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Cleavage of bovine brain microtubule-associated protein-2 by human immunodeficiency virus proteinase
1Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville 32610-0245.
Abstract:
The high-molecular-weight dendritic cytoskeletal protein known as microtubule-associated protein (MAP)-2 displays the capacity to stimulate tubulin polymerization and to associate with microtubules. Serine proteases cleave MAP-2 into a C-terminal M(r) 28,000-35,000 microtubule-binding fragment and a larger N-terminal M(r) 240,000 projection-arm region. We now show that human immunodeficiency virus (HIV) proteinase also progressively degrades purified MAP-2 in vitro. This proteolysis reaction is characterized by transient accumulation of at least six intermediates, and most abundant of these is an M(r) 72,000 species that retains the ability to associate with taxol-stabilized microtubules. Treatment of this M(r) 72,000 species with thrombin releases the same M(r) 28,000 component as that derived from thrombin action on intact high-molecular-weight MAP-2, indicating that the viral aspartoproteinase action preferentially occurs further toward the N-terminus. The association of the M(r) 72,000 component with microtubules can be disrupted by the presence of a 21-amino acid peptide analogue of the second repeated sequence in the MAP-2 microtubule-binding region. We also studied HIV proteinase action on MAP-2 in the presence of tubulin and other MAPs that recycle with tubulin, and contrary to other published studies we found no effect of such treatment on microtubule self-assembly behavior. Cleavage of isolated MAP-2 by the HIV enzyme at high salt concentrations, followed by desalting and addition of tubulin, also resulted in microtubule assembly, albeit with slightly reduced efficiency.
Insights
Human immunodeficiency virus (HIV) proteinase degrades microtubule-associated protein (MAP)-2, generating intermediates that bind microtubules. HIV protease cleavage of MAP-2 does not affect microtubule assembly, even in the presence of tubulin.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Microtubule-associated protein (MAP)-2 is a high-molecular-weight cytoskeletal protein crucial for tubulin polymerization and microtubule association.
- Serine proteases cleave MAP-2 into distinct N-terminal and C-terminal fragments, with the C-terminal fragment binding microtubules.
Purpose of the Study:
- To investigate the in vitro degradation of MAP-2 by human immunodeficiency virus (HIV) proteinase.
- To characterize the resulting fragments and their interaction with microtubules.
- To assess the impact of HIV proteinase-mediated MAP-2 cleavage on microtubule self-assembly.
Main Methods:
- Purified MAP-2 was subjected to in vitro degradation by purified HIV proteinase.
- Proteolysis products were analyzed for intermediate accumulation and molecular weight.
- The microtubule-binding ability of degradation products was assessed using taxol-stabilized microtubules.
- The effect of HIV proteinase on MAP-2 was studied in the presence of tubulin and other MAPs.
Main Results:
- HIV proteinase progressively degrades MAP-2, forming at least six transient intermediates, with an M(r) 72,000 species being most abundant.
- The M(r) 72,000 MAP-2 fragment retains the ability to associate with microtubules.
- Thrombin cleavage of the M(r) 72,000 fragment yields the same C-terminal microtubule-binding component as thrombin cleavage of intact MAP-2, indicating preferential N-terminal cleavage by HIV proteinase.
- A peptide analogue of the MAP-2 microtubule-binding region disrupts the association of the M(r) 72,000 fragment with microtubules.
- Contrary to previous findings, HIV proteinase action on MAP-2 in the presence of tubulin and other MAPs did not affect microtubule self-assembly.
- Cleavage of isolated MAP-2 by HIV enzyme followed by addition of tubulin still resulted in microtubule assembly.
Conclusions:
- HIV proteinase effectively degrades MAP-2 in vitro, generating microtubule-binding fragments.
- The M(r) 72,000 fragment generated by HIV proteinase cleavage retains microtubule-binding capacity.
- HIV proteinase-mediated cleavage of MAP-2 does not inhibit microtubule self-assembly, even in the presence of other microtubule-associated proteins and tubulin.
