linking zinc deficiency with Hedgehog activation.

Zinc is an essential trace element with wide-ranging biological functions, whereas the Hedgehog (Hh) signaling pathway plays crucial roles in both development and disease. Here we show that there is a mechanistic link between zinc and Hh signaling. The upstream activator of Hh signaling, the Hh ligand, originates from Hh autoprocessing, which converts the Hh precursor protein to the Hh ligand. In an in vitro Hh autoprocessing assay we show that zinc inhibits Hh autoprocessing with a Ki of 2 µm. We then demonstrate that zinc inhibits Hh autoprocessing in a cellular environment with experiments in primary rat astrocyte culture. Solution NMR reveals that zinc binds the active site residues of the Hh autoprocessing domain to inhibit autoprocessing, and isothermal titration calorimetry provided the thermodynamics of the binding. In normal physiology, zinc likely acts as a negative regulator of Hh autoprocessing and inhibits the generation of Hh ligand and Hh signaling. In many diseases, zinc deficiency and elevated level of Hh ligand co-exist, including prostate cancer, lung cancer, ovarian cancer, and autism. Our data suggest a causal relationship between zinc deficiency and the overproduction of Hh ligand.

Masking autoprocessing of Clostridium difficile toxin A by the C-terminus combined repetitive oligo peptides.

Clostridium difficile toxin A and B (TcdA and TcdB) are the major virulence factors of the bacterium, both of which consist of two enzymatic domains: an effector glucosyltransferase domain (GTD) and a cysteine protease domain (CPD) responsible for autocleavage and release of GTD. Although the CPDs from both toxins share a similar structure and mechanism of hexakisphosphate (InsP6)-induced activation, TcdA is substantially less sensitive to the autocleavage as compared with TcdB. In this study, we provided evidence of inter-domain regulation of CPD activity of TcdA and its autoprocessing. The C-terminus combined repetitive oligo peptides (CROPs) of TcdA reduced the accessibility of TcdB CPD to its substrate in a chimeric toxin TxB-Ar, consequently blocking autoprocessing. Moreover, interference of antibodies with the CROPs of full-length TcdA efficiently enhanced its GTD release. In conclusion, by utilizing chimeric toxins and specific antibodies, we identified that the CROPs of TcdA plays a crucial role in controlling the InsP6-mediated activation of CPD and autocleavage of GTD. Our data provides insights on the molecular mode of action of the C. difficile toxins.

Active site targeting of hedgehog precursor protein with phenylarsine oxide.

Hedgehog proteins, signaling molecules implicated in human embryo development and cancer, can be inhibited at the stage of autoprocessing by the trivalent arsenical phenyl arsine oxide (PhAs(III) ). The interaction (apparent Ki , 4 × 10(-7) M) is characterized by an optical binding assay and by NMR spectroscopy. PhAs(III) appears to be the first validated inhibitor of hedgehog autoprocessing, which is unique to hedgehog proteins and essential for biological activity.

Paracatalytic induction: Subverting specificity in hedgehog protein autoprocessing with small molecules.

Paracatalytic inducers are antagonists that shift the specificity of biological catalysts, resulting in non-native transformations. In this Chapter we describe methods to discover paracatalytic inducers of Hedgehog (Hh) protein autoprocessing. Native autoprocessing uses cholesterol as a substrate nucleophile to assist in cleaving an internal peptide bond within a precursor form of Hh. This unusual reaction is brought about by HhC, an enzymatic domain that resides within the C-terminal region of Hh precursor proteins. Recently, we reported paracatalytic inducers as a novel class of Hh autoprocessing antagonists. These small molecules bind HhC and tilt the substrate specificity away from cholesterol in favor of solvent water. The resulting cholesterol-independent autoproteolysis of the Hh precursor generates a non-native Hh side product with substantially reduced biological signaling activity. Protocols are provided for in vitro FRET-based and in-cell bioluminescence assays to discover and characterize paracatalytic inducers of Drosophila and human hedgehog protein autoprocessing, respectively.

A standard data format for 3DED/MicroED.

Electron diffraction from three dimensional crystals, as a technique for solving molecular structures, is rapidly increasing in popularity. The development of methodology and software has borrowed, to great effect, from macromolecular X-ray crystallography. However, standardization lags behind the development of the technique, and practitioners are forced to work with inadequate data formats that are unable to capture a full description of their experiments. This creates obstacles that are increasingly difficult to overcome as experiments become ever faster and the need for data autoprocessing becomes more pressing. We present a data format standard based on best practice from macromolecular crystallography and demonstrate how the adoption of this standard enabled autoprocessing of datasets collected with a high-throughput detector system.

Biochemical and Bioinformatic Characterization of Type II Metacaspase Protein (TaeMCAII) from Wheat.

The biochemical analysis and homology modeling of a tertiary structure of a cereal type II metacaspase protein from wheat (Triticum aestivum), TaeMCAII, are presented. The biochemical characterization of synthetic oligopeptides and protease inhibitors of Escherichia coli-produced and purified recombinant TaeMCAII revealed that this metacaspase protein, similar to other known plant metacaspases, is an arginine/lysine-specific cysteine protease. Thus, a model of a plant type II metacaspase structure based on newly identified putative metacaspase-like template was proposed. Homology modeling of the TaeMCAII active site tertiary structure showed two cysteine residues, Cys140 and 23, in close proximity to the catalytic histidine, most likely participating in proton exchange during the catalytic process. The autoprocessing that leads to activation of TaeMCAII was highly dependent on Cys140. TaeMCAII required high levels of calcium ions for activity, which could indicate its involvement in stress signaling pathways connected to programmed cell death.

