Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Dynein mutations associated with hereditary motor neuropathies impair mitochondrial morphology and function with age.

Mutations in the DYNC1H1 gene encoding for dynein heavy chain cause two closely related human motor neuropathies, dominant spinal muscular atrophy with lower extremity predominance (SMA-LED) and axonal Charcot-Marie-Tooth (CMT) disease, and lead to sensory neuropathy and striatal atrophy in mutant mice. Dynein is the molecular motor carrying mitochondria retrogradely on microtubules, yet the consequences of dynein mutations on mitochondrial physiology have not been explored. Here, we show that mouse fibroblasts bearing heterozygous or homozygous point mutation in Dync1h1, similar to human mutations, show profoundly abnormal mitochondrial morphology associated with the loss of mitofusin 1. Furthermore, heterozygous Dync1h1 mutant mice display progressive mitochondrial dysfunction in muscle and mitochondria progressively increase in size and invade sarcomeres. As a likely consequence of systemic mitochondrial dysfunction, Dync1h1 mutant mice develop hyperinsulinemia and hyperglycemia and progress to glucose intolerance with age. Similar defects in mitochondrial morphology and mitofusin levels are observed in fibroblasts from patients with SMA-LED. Last, we show that Dync1h1 mutant fibroblasts show impaired perinuclear clustering of mitochondria in response to mitochondrial uncoupling. Our results show that dynein function is required for the maintenance of mitochondrial morphology and function with aging and suggest that mitochondrial dysfunction contributes to dynein-dependent neurological diseases, such as SMA-LED.

Distinctive genetic and clinical features of CMT4J: a severe neuropathy caused by mutations in the PI(3,5)P2 phosphatase FIG4.

Charcot-Marie-Tooth disease is a genetically heterogeneous group of motor and sensory neuropathies associated with mutations in more than 30 genes. Charcot-Marie-Tooth disease type 4J (OMIM 611228) is a recessive, potentially severe form of the disease caused by mutations of the lipid phosphatase FIG4. We provide a more complete view of the features of this disorder by describing 11 previously unreported patients with Charcot-Marie-Tooth disease type 4J. Three patients were identified from a small cohort selected for screening because of their early onset disease and progressive proximal as well as distal weakness. Eight patients were identified by large-scale exon sequencing of an unselected group of 4000 patients with Charcot-Marie-Tooth disease. In addition, 34 new FIG4 variants were detected. Ten of the new CMT4J cases have the compound heterozygous genotype FIG4(I41T/null) described in the original four families, while one has the novel genotype FIG4(L17P/nul)(l). The population frequency of the I41T allele was found to be 0.001 by genotyping 5769 Northern European controls. Thirty four new variants of FIG4 were identified. The severity of Charcot-Marie-Tooth disease type 4J ranges from mild clinical signs to severe disability requiring the use of a wheelchair. Both mild and severe forms have been seen in patients with the same genotype. The results demonstrate that Charcot-Marie-Tooth disease type 4J is characterized by highly variable onset and severity, proximal as well as distal and asymmetric muscle weakness, electromyography demonstrating denervation in proximal and distal muscles, and frequent progression to severe amyotrophy. FIG4 mutations should be considered in Charcot-Marie-Tooth patients with these characteristics, especially if found in combination with sporadic or recessive inheritance, childhood onset and a phase of rapid progression.

Isolation of a gene encoding a cellulolytic enzyme from swamp buffalo rumen metagenomes and its cloning and expression in Escherichia coli.

Ruminants are capable of hydrolyzing lignocellulosic residues to absorbable sugars by virtue of the microbial communities residing in their rumen. However, large sections of such microbial communities are not yet culturable using conventional laboratory techniques. Therefore in the present study, the metagenomic DNA of swamp buffalo (Bubalus bubalis) rumen contents was explored using culture-independent techniques. The consensus regions of glycosyl hydrolase 5 (GH5) family of cellulases were used as primers for PCR amplification. A full-length metagenomic cellulase gene, Umcel5B29, with a complete open reading frame (ORF) of 1611 bp was identified. The similarity search analysis revealed that Umcel5B29 is closely related to the cellulases (73% to 98% similarity) of ruminal unculturable microorganisms, indicating its phylogenetic origin. Further analysis indicated that Umcel5B29 does not contain a carbohydrate binding module (CBM). Subsequently, Umcel5B29 was overexpressed in Escherichia coli. The recombinant enzyme worked optimally at pH 5.5 and 45°C, a condition similar to the buffalo's rumen. However, the enzyme retained more than 70% of its maximal activity after incubation at pH 4-7 and more than 50% maximal activity after incubation at 30-60°C for 30 min. These characteristics render Umcel5B29 as a potential candidate for the bio-stoning process of denim.

Pseudohyperphosphorylation has differential effects on polymerization and function of tau isoforms.

