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A pH-sensitive luminal His-cluster promotes interaction of PAM with V-ATPase along the secretory and endocytic pathways of peptidergic cells.

ABSTRACT: The biosynthetic and endocytic pathways of secretory cells are characterized by progressive luminal acidification, a process which is crucial for posttranslational modifications and membrane trafficking. This progressive fall in luminal pH is mainly achieved by the vacuolar-type-H+ ATPase (V-ATPase). V-ATPases are large, evolutionarily ancient rotary proton pumps that consist of a peripheral V1 complex, which hydrolyzes ATP, and an integral membrane V0 complex, which transports protons from the cytosol into the lumen. Upon sensing the desired luminal pH, V-ATPase activity is regulated by reversible dissociation of the complex into its V1 and V0 components. Molecular details of how intraluminal pH is sensed and transmitted to the cytosol are not fully understood. Peptidylglycine ?-amidating mono-oxygenase (PAM; EC 1.14.17.3), a secretory pathway membrane enzyme which shares similar topology with two V-ATPase accessory proteins (Ac45 and prorenin receptor), has a pH-sensitive luminal linker region. Immunofluorescence and sucrose gradient analysis of peptidergic cells (AtT-20) identified distinct subcellular compartments exhibiting spatial co-occurrence of PAM and V-ATPase. In vitro binding assays demonstrated direct binding of the cytosolic domain of PAM to V1H. Blue native PAGE identified heterogeneous high-molecular weight complexes of PAM and V-ATPase. A PAM-1 mutant (PAM-1/H3A) with altered pH sensitivity had diminished ability to form high-molecular weight complexes. In addition, V-ATPase assembly status was altered in PAM-1/H3A expressing cells. Our analysis of the secretory and endocytic pathways of peptidergic cells supports the hypothesis that PAM serves as a luminal pH-sensor, regulating V-ATPase action by altering its assembly status.

SUBMITTER: Rao VK

PROVIDER: S-EPMC6395498 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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A pH-sensitive luminal His-cluster promotes interaction of PAM with V-ATPase along the secretory and endocytic pathways of peptidergic cells.

Rao Vishwanatha K VK Zavala Gerardo G Deb Roy Abhijit A Mains Richard E RE Eipper Betty A BA

Journal of cellular physiology 20181014 6

The biosynthetic and endocytic pathways of secretory cells are characterized by progressive luminal acidification, a process which is crucial for posttranslational modifications and membrane trafficking. This progressive fall in luminal pH is mainly achieved by the vacuolar-type-H<sup>+</sup> ATPase (V-ATPase). V-ATPases are large, evolutionarily ancient rotary proton pumps that consist of a peripheral V1 complex, which hydrolyzes ATP, and an integral membrane V0 complex, which transports proton ...[more]

PMID: 30317586

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Project description:Human noroviruses (NoVs) are the main cause of epidemic and sporadic gastroenteritis. Phylogenetically, noroviruses are divided into seven genogroups, with each divided into multiple genotypes. NoVs belonging to genogroup II and genotype 4 (GII.4) are globally most prevalent. Genetic diversity among the NoVs and the periodic emergence of novel strains present a challenge for the development of vaccines and antivirals to treat NoV infection. NoV protease is essential for viral replication and is an attractive target for the development of antivirals. The available structure of GI.1 protease provided a basis for the design of inhibitors targeting the active site of the protease. These inhibitors, although potent against the GI proteases, poorly inhibit the GII proteases, for which structural information is lacking. To elucidate the structural basis for this difference in the inhibitor efficiency, we determined the crystal structure of a GII.4 protease. The structure revealed significant changes in the S2 substrate-binding pocket, making it noticeably smaller, and in the active site, with the catalytic triad residues showing conformational changes. Furthermore, a conserved arginine is found inserted into the active site, interacting with the catalytic histidine and restricting substrate/inhibitor access to the S2 pocket. This interaction alters the relationships between the catalytic residues and may allow for a pH-dependent regulation of protease activity. The changes we observed in the GII.4 protease structure may explain the reduced potency of the GI-specific inhibitors against the GII protease and therefore must be taken into account when designing broadly cross-reactive antivirals against NoVs.IMPORTANCE Human noroviruses (NoVs) cause sporadic and epidemic gastroenteritis worldwide. They are divided into seven genogroups (GI to GVII), with each genogroup further divided into several genotypes. Human NoVs belonging to genogroup II and genotype 4 (GII.4) are the most prevalent. Currently, there are no vaccines or antiviral drugs available for NoV infection. The protease encoded by NoV is considered a valuable target because of its essential role in replication. NoV protease structures have only been determined for the GI genogroup. We show here that the structure of the GII.4 protease exhibits several significant changes from GI proteases, including a unique pairing of an arginine with the catalytic histidine that makes the proteolytic activity of GII.4 protease pH sensitive. A comparative analysis of NoV protease structures may provide a rational framework for structure-based drug design of broadly cross-reactive inhibitors targeting NoVs.

Dataset Information

A pH-sensitive luminal His-cluster promotes interaction of PAM with V-ATPase along the secretory and endocytic pathways of peptidergic cells.

Publications

A pH-sensitive luminal His-cluster promotes interaction of PAM with V-ATPase along the secretory and endocytic pathways of peptidergic cells.

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