Phosphorylation-Induced Mechanical Regulation of Intrinsically Disordered Neurofilament Proteins (original) (raw)
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The journal of physical chemistry. B, 2015
Microtubules and actin filaments are biopolymer molecules that are major components of cytoskeleton networks in biological cells. They play important roles in supporting fundamental cellular processes such as cell division, signalling, locomotion and intracellular transport. In cells cytoskeleton proteins function under nonequilibrium conditions that are powered by hydrolysis of adenosine triphosphate (ATP) or guanosine triphosphate (GTP) molecules attached to them. Although these biopolymers are critically important for all cellular processes, the mechanisms that govern their complex dynamics and force generation remain not well explained. One of the most difficult fundamental issues is to understand how different components of cytoskeleton proteins interact together. We develop an approximate theoretical approach for analyzing complex processes in cytoskeleton proteins that takes into account the multifilament structure, lateral interactions between parallel protofilaments, and th...
Frontiers in Molecular Neuroscience
Cellular localization, assembly and abnormal aggregation of neurofilaments depend on phosphorylation. Pathological processes associated with neurodegeneration exhibit aberrant accumulation of microtubule associated aggregated forms of hyperphosphorylated neuronal protein tau in cell bodies. These processes are critical for the disease progression in patients suffering from Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. In healthy cells, tau is localized in axons. Topographic regulation suggests that whereas the sites of synthesis of kinases and neurofilaments are the cell bodies, and sites of their functional assemblies are axons, phosphorylation/dephosphorylation are the key processes that arrange the molecules at their precise locations. Phosphorylation sites in the dynamic developmental and degenerative processes differ. Not all these processes are well understood. New advancements identify epigenetic factors involved in AD which account for the influence of age-related environment/genome interactions leading to the disease. Progress in proteomics highlights previously found major proteins and adds more to the list of those involved in AD. New key elements of specificity provide determinants of molecular recognition important for the assembly of macromolecular complexes. In this review, we discuss aberrant spatial distribution of neuronal polypeptides observed in neuropathies: aggregation, association with proteins of the neuronal cytoskeleton, and phosphorylation dependent dynamics. Particularly, we emphasize recent advancements in understanding the function and determinants of specific association of molecules involved in Alzheimer's disease with respect to the topographic regulation of phosphorylation in neuronal cytoskeleton and implications for the design of new therapies. Further, we address the role of various filament systems in maintenance of the shape, rigidity and dynamics of the cytoskeleton.
Modulation of repulsive forces between neurofilaments by sidearm phosphorylation
Biochemical and Biophysical Research Communications, 2004
Recent studies have advanced the notion that the axonal organization of neurofilaments (NFs) is based on mutual steric repulsion between the unstructured ''sidearm'' domains of adjacent NFs. Here, we present experimental evidence that these repulsive forces are modulated by the degree of sidearm phosphorylation. When NFs are sedimented into a gelatinous pellet, pellet volume falls with increasing ionic strength and enzymatic dephosphorylation; sedimentation of phosphorylated NFs in the presence of divalent cations also dramatically reduces pellet volume. Further, atomic force microscopy imaging of isolated mammalian NFs reveals robust exclusion of colloidal particles from the NF backbone that is reduced at high ionic strength and attenuated when the filaments are enzymatically dephosphorylated. Phosphate-phosphate repulsion on the NF sidearm appears to modulate NF excluded volume in a graded fashion, thereby controlling axonal NF organization through interfilament forces.
Nature Materials, 2010
NF-H (high), assembled to form mature filaments with protruding unstructured C-terminus side arms 1-5 . Liquidcrystal gel networks of side-arm-mediated neurofilament assemblies have a key role in the mechanical stability of neuronal processes. Disruptions of the neurofilament network, owing to neurofilament over-accumulation or incorrect sidearm interactions, are a hallmark of motor-neuron diseases including amyotrophic lateral sclerosis 3-9 . Using synchrotron X-ray scattering, we report on a direct measurement of forces in reconstituted neurofilament gels under osmotic pressure (P). With increasing pressure near physiological salt and average phosphorylation conditions, NF-LMH, comprising the three subunits near in vivo composition, or NF-LH gels, undergo for P > P c ≈ 10 kPa, an abrupt non-reversible gel-expanded to gel-condensed transition. The transition indicates side-armmediated attractions between neurofilaments consistent with an electrostatic model of interpenetrating chains. In contrast, NF-LM gels remain in a collapsed state for P < P c and transition to the gel-condensed state at P > P c . These findings, which delineate the distinct roles of NF-M and NF-H in regulating neurofilament interactions, shed light on possible mechanisms for disruptions of optimal mechanical network properties.
Protein flexibility and electrostatic interactions
IBM Journal of Research and Development, 2000
In this paper we address the interrelationship between electrostatic interactions and protein flexibility. Protein flexibility may imply small conformational changes due to the movement of backbone and of side-chain atoms, and/or large-scale molecular motions, in which parts of the protein move as rigid bodies with respect to one another. In particular, we focus on oppositely charged side chains interacting to form salt bridges. The paper has two parts: In the first, we illustrate that the majority of the salt bridges are formed within the independently folding, compact hydrophobic units (HFUs) of the proteins. On the other hand, salt bridges forming across the HFUs, where one amino acid resides in one HFU and its pairing "spouse" in a second, appear to be avoided. In the second part of the paper, we address electrostatic interactions in conformational isomers around the native state. We pick the protein Cyanovirin-N as an example. We show that salt bridges and ion pairs, with less optimal geometry, often interconvert between being stabilizing and destabilizing. We conclude that the stabilizing, or destabilizing, contribution of a salt bridge to protein structure is conformer-dependent.
Effects of phosphorylation of the neurofilament L protein on filamentous structures
Cell Regulation, 1990
Effects of phosphorylation of the neurofilament L protein (NF-L) on the reassembly system were studied by both sedimentation experiments and low-angle rotary shadowing. Bovine spinal cord NF-L was phosphorylated with 3-4 mol/mol protein by either the catalytic subunit of cAMP-dependent protein kinase or protein kinase C. Phosphorylated NF-L could not assemble into filaments. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C inhibited the same step of the reassembly process. Phosphorylated NF-L remained as an 8-chain complex even in favorable conditions for reassembly. The extent of the effect of phosphorylation on the filamentous structure of NF-L was also investigated by using the catalytic subunit of cAMP-dependent protein kinase. The amount of unassembled NF-L increased linearly with increased phosphorylation in the sedimentation experiments. Structural observations indicated that 1 or 2 mol of phosphorylation is enough to inhibit reassembly and to induc...
Structures and Interactions in Neurofilament: Gel Expanded To Gel Condensed Transition
2010
NF-H (high), assembled to form mature filaments with protruding unstructured C-terminus side arms 1-5 . Liquidcrystal gel networks of side-arm-mediated neurofilament assemblies have a key role in the mechanical stability of neuronal processes. Disruptions of the neurofilament network, owing to neurofilament over-accumulation or incorrect sidearm interactions, are a hallmark of motor-neuron diseases including amyotrophic lateral sclerosis 3-9 . Using synchrotron X-ray scattering, we report on a direct measurement of forces in reconstituted neurofilament gels under osmotic pressure (P). With increasing pressure near physiological salt and average phosphorylation conditions, NF-LMH, comprising the three subunits near in vivo composition, or NF-LH gels, undergo for P > P c ≈ 10 kPa, an abrupt non-reversible gel-expanded to gel-condensed transition. The transition indicates side-armmediated attractions between neurofilaments consistent with an electrostatic model of interpenetrating chains. In contrast, NF-LM gels remain in a collapsed state for P < P c and transition to the gel-condensed state at P > P c . These findings, which delineate the distinct roles of NF-M and NF-H in regulating neurofilament interactions, shed light on possible mechanisms for disruptions of optimal mechanical network properties.
Molecular architecture of the neurofilament
Journal of Molecular Biology, 1990
Using the smallest subunit (NF-L) of a neurofilament and a glial fibrillary acidic protein, the subunit arrangement in intermediate filaments was studied by low-angle rotary shadowing. NF-L formed a pair of 70 to 80 nm rods in a low ionic strength solution at pH 6.8. Two 70 to 80 nm rods appeared to associate in an antiparallel manner with an overlap of about 55 nm, almost the same length as the a-helix-rich central rod domain of intermediate filament proteins. The overlap extended for three-beaded segments, present at 22 nm intervals along the pairs of rods. The observations that (1) 70 to 80 nm rods were a predominant structure in a low ionic strength solution at pH 85, (2) the molecular weights of the rod and the pair were measured by sedimentation equilibrium as 190,000 and 37,000 respectively, and (3) the rods formed from the trypsin-digested NF-L had a length of about 47 nm, indicated that the 70 to 80 nm rod is the four-chain complex and the pair of rods is the eight-chain complex. Similar structures were observed with glial fibrillary acidic protein, indicating that these oligomeric structures are common to other intermediate filament proteins. NF-I, assembled into short intermediate-sized filaments upon dialysis against a low-salt solution containing 1 to 2 mM-MgCl, at 4°C. The majority of these short filaments possessed four or five-beaded segments, suggesting that the pair of rods were arranged in a half-staggered fashion in neurofilaments. On the basis of these observations, we propose the following model for the intermediate filament subunit arrangement. (1) The four-chain complex is the 70 to 80 nm rod, in which two coiled-coil molecules align in parallel and in register.