Oligomeric structure of brain abundant proteins GAP-43 and BASP1.

Brain abundant proteins GAP-43 and BASP1 participate in the regulation of actin cytoskeleton dynamics in neuronal axon terminals. The proposed mechanism suggests that the proteins sequester phosphatidylinositol-4,5-diphosphate (PIP(2)) in the inner leaflet of the plasma membrane. We found that model anionic phospholipid membranes in the form of liposomes induce rapid oligomerization of GAP-43 and BASP1 proteins. Multiply charged phosphoinositides produced the most potent effect. Anionic detergent sodium dodecyl sulfate (SDS) at submicellar concentration stimulated formation of similar oligomers in solution. BASP1, but not GAP-43, also formed oligomers at sufficiently high concentration in the absence of lipids and SDS. Electron microscopy study demonstrated that the oligomers have disk-shaped or annular structure of 10-30nm in diameter. BASP1 also formed higher aggregates of linear rod-like structure, with average length of about 100nm. In outward appearance, the oligomers and linear aggregates are reminiscent of oligomers and protofibrils of amyloid proteins. Both the synthetic N-terminal peptide GAP-43(1-40) and the brain-derived fragment GAP-43-3 preserved the ability to oligomerize under the action of acidic phospholipids and SDS. On the contrary, BASP1 fragment truncated by the short N-terminal myristoylated peptide was unable to form oligomers. GAP-43 and BASP1 oligomerization can be regulated by calmodulin, which disrupts the oligomers and displaces the proteins from the membrane. We suggest that in vivo, the role of membrane-bound GAP-43 and BASP1 oligomers consists in accumulation of PIP(2) in functional clusters, which become accessible for other PIP(2)-binding proteins after dissociation of the oligomers.

High-order oligomers of intrinsically disordered brain proteins BASP1 and GAP-43 preserve the structural disorder.

Brain acid-soluble protein-1 (BASP1) and growth-associated protein-43 (GAP-43) are presynaptic membrane proteins participating in axon guidance, neuroregeneration and synaptic plasticity. They are presumed to sequester phosphatidylinositol-4,5-bisphosphate (PIP2 ) in lipid rafts. Previously we have shown that the proteins form heterogeneously sized oligomers in the presence of anionic phospholipids or SDS at submicellar concentration. BASP1 and GAP-43 are intrinsically disordered proteins (IDPs). In light of this, we investigated the structure of their oligomers. Using partial cross-linking of the oligomers with glutaraldehyde, the aggregation numbers of BASP1 and GAP-43 were estimated as 10-14 and 6-7 monomer subunits, respectively. The cross-linking pattern indicated that the subunits are circularly arranged. The circular dichroism (CD) spectra of the monomers were characteristic of coil-like IDPs showing unordered structure with a high population of polyproline-II conformation. The oligomerization was accompanied by a minor CD spectral change attributable to formation of a small amount of α-helix. The number of residues in the α-helical conformation was estimated as 13 in BASP1 and 18 in GAP-43. However, the overall structure of the oligomers remained disordered, indicating a high degree of 'fuzziness'. This was confirmed by measuring the hydrodynamic dimensions of the oligomers using polyacrylamide gradient gel electrophoresis and size-exclusion chromatography, and by assaying their sensitivity to proteolytic digestion. There is evidence that the observed α-helical folding occurs within the basic effector domains, which are presumably tethered together via anionic molecules of SDS or PIP2 . We conclude that BASP1 and GAP-43 oligomers preserve a mostly disordered structure, which may be of great importance for their function in PIP2 signaling pathway.

Natural N-terminal fragments of brain abundant myristoylated protein BASP1.