The microtubule-associated protein tau exists as six isoforms created through the splicing of the second, third, and tenth exons. The isoforms are classified by their number of N-terminal exons (0N, 1N, or 2N) and by their number of microtubule-binding repeat regions (3R or 4R). Hyperphosphorylated isoforms accumulate in insoluble aggregates in Alzheimer's disease and other tauopathies. These neurodegenerative diseases can be categorized based on the isoform content of the aggregates they contain. Hyperphosphorylated tau has the general characteristics of an upward electrophoretic shift, decreased microtubule binding, and an association with aggregation. Previously we have shown that a combination of seven pseudophosphorylation mutations at sites phosphorylated by GSK-3ß, referred to as 7-Phos, induced several of these characteristics in full-length 2N4R tau and led to the formation of fewer but longer filaments. We sought to determine whether the same phosphorylation pattern could cause differential effects in the other tau isoforms, possibly through varied conformational effects. Using in vitro techniques, we examined the electrophoretic mobility, aggregation properties, and microtubule stabilization of all isoforms and their pseudophosphorylated counterparts. We found that pseudophosphorylation affected each isoform, but in several cases certain isoforms were affected more than others. These results suggest that hyperphosphorylation of tau isoforms could play a major role in determining the isoform composition of tau aggregates in disease.

Auditory nerve is affected in one of two different point mutations of the neurofilament light gene.

OBJECTIVE: To define auditory nerve and cochlear functions in two families with autosomal dominant axonal Charcot-Marie-Tooth (CMT). METHODS: Affected members in two families with different point mutations of NF-L gene were screened with auditory brainstem responses (ABRs). Those with abnormal ABRs were further investigated with clinical, neurophysiological and audiological procedures. The point mutations of NF-L gene involved were Glu397Lys in 8 affected members of the family with AN, and Pro22Ser in 9 affected members of the family without AN. RESULTS: ABRs and stapedial muscle reflexes were absent or abnormal in affected members of only one family consistent with auditory neuropathy (AN). In them, audiograms, otoacoustic emissions, and speech comprehension were normal. Absent or abnormal ABRs were consistent with slowing of conduction along auditory nerve and/or brainstem auditory pathway. Wave I when present was of normal latency. CONCLUSIONS: Auditory nerve involvement in the presence of normal cochlear outer hair cell activity is asymptomatic in one of two families with CMT disorder with different point mutations of the NF-L gene. The nerve disorder is consistent with altered synchrony and slowed conduction. SIGNIFICANCE: The absence of "deafness" may reflect the ability of central mechanisms to compensate for the slowly developing auditory nerve abnormalities.

FGFR3 dimer stabilization due to a single amino acid pathogenic mutation.

Mutations in the transmembrane (TM) domains of receptor tyrosine kinases (RTKs) have been implicated in the induction of pathological phenotypes. These mutations are believed to stabilize the RTK dimers, and thus promote unregulated signaling. However, the energetics behind the pathology induction has not been determined. An example of a TM domain pathogenic mutation is the Ala391-->Glu mutation in fibroblast growth factor receptor 3 (FGFR3), linked to Crouzon syndrome with acanthosis nigricans, as well as to bladder cancer. Here, we determine the free energy of dimerization of wild-type and mutant FGFR3 TM domain in lipid bilayers using Förster resonance energy transfer, and we show that hydrogen bonding between Glu391 and the adjacent helix in the dimer is a feasible mechanism for dimer stabilization. The measured change in the free energy of dimerization due to the Ala391-->Glu pathogenic mutation is -1.3 kcal/mol, consistent with previous reports of hydrogen bond strengths in proteins. This is the first quantitative measurement of mutant RTK stabilization in a membrane environment. We show that this seemingly modest value can lead to a large increase in dimer fraction and thus profoundly affect RTK-mediated signal transduction.

Endothelial nitric oxide synthase Glu298Asp gene polymorphism in periodontal diseases.

BACKGROUND: Endothelial nitric oxide synthase (eNOS) is involved in key steps of immune response. The aim of the present study was to evaluate genotype distribution and genotype-phenotype association in periodontal disease regarding Glu298Asp polymorphism of the eNOS gene. METHODS: A total of 272 subjects were included into the study. Genomic DNA was obtained from the peripheral blood of 51 chronic periodontitis (CP) patients, 48 generalized aggressive periodontitis (GAgP), and 173 reference controls. Polymerase chain reaction (PCR) amplification and subsequent BanII restriction fragment length polymorphism (RFLP) analysis were used to detect eNOS Glu298Asp polymorphism. Probing depth, clinical attachment loss, plaque accumulation, and bleeding on probing (BOP) were recorded. The data were analyzed by the chi2 test, logistic regression, and Mann-Whitney U test. RESULTS: The distributions of eNOS Glu298Asp genotypes and alleles were similar among study groups. Subjects with the Asp allele (Asp+) were statistically higher in the CP group compared to the control group (odds ratio [OR] = 1.957; 95% confidence interval [95% CI] = 1.038 to 3.689). In the GAgP group, BOP (%) was significantly higher in patients with the 298Asp allele (Asp+) compared to patients without the Asp allele (Asp-) (P = 0.015). CONCLUSIONS: The present study showed that eNOS Glu298Asp polymorphism is associated with BOP in GAgP patients. Moreover, the 298Asp allele of the eNOS gene might be related to CP in the Turkish population.