The C-Terminal Domain of RNase H and the C-Terminus Amino Acid Residue Regulate Virus Release and Autoprocessing of a Defective HIV-1 Possessing M50I and V151I Changes in Integrase.

Previously, we reported that an HIV-1 variant containing Met-to-Ile change at codon 50 and Val-to-Ile mutation at codon 151 of integrase (IN), HIV(IN:M50I/V151I), was an impaired virus. Despite the mutations being in IN, the virus release was significantly suppressed (p < 0.0001) and the initiation of autoprocessing was inhibited; the mechanism of the defect remains unknown. In the current study, we attempted to identify the critical domains or amino acid (aa) residue(s) that promote defects in HIV(IN:M50I/V151I), using a series of variants, including truncated or aa-substituted RNase H (RH) or IN. The results demonstrated that virus release and the initiation of autoprocessing were regulated by the C-terminal domains (CTDs) of RH and IN. Further studies illustrated that Asp at codon 109 of RH CTD and Asp at the C terminus of IN induces the defect. This result indicated that the CTDs of RH and IN in GagPol and particular aa positions in RH and IN regulated the virus release and the initiation of autoprocessing, and these sites could be potential targets for the development of new therapies.

Hedgehog Autoprocessing: From Structural Mechanisms to Drug Discovery.

Hedgehog (Hh) signaling plays pivotal roles in embryonic development. In adults, Hh signaling is mostly turned off but its abnormal activation is involved in many types of cancer. Hh signaling is initiated by the Hh ligand, generated from the Hh precursor by a specialized autocatalytic process called Hh autoprocessing. The Hh precursor consists of an N-terminal signaling domain (HhN) and a C-terminal autoprocessing domain (HhC). During Hh autoprocessing, the precursor is cleaved between N- and C-terminal domain followed by the covalent ligation of cholesterol to the last residue of HhN, which subsequently leads to the generation of Hh ligand for Hh signaling. Hh autoprocessing is at the origin of canonical Hh signaling and precedes all downstream signaling events. Mutations in the catalytic residues in HhC can lead to congenital defects such as holoprosencephaly (HPE). The aim of this review is to provide an in-depth summary of the progresses and challenges towards an atomic level understanding of the structural mechanisms of Hh autoprocessing. We also discuss drug discovery efforts to inhibit Hh autoprocessing as a new direction in cancer therapy.

Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-Target Considerations.

Protein splicing is a post-translational process by which an intervening polypeptide, or intein, catalyzes its own removal from the flanking polypeptides, or exteins, concomitant with extein ligation. Although inteins are highly abundant in the microbial world, including within several human pathogens, they are absent in the genomes of metazoans. As protein splicing is required to permit function of essential proteins within pathogens, inteins represent attractive antimicrobial targets. Here we review key proteins interrupted by inteins in pathogenic mycobacteria and fungi, exciting discoveries that provide proof of concept that intein activity can be inhibited and that this inhibition has an effect on the host organism's fitness, and bioanalytical methods that have been used to screen for intein activity. We also consider potential off-target inhibition of hedgehog signaling, given the similarity in structure and function of inteins and hedgehog autoprocessing domains.

A Combination of M50I and V151I Polymorphic Mutations in HIV-1 Subtype B Integrase Results in Defects in Autoprocessing.

We have recently reported that a recombinant HIV-1NL4.3 containing Met-to-Ile change at codon 50 of integrase (IN) (IN:M50I) exhibits suppression of the virus release below 0.5% of WT HIV, and the released viral particles are replication-incompetent due to defects in Gag/GagPol processing by inhibition of the initiation of autoprocessing of GagPol polyproteins in the virions and leads to replication-incompetent viruses. The coexisting Ser-to-Asn change at codon 17 of IN or Asn-to-Ser mutation at codon 79 of RNaseH (RH) compensated the defective IN:M50I phenotype, suggesting that both IN and RH regulate an HIV infectability. In the current study, to elucidate a distribution of the three mutations during anti-retroviral therapy among patients, we performed a population analysis using 529 plasma virus RNA sequences obtained through the MiSeq. The result demonstrated that 14 plasma HIVs contained IN:M50I without the compensatory mutations. Comparing the sequences of the 14 viruses with that of the defective virus illustrated that only Val-to-Ile change at codon 151 of IN (IN:V151I) existed in the recombinant virus. This IN:V151I is known as a polymorphic mutation and was derived from HIVNL4.3 backbone. A back-mutation at 151 from Ile-to-Val in the defective virus recovered HIV replication capability, and Western Blotting assay displayed that the back-mutation restored Gag/GagPol processing in viral particles. These results demonstrate that a combination of IN:M50I and IN:V151I mutations, but not IN:M50I alone, produces a defective virus.

Human L­asparaginase: Acquiring knowledge of its activation (Review).

L­asparaginase enzymes have been a vital component of acute lymphoblastic leukemia therapy for >40 years. L­asparaginase acts by depleting plasma L­asparagine, which is essential to the survival of leukemia cells. In contrast to normal cells, tumor cells cannot synthesize L­asparagine and thus depend on its external uptake for growth. Currently, three bacterial L­asparaginases are used in therapy; however, they are associated with severe side­effects related to high toxicity and immunogenicity. The introduction of human L­asparaginase­like protein 1 in acute lymphoblastic leukemia treatment would avoid the problems caused by the bacterial enzymes; however, a major difficulty in the therapeutic use of the human enzyme comes from the fact that human L­asparaginase must be activated through an autoprocessing step, which is a low­efficiency process in vitro that results in reduced enzymatic activity. The present review article aimed to contribute to the understanding of the enzyme self­activation process and focuses on the efforts made for the development of a therapeutic variant of human L­asparaginase.