BASP1 (also known as CAP-23 and NAP-22) is a novel myristoylated calmodulin-binding protein, abundant in nerve terminals. It is considered as a signal protein participating in neurite outgrowth and synaptic plasticity. BASP1 is also present in significant amounts in kidney, testis, and lymphoid tissues. In this study, we show that BASP1 is accompanied by at least six BASP1 immunologically related proteins (BIRPs), which are present in all animal species studied (rat, bovine, human, chicken). BIRPs have lower molecular masses than that of BASP1. Similarly to BASP1, they are myristoylated. Peptide mapping and partial sequencing have shown that BIRPs represent a set of BASP1 N-terminal fragments devoid of C-terminal parts of different length. In a definite species, the same set of BASP1 fragments is present in both brain and other tissues. The sum amount of the fragments is about 50% of the BASP1 amount in a tissue. Obligatory accompanying of BASP1 by a set of specific fragments indicates that these fragments are of physiological significance.

Ion channel activity of brain abundant protein BASP1 in planar lipid bilayers.

BASP1 (also known as CAP-23 and NAP-22) is a brain abundant myristoylated protein localized at the inner surface of the presynaptic plasma membrane. Emerging evidence suggests that BASP1 is critically involved in various cellular processes, in particular, in the accumulation of phosphatidylinositol-4,5-diphosphate (PIP(2)) in lipid raft microdomains. We have recently shown that BASP1 forms heterogeneously-sized oligomers and higher aggregates with an outward similarity to oligomers and protofibrils of amyloid proteins. However, BASP1 is not known to be related to any amyloid disease. In the present study, we show that BASP1 induces single channel currents across negatively-charged planar lipid bilayers (containing phosphatidylserine or PIP(2)) bathed in 0.1-0.2 M KCl (pH 7.5). By their characteristics, BASP1 channels are similar to amyloid protein channels. BASP1 channels exhibit multiple conductance levels, in the range 10-3000 pS, with the most frequently observed conductance state of approximately 50 pS. The channels demonstrate a linear current-voltage relationship and voltage-independent kinetics of opening and closing. Their K(+) to Cl(-) permeability ratio is approximately 14, indicating that BASP1 channels are cation-selective. The ion channel activity of BASP1 is in accordance with the pore-like structure of BASP1 oligomers observed by electron microscopy on a lipid monolayer. Neuronal protein GAP-43, which is functionally related to BASP1 and also forms oligomers, elicited no ion channel currents under the conditions used in the present study. Elucidation of the physiological or pathological roles of ion channel activity of membrane-bound BASP1 oligomers will help to define the precise mechanism of amyloid protein toxicity.

M-calpain-mediated cleavage of GAP-43 near Ser41 is negatively regulated by protein kinase C, calmodulin and calpain-inhibiting fragment GAP-43-3.

Neuronal protein GAP-43 performs multiple functions in axon guidance, synaptic plasticity and regulation of neuronal death and survival. However, the molecular mechanisms of its action in these processes are poorly understood. We have shown that in axon terminals GAP-43 is a substrate for calcium-activated cysteine protease m-calpain, which participates in repulsion of axonal growth cones and induction of neuronal death. In pre-synaptic terminals in vivo, in synaptosomes, and in vitro, m-calpain cleaved GAP-43 in a small region near Ser41, on either side of this residue. In contrast, micro-calpain cleaved GAP-43 in vitro at several other sites, besides Ser41. Phosphorylation of Ser41 by protein kinase C or GAP-43 binding to calmodulin strongly suppressed GAP-43 proteolysis by m-calpain. A GAP-43 fragment, lacking about forty N-terminal residues (named GAP-43-3), was produced by m-calpain-mediated cleavage of GAP-43 and inhibited m-calpain, but not micro-calpain. This fragment prevented complete cleavage of intact GAP-43 by m-calpain as a negative feedback. GAP-43-3 also blocked m-calpain activity against casein, a model calpain substrate. This implies that GAP-43-3, which is present in axon terminals in high amount, can play important role in regulation of m-calpain activity in neurons. We suggest that GAP-43-3 and another (N-terminal) GAP-43 fragment produced by m-calpain participate in modulation of neuronal response to repulsive and apoptotic signals.