The Outer Pore and Selectivity Filter of TRPA1.

TRPA1 (transient-receptor-potential-related ion channel with ankyrin domains) is a direct receptor or indirect effector for a wide variety of nociceptive signals, and thus is a compelling target for development of analgesic pharmaceuticals such as channel blockers. Recently, the structure of TRPA1 was reported, providing insights into channel assembly and pore architecture. Here we report whole-cell and single-channel current recordings of wild-type human TRPA1 as well as TRPA1 bearing point mutations of key charged residues in the outer pore. These measurements demonstrate that the glutamate at position 920 plays an important role in collecting cations into the mouth of the pore, by changing the effective surface potential by ~16 mV, while acidic residues further out have little effect on permeation. Electrophysiology experiments also confirm that the aspartate residue at position 915 represents a constriction site of the TRPA1 pore and is critical in controlling ion permeation.

Glutamate signals through mGluR2 to control Schwann cell differentiation and proliferation.

Rapid saltatory nerve conduction is facilitated by myelin structure, which is produced by Schwann cells (SC) in the peripheral nervous system (PNS). Proper development and degeneration/regeneration after injury requires regulated phenotypic changes of SC. We have previously shown that glutamate can induce SC proliferation in culture. Here we show that glutamate signals through metabotropic glutamate receptor 2 (mGluR2) to induce Erk phosphorylation in SC. mGluR2-elicited Erk phosphorylation requires ErbB2/3 receptor tyrosine kinase phosphorylation to limit the signaling cascade that promotes phosphorylation of Erk, but not Akt. We found that Gßγ and Src are involved in subcellular signaling downstream of mGluR2. We also found that glutamate can transform myelinating SC to proliferating SC, while inhibition of mGluR2 signaling can inhibit demyelination of injured nerves in vivo. These data suggest pathophysiological significance of mGluR2 signaling in PNS and its possible therapeutic importance to combat demyelinating disorders including Charcot-Marie-Tooth disease.

Mutation analysis of CLPTM 1 and PVRL 1 genes in patients with non-syndromic clefts of lip, alveolus and palate.

INTRODUCTION: Non-syndromic cleft lip with or without cleft palate (CL/P), is one of the most common birth defects, but its aetiology is largely unknown. The aim of this study was to determine the sequence changes of the Cleft Lip and Palate Transmembrane Protein 1 (CLPTM 1) and Poliovirus Receptor Related 1 (PVRL 1) genes in patients with non-syndromic complete clefts of lip, alveolus and palate and to correlate these findings with clinical features. PATIENTS AND METHODS: 25 patients were analysed (14 male and 11 female, aged 4-10 years) of European descent (9 patients with right, 9 with left and 7 patients with bilateral CLAP) and 25 controls, respectively. Exons 2-14 of the CLPTM1 and exons 1-6 of the PVRL1 gene were analysed by a direct sequencing method using DNA extracted from whole blood. RESULTS: A novel in frame Glu441-Gly442 ins Glu mutation of the PVRL 1 gene in combination with novel exon mutations Gly331Gly, Ala88Ala, Pro309Pro and intron change IVS7-10G/A of the CLPTM 1 gene were found in 9 patients. The Glu441-Gly442 ins Glu mutation and the intron change IVS7-10G/A were not detected in 25 controls. CONCLUSION: These results suggest that a simultaneous occurrence of PVRL1 and CLPTM 1 gene mutations in cleft patients does not correlate with the type of cleft (left, right, bilateral) or the gender of the patients. If a combination of the intron change IVS7-10G/A, exon changes Gly331Gly, Ala88Ala and Pro309Pro of the CLMPT 1 gene and Glu441-Gly442 ins Glu mutation of the PVRL 1 gene could be a genetic factor for non-syndromic clefts of the primary and the secondary palates, it is important to investigate more patients and controls.

Glu-256 is a main structural determinant for oligomerisation of human arginase I.

One determinant that could play a role in the quaternary structure of human arginase is the pair of salt links between the strictly conserved residues R255 from one monomer and E256 from every adjacent subunit. In this work, the ionic interaction between monomers was disrupted by expressing a human arginase where Glu-256 had been substituted by Gln. Biochemical analyses of the mutant protein showed that: (i) it shares the wild-type kinetic parameters of the arginine substrate; (ii) E256Q arginase behaves as a monomer by gel filtration; (iii) it is drastically inactivated by dialysis in the presence of EDTA, an inhibitory effect which is reversed by addition of Mn(2+); and (iv) the mutant enzyme loses thermal stability. The lack of oligomerisation for E256Q arginase and the conservation of E256 throughout evolution of the protein family suggest that this residue is involved in the quaternary structure of arginases.

The novel neurofilament light (NEFL) mutation Glu397Lys is associated with a clinically and morphologically heterogeneous type of Charcot-Marie-Tooth neuropathy.

Charcot-Marie-Tooth disease comprises a heterogeneous group of hereditary neