N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. Here, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile. By solving the NAALADase L x-ray structure, we provide the first experimental evidence that it is a zinc-dependent metallopeptidase with a catalytic mechanism similar to that of glutamate carboxypeptidase II yet distinct substrate specificity. A proteome-based assay revealed that the NAALADL1 gene product possesses previously unrecognized aminopeptidase activity but no carboxy- or endopeptidase activity. These findings were corroborated by site-directed mutagenesis and identification of bestatin as a potent inhibitor of the enzyme. Analysis of NAALADL1 gene expression at both the mRNA and protein levels revealed the small intestine as the major site of protein expression and points toward extensive alternative splicing of the NAALADL1 gene transcript. Taken together, our data imply that the NAALADL1 gene product's primary physiological function is associated with the final stages of protein/peptide digestion and absorption in the human digestive system. Based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP).

• Klíčová slova
• Aminopeptidase, DPP IV Activity, Human Ileal Aminopeptidase, Intestinal Metabolism, Metalloprotease, Molecular Evolution, PICS, Protein Degradation, X-ray Crystallography,
• MeSH
• dipeptidylpeptidasa 4 metabolismus   MeSH
• endopeptidasy metabolismus   MeSH
• glutamátkarboxypeptidasa II chemie   genetika   metabolismus   MeSH
• krysa rodu Rattus MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• regulace genové exprese enzymů MeSH
• sekvence aminokyselin MeSH
• střeva enzymologie   MeSH
• terciární struktura proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• dipeptidylpeptidasa 4 MeSH
• endopeptidasy MeSH
• glutamátkarboxypeptidasa II MeSH

High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of -32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 μM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.

• MeSH
• anilin-naftalen sulfonáty metabolismus   MeSH
• atmosférický tlak MeSH
• darunavir metabolismus   MeSH
• dimerizace MeSH
• fluorescenční spektrometrie MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• inhibitory HIV-proteasy metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• sbalování proteinů * MeSH
• stabilita proteinů účinky léků   MeSH
• termodynamika MeSH
• tryptofan metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-anilino-8-naphthalenesulfonate MeSH Prohlížeč
• anilin-naftalen sulfonáty MeSH
• darunavir MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Prohlížeč
• tryptofan MeSH

Insertions in the protease (PR) region of human immunodeficiency virus (HIV) represent an interesting mechanism of antiviral resistance against HIV PR inhibitors (PIs). Here, we demonstrate the improved ability of a phosphonate-containing experimental HIV PI, GS-8374, relative to that of other PIs, to effectively inhibit patient-derived recombinant HIV strains bearing PR insertions and numerous other mutations. We correlate enzyme inhibition with the catalytic activities of corresponding recombinant PRs in vitro and provide a biochemical and structural analysis of the PR-inhibitor complex.

• MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 chemie   účinky léků   enzymologie   genetika   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   farmakologie   MeSH
• inzerční mutageneze * MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• organofosfonáty analýza   MeSH
• sekvence aminokyselin MeSH
• vazebná místa MeSH
• virová léková rezistence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• organofosfonáty MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Prohlížeč

During the last few decades, the treatment of HIV-infected patients by highly active antiretroviral therapy, including protease inhibitors (PIs), has become standard. Here, we present results of analysis of a patient-derived, multiresistant HIV-1 CRF02_AG recombinant strain with a highly mutated protease (PR) coding sequence, where up to 19 coding mutations have accumulated in the PR. The results of biochemical analysis in vitro showed that the patient-derived PR is highly resistant to most of the currently used PIs and that it also exhibits very poor catalytic activity. Determination of the crystal structure revealed prominent changes in the flap elbow region and S1/S1' active site subsites. While viral loads in the patient were found to be high, the insertion of the patient-derived PR into a HIV-1 subtype B backbone resulted in reduction of infectivity by 3 orders of magnitude. Fitness compensation was not achieved by elevated polymerase (Pol) expression, but the introduction of patient-derived gag and pol sequences in a CRF02_AG backbone rescued viral infectivity to near wild-type (wt) levels. The mutations that accumulated in the vicinity of the processing sites spanning the p2/NC, NC/p1, and p6pol/PR proteins lead to much more efficient hydrolysis of corresponding peptides by patient-derived PR in comparison to the wt enzyme. This indicates a very efficient coevolution of enzyme and substrate maintaining high viral loads in vivo under constant drug pressure.

• MeSH
• buněčné linie MeSH
• genové produkty gag - virus lidské imunodeficience genetika   MeSH
• genové produkty pol - virus lidské imunodeficience genetika   MeSH
• geny gag MeSH
• geny pol MeSH
• HEK293 buňky MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 genetika   izolace a purifikace   fyziologie   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy terapeutické užití   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• peptidové fragmenty genetika   MeSH
• virová léková rezistence genetika   MeSH
• virová nálož MeSH
• vysoce aktivní antiretrovirová terapie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• gag protein p1, Human immunodeficiency virus MeSH Prohlížeč
• genové produkty gag - virus lidské imunodeficience MeSH
• genové produkty pol - virus lidské imunodeficience MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• p2 gag peptide, Human immunodeficiency virus 1 MeSH Prohlížeč
• peptidové fragmenty MeSH

The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed.

• Klíčová slova
• HAART, HIV protease, alternative inhibitors, pharmacokinetic boosting, protease dimerization, protease inhibitors, resistance development,
• Publikační typ
• časopisecké články MeSH

Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2' binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

• MeSH
• darunavir MeSH
• genové produkty env - virus lidské imunodeficience metabolismus   MeSH
• genové produkty gag - virus lidské imunodeficience metabolismus   MeSH
• HIV infekce virologie   MeSH
• HIV-1 účinky léků   izolace a purifikace   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• missense mutace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• polyproteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• sulfonamidy farmakologie   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virová léková rezistence * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• darunavir MeSH
• genové produkty env - virus lidské imunodeficience MeSH
• genové produkty gag - virus lidské imunodeficience MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Prohlížeč
• polyproteiny MeSH
• sulfonamidy MeSH