Koichi Uegaki

Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survival

BackgroundProneurotrophins and mature neurotrophins elicit opposite effects via the p75 neurotrophin receptor (p75NTR) and Trk tyrosine kinase receptors, respectively; however the molecular roles of proneurotrophins in the CNS are not fully understood.ResultsBased on two rare single nucleotide polymorphisms (SNPs) of the human brain-derived neurotrophic factor (BDNF) gene, we generated R125M-, R127L- and R125M/R127L-BDNF, which have amino acid substitution(s) near the cleavage site between the pro- and mature-domain of BDNF. Western blot analyses demonstrated that these BDNF variants are poorly cleaved and result in the predominant secretion of proBDNF. Using these cleavage-resistant proBDNF (CR-proBDNF) variants, the molecular and cellular roles of proBDNF on the CNS neurons were examined. First, CR-proBDNF showed normal intracellular distribution and secretion in cultured hippocampal neurons, suggesting that inhibition of proBDNF cleavage does not affect intracellular transportation and secretion of BDNF. Second, we purified recombinant CR-proBDNF and tested its biological effects using cultured CNS neurons. Treatment with CR-proBDNF elicited apoptosis of cultured cerebellar granule neurons (CGNs), while treatment with mature BDNF (matBDNF) promoted cell survival. Third, we examined the effects of CR-proBDNF on neuronal morphology using more than 2-week cultures of basal forebrain cholinergic neurons (BFCNs) and hippocampal neurons. Interestingly, in marked contrast to the action of matBDNF, which increased the number of cholinergic fibers and hippocampal dendritic spines, CR-proBDNF dramatically reduced the number of cholinergic fibers and hippocampal dendritic spines, without affecting the survival of these neurons.ConclusionThese results suggest that proBDNF has distinct functions in different populations of CNS neurons and might be responsible for specific physiological cellular processes in the brain.

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala49 and Ile70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 °C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala49 and Ile70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.

Structure of the heterodimeric complex between CAD domains of CAD and ICAD.

We present here the structure of the complex between the CAD domain of caspase activated deoxyribonuclease (CAD) and the CAD domain of its inhibitor (ICAD), determined by nuclear magnetic resonance spectroscopy. The two domains adopt a very similar fold, which consists of an α-helix and a β-sheet, and are aligned side by side in the complex. Notably, the positive charges on the strand β2 at one end of the β-sheet of CAD and negative charges around the opposite end of the β-sheet of ICAD are paired in the complex. Point mutations of the charged amino acids at this interface, on either CAD or ICAD, prevented formation of the functional CAD–ICAD complex. This implies that the interaction between the CAD domains of CAD and ICAD is an essential step in the correct folding of CAD in the complex.

N-Terminal Phosphorylation of HP1 Promotes Its Chromatin Binding

ABSTRACT The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1α plays a central role in its targeting to chromatin. Recombinant HP1α prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1α was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1αs affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1α to H3K9me by mediating the interaction between HP1α and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1α exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1αs N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications.

Critical amino acid residues of AIP, a highly specific inhibitory peptide of calmodulin-dependent protein kinase II

The importance of the individual amino acid residues of AIP (KKALRRQEAVDAL), a highly specific inhibitor of calmodulin‐dependent protein kinase II (CaMKII), was studied. Replacement of Arg6, Gln7, or Ala9 by other amino acid residues produced a marked increase in the IC50 value. Leu4 and Val10 were also sensitive to replacement, but some hydrophobic amino acids could substitute for these residues. Although replacement of Ala3, Glu8, Ala12, and Leu13 by other residues produced no significant increase in the IC50, the substitution of Lys for Ala3 decreased the IC50. An AIP analog (KK LRRQEA DAY), in which Ala3 and Val10 were replaced with Lys and Phe, respectively, showed an IC50 value as low as 4 nM, suggesting that it is a useful tool for studying the physiological roles of CaMKII.

Improvement of the enzymatic activity of the hyperthermophilic cellulase from Pyrococcus horikoshii

A hyperthermophilic β-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type EGPh toward both CMC and crystalline cellulose (Avicel).

Tertiary Structure and Carbohydrate Recognition by the Chitin-Binding Domain of a Hyperthermophilic Chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both alpha and beta crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded beta-sheets, was composed of a typical beta-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.

