A heterotypic assembly mechanism regulates CHIP E3 ligase activity

CHIP (C‐terminus of Hsc70‐interacting protein) and its worm ortholog CHN‐1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin‐proteasome system (UPS). CHN‐1 can cooperate with UFD‐2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN‐1–UFD‐2 complex in Caenorhabditis elegans. Our data show that UFD‐2 binding promotes the cooperation between CHN‐1 and ubiquitin‐conjugating E2 enzymes by stabilizing the CHN‐1 U‐box dimer. However, HSP70/HSP‐1 chaperone outcompetes UFD‐2 for CHN‐1 binding, thereby promoting a shift to the autoinhibited CHN‐1 state by acting on a conserved residue in its U‐box domain. The interaction with UFD‐2 enables CHN‐1 to efficiently ubiquitylate and regulate S‐adenosylhomocysteinase (AHCY‐1), a key enzyme in the S‐adenosylmethionine (SAM) regeneration cycle, which is essential for SAM‐dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN‐1 and UFD‐2 in substrate ubiquitylation.     

Partial purification of neurofilament subunits from bovine brains and studies on neurofilament assembly

The 200,000-dalton neurofilament subunit (P200) and the 160,000-dalton (P160) and 78,000-dalton (P78) neurofilament subunits were partially purified from bovine brain. Intact neurofilaments were prepared by high- speed and sucrose-zone centrifugation. The crude neurofilament was solubilized in 8 M urea solution containing pyridine, formic acid, and 2-mercaptoethanol. The solubilized neurofilament was purified by carboxymethyl (CM) cellulose column and hydroxylapatite column chromatography. The P200 was purified as separate from P160 and P78, but the P160 and P78 subunits were copurified on CM cellulose, hydroxylapatite, Bio-Gel A150m, and Sephadex G-150 column chromatography. Electron microscopy of these purified neurofilament subunits revealed the P200 subunit as a globular structure, and the P160 and P78 subunits as a rod-shaped structure extending up to 120 nm with a 8- to 12-nm width. In the presence of 200 mM KCl, 15 mM MgCl2, and 1 mM ATP, the purified subunits assembled into long filaments. Under the assembly condition, P160 and P78 subunits elongated up to 500 nm, but the longer filament formation required the presence of P200 subunits. The filaments formed in vitro were of two types: long straight filaments and intertwined knobby-type filaments. From these results, we have suggested that P160 and P78 form the neurofilament backbone structure and P200 facilitates the assembly of the backbone units into longer filaments.     

Subcellular compartmentalization of phosphorylated neurofilament polypeptides in neurons

Immunocytochemistry and polyacrylamide gel electrophoresis have been used to study the distribution of phosphorylated forms of neurofilament antigens in rat brain. Immunostaining of tissue with an antisera produced against phosphatase-sensitive domains of the 200-kilodalton (kd) neurofilament polypeptide showed that phosphorylated forms of this polypeptide were present in virtually all axons and certain somata and dendrites of neurons in different brain regions. Immunoblots of whole brain homogenate or a neurofilament preparation from rat revealed that the affinity-purified anti-200-kd sera used to immunostain tissue labeled the neurofilament-associated 200-kd band in a phosphatase-sensitive manner. Fine structural analysis of this immunoreactivity in tissue showed that whenever the labeled organelle could be identified, it was a microtubule. In contrast, immunoblot analysis of twice-cycled microtubules from porcine brain revealed that microtubules in vitro did not possess the 200-kd antigen that was observed in situ. The results suggest that our antibody recognizes a phosphorylated domain on the neurofilament involved in cross-linking neurofilaments and microtubules, and that in vivo, phosphorylated epitopes of the 200-kd neurofilament polypeptide are capable of associating with microtubules.     

A CDK-related kinase regulates the length and assembly of flagella in Chlamydomonas

Little is known about how cells regulate the size of their organelles. In this study, we find that proper flagellar length control in Chlamydomonas reinhardtii requires the activity of a new member of the cyclin-dependent kinase (CDK) family, which is encoded by the LF2 (long flagella 2) gene. This novel CDK contains all of the important residues that are essential for kinase activity but lacks the cyclin-binding motif PSTAIRE. Analysis of genetic lesions in a series of lf2 mutant alleles and site-directed mutagenesis of LF2p reveals that improper flagellar length and defective flagellar assembly correlate with the extent of disruption of conserved kinase structures or residues by mutations. LF2p appears to interact with both LF1p and LF3p in the cytoplasm, as indicated by immunofluorescence localization, sucrose density gradients, cell fractionation, and yeast two-hybrid experiments. We propose that LF2p is the catalytic subunit of a regulatory kinase complex that controls flagellar length and flagellar assembly.     

Phosphorylation regulates the assembly of chloroplast import machinery

Chloroplast function depends on the translocation of cytosolically synthesized precursor proteins into the organelle. The recognition and transfer of most precursor proteins across the outer membrane depend on a membrane inserted complex. Two receptor components of this complex, Toc34 and Toc159, are GTPases, which can be phosphorylated by kinases present in the hosting membrane. However, the physiological function of phosphorylation is not yet understood in detail. It is demonstrated that both receptors are phosphorylated within their G-domains. In vitro, the phosphorylation of Toc34 disrupts both homo- and heterodimerization of the G-domains as determined using a phospho-mimicking mutant. In endogenous membranes this mutation or phosphorylation of the wild-type receptor disturbs the association of Toc34, but not of Toc159 with the translocation pore. Therefore, phosphorylation serves as an inhibitor for the association of Toc34 with other components of the complex and phosphorylation can now be discussed as a mechanism to exchange different isoforms of Toc34 within this ensemble.     

Mts1 regulates the assembly of nonmuscle myosin-IIA

The formation of myosin-II filaments is fundamental to contractile and motile processes in nonmuscle cells, and elucidating the mechanisms controlling filament assembly is essential for understanding how myosin-II rapidly responds to changing conditions within the cell. Several proteins including KRP and a novel 38 kDa protein (1, 2) have been shown to modulate filament assembly through the stabilization of myosin-II assemblies. In contrast, we demonstrate that mts1, a member of the Ca(2+)-regulated S100 family of proteins, may regulate the monomeric, unassembled state in an isoform-specific manner. Biochemical analyses demonstrate that mts1 has a 9-fold higher affinity for myosin-IIA filaments than for myosin-IIB filaments. At stoichiometric levels, mts1 inhibits the assembly of myosin-IIA monomers into filaments and promotes the disassembly of myosin-IIA filaments into monomers; however, mts1 has little effect on the assembly properties of myosin-IIB. Using a solution based-assay, we have demonstrated that mts1 binds to residues 1909-1924 of the myosin-IIA heavy chain, which is near the C-terminal tip of the alpha-helical coiled-coil. The observation that mts1 binds a linear sequence of approximately 16 amino acids is consistent with other S100 family members, which bind linear sequences of 13-22 residues in their protein targets. In addition, mts1 increases the critical monomer concentration for myosin-IIA filament assembly by approximately 11-fold. Kinetic assembly assays indicate that the elongation rate and the extent of polymerization depend on the initial myosin-IIA concentration; however, mts1 had only a small affect on the half-time for assembly and predominately affected the extent of myosin IIA polymerization. Altogether, these observations are consistent with mts1 regulating myosin IIA assembly by monomer sequestration and suggest that mts1 regulates cell shape and motility through the modulation of myosin-IIA function.     

Defective axonal transport of neurofilament proteins in neurons overexpressing peripherin

Peripherin is a type III neuronal intermediate filament detected in motor neuron inclusions of amyotrophic lateral sclerosis (ALS) patients. We previously reported that overexpression of peripherin provokes late-onset motor neuron dysfunction in transgenic mice. Here, we show that peripherin overexpression slows down axonal transport of neurofilament (NF) proteins, and that the transport defect precedes by several months the appearance of axonal spheroids in adult mice. Defective NF transport by peripherin up-regulation was further confirmed with dorsal root ganglia (DRG) neurons cultured from peripherin transgenic embryos. Immunofluorescence microscopy and western blotting revealed that excess peripherin provokes reduction in levels of hyperphosphorylated NF-H species in DRG neurites. Similarly the transport of a green fluorescent protein (GFP)-tagged NF-M, delivered by means of a lentiviral construct, was impaired in DRG neurites overexpressing peripherin. These results demonstrate that peripherin overexpression can cause defective transport of type IV NF proteins, a phenomenon that may account for the progressive formation of ALS-like spheroids in axons.     

Benzodiazepine pharmacology of cultured mammalian CNS neurons

Many neurons cultured from the embryonic mammalian central nervous system (CNS) express benzodiazepine receptors while some neurons differentiate specific transmitter phenotypes like glutamic acid decarboxylase (GAD), the synthetic enzyme for gamma-aminobutyric acid (GABA). The benzodiazepine receptors in these cultured neurons are often, if not always coupled to a practically ubiquitous GABA-mediated function, activation of Cl- ion conductance. The transmitter signal serves to inhibit neuronal excitability and is facilitated by clinically important benzodiazepines. Here we review some details regarding the pharmacological actions of benzodiazepines on membrane excitability.     

On the assembly mechanism of neurofilaments

1. Isolated individual components of the triplet of neurofilaments from bovine brain can reassemble to make filaments with a specific structure, contrary to the already reported result that NF-H and NF-M cannot make filaments alone but can only make filaments by co-polymerization with NF-L. 2. This result suggests an alternative mechanism of assembly of the neurofilaments in which individual components of the triplet make filaments first, and then these aggregate to form the intact neurofilaments. 3. The triplet components of neurofilaments are reduced to a monomeric form in 8 M urea and 1% beta-mercaptoethanol (beta-ME) solution. However, in the absence of beta-ME, a part of each component of the triplet was preserved as oligomeric forms. 4. Among them, a stable tetramer of NF-L was isolated by DE-52 column chromatography using 6 M urea solution in the absence of beta-ME. 5. This results supports the hypothesis that this tetramer can be considered as a protofilament of the neurofilament structure.     

A combinatorial semaphorin code instructs the initial steps of sensory circuit assembly in the Drosophila CNS

Longitudinal axon fascicles within the Drosophila embryonic CNS provide connections between body segments and are required for coordinated neural signaling along the anterior-posterior axis. We show here that establishment of select CNS longitudinal tracts and formation of precise mechanosensory afferent innervation to the same CNS region are coordinately regulated by the secreted semaphorins Sema-2a and Sema-2b. Both Sema-2a and Sema-2b utilize the same neuronal receptor, plexin B (PlexB), but serve distinct guidance functions. Localized Sema-2b attraction promotes the initial assembly of a subset of CNS longitudinal projections and subsequent targeting of chordotonal sensory afferent axons to these same longitudinal connectives, whereas broader Sema-2a repulsion serves to prevent aberrant innervation. In the absence of Sema-2b or PlexB, chordotonal afferent connectivity within the CNS is severely disrupted, resulting in specific larval behavioral deficits. These results reveal that distinct semaphorin-mediated guidance functions converge at PlexB and are critical for functional neural circuit assembly.     

Neuropeptide messenger plasticity in the CNS neurons following axotomy

Neuronal peptides exert neurohormonal and neurotransmitter (neuromodulator) functions in the central nervous system (CNS). Besides these functions, a group of neuropeptides may have a capacity to create cell proliferation, growth, and survival. Axotomy induces transient (1–21 d) upregulation of synthesis and gene expression of neuropeptides, such as galanin, corticotropin releasing factor, dynorphin, calcitonin gene-related peptide, vasoactive intestinal polypeptide, cholecystokinin, angiotensin II, and neuropeptide Y. These neuropeptides are colocalized with “classic” neurotransmitters (acetylcholine, aspartate, glutamate) or neurohormones (vasopressin, oxytocin) that are downregulated by axotomy in the same neuronal cells. It is more likely that neuronal cells, in response to axotomy, increase expression of neuropeptides that promote their survival and regeneration, and may downregulate substances related to their transmitter or secretory activities.     

Phosphorylation regulates assembly of the caspase-6 substrate-binding groove

Caspases, a family of apoptotic proteases, are increasingly recognized as being extensively phosphorylated, usually leading to inactivation. To date, no structural mechanism for phosphorylation-based caspase inactivation is available, although this information may be key to achieving caspase-specific inhibition. Caspase-6 has recently been implicated in neurodegenerative conditions including Huntington's and Alzheimer's diseases. A full understanding of caspase-6 regulation is crucial to caspase-6-specific inhibition. Caspase-6 is phosphorylated by ARK5 kinase at serine 257 leading to suppression of cell death via caspase-6 inhibition. Our structure of the fully inactive phosphomimetic S257D reveals that phosphorylation results in a steric clash with P201 in the L2' loop. Removal of the proline side chain alleviates the clash resulting in nearly wild-type activity levels. This phosphomimetic-mediated steric clash causes misalignment of the substrate-binding groove, preventing substrate binding. Substrate-binding loop misalignment appears to be a widely used regulatory strategy among caspases and may present a new paradigm for caspase-specific control.     

The serine protease inhibitor neuroserpin regulates the growth and maturation of hippocampal neurons through a non-inhibitory mechanism

Neuroserpin is a brain-specific serine protease inhibitor that is expressed in the developing and adult nervous system. Its expression profile led to suggestions that it played roles in neuronal growth and connectivity. In this study, we provide direct evidence to support a role for neuroserpin in axon and dendritic growth. We report that axon growth is enhanced while axon and dendrite diameter are reduced following neuroserpin treatment of hippocampal neurons. More complex effects are seen on dendritic growth and branching with neuroserpin-stimulating dendritic growth and branching in young neurons but switching to an inhibitory response in older neurons. The protease inhibitory activity of neuroserpin is not required to activate changes in neuronal morphology and a proportion of responses are modulated by an antagonist to the LRP1 receptor. Collectively, these findings support a key role for neuroserpin as a regulator of neuronal development through a non-inhibitory mechanism and suggest a basis for neuroserpin's effects on complex emotional behaviours and recent link to schizophrenia.     

Specification of dopaminergic and serotonergic neurons in the vertebrate CNS

The early specification of dopaminergic and serotonergic neurons during vertebrate CNS development relies on signals produced by a small number of organizing centers. Recent studies have characterized these early organizing centers, the manner in which they may be established, the inductive signals they produce, and candidate signaling systems that control the later development of the dopaminergic system.