BDNF pro-peptide actions facilitate hippocampal LTD and are altered by the common BDNF polymorphism Val66Met

Significance Brain-derived neurotrophic factor (BDNF) is a neurotrophin that elicits biological effects on synaptic plasticity. BDNF is initially synthesized as precursor proBDNF, and then the BDNF pro-peptide is simultaneously produced from the precursor protein. However, the physiological functions of the pro-peptide are largely unknown. Here, we demonstrate that the BDNF pro-peptide is a facilitator of hippocampal long-term depression (LTD), requiring the activation of GluN2B-containing NMDA-type receptors and the pan-neurotrophin receptor p75NTR. Second, a common BDNF polymorphism substitutes valine for methionine at amino acid position 66 (Val66Met) in the pro-peptide of BDNF and impairs memory function. Unexpectedly, the pro-peptide with Met mutation completely inhibits hippocampal LTD. These findings provide insights into the physiological role of the BDNF pro-peptide in the brain. Most growth factors are initially synthesized as precursor proteins and subsequently processed into their mature form by proteolytic cleavage, resulting in simultaneous removal of a pro-peptide. However, compared with that of mature form, the biological role of the pro-peptide is poorly understood. Here, we investigated the biological role of the pro-peptide of brain-derived neurotrophic factor (BDNF) and first showed that the pro-peptide is expressed and secreted in hippocampal tissues and cultures, respectively. Interestingly, we found that the BDNF pro-peptide directly facilitates hippocampal long-term depression (LTD), requiring the activation of GluN2B-containing NMDA receptors and the pan-neurotrophin receptor p75NTR. The BDNF pro-peptide also enhances NMDA-induced α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor endocytosis, a mechanism crucial for LTD expression. Thus, the BDNF pro-peptide is involved in synaptic plasticity that regulates a mechanism responsible for promoting LTD. The well-known BDNF polymorphism valine for methionine at amino acid position 66 (Val66Met) affects human memory function. Here, the BDNF pro-peptide with Met mutation completely inhibits hippocampal LTD. These findings demonstrate functional roles for the BDNF pro-peptide and a naturally occurring human BDNF polymorphism in hippocampal synaptic depression.

Intrinsic nucleic Acid-binding activity of chp1 chromodomain is required for heterochromatic gene silencing

Centromeric heterochromatin assembly in fission yeast requires the RNAi pathway. Chp1, a chromodomain (CD) protein, forms the Ago1-containing RNA-induced transcriptional silencing (RITS) complex and recruits siRNA-bound RITS to methylated histone H3 lysine 9 (H3K9me) via its CD. Here, we show that the CD of Chp1 (Chp1-CD) possesses unique nucleic acid-binding activities that are essential for heterochromatic gene silencing. Detailed electrophoretic-mobility shift analyses demonstrated that Chp1 binds to RNA via the CD in addition to its central RNA-recognition motif. Interestingly, robust RNA- and DNA-binding activity of Chp1-CD was strongly enhanced when it was bound to H3K9me, which was revealed to involve a positively charged domain within the Chp1-CD by structural analyses. These results demonstrate a role for the CD that provides a link between RNA, DNA, and methylated histone tails to ensure heterochromatic gene silencing.

Kinetic and crystallographic analyses of the catalytic domain of chitinase from Pyrococcus furiosus- the role of conserved residues in the active site.

The hyperthermostable chitinase from the hyperthermophilic archaeon Pyrococcus furiosus has a unique multidomain structure containing two chitin‐binding domains and two catalytic domains, and exhibits strong crystalline chitin hydrolyzing activity at high temperature. In order to investigate the structure–function relationship of this chitinase, we analyzed one of the catalytic domains (AD2) using mutational and kinetic approaches, and determined the crystal structure of AD2 complexed with chito‐oligosaccharide substrate. Kinetic studies showed that, among the acidic residues in the signature sequence of family 18 chitinases (DXDXE motif), the second Asp (D2) and Glu (E) residues play critical roles in the catalysis of archaeal chitinase. Crystallographic analyses showed that the side‐chain of the catalytic proton‐donating E residue is restrained into the favorable conformer for proton donation by a hydrogen bond interaction with the adjacent D2 residue. The comparison of active site conformations of family 18 chitinases provides a new criterion for the subclassification of family 18 chitinase based on the conformational change of the D2 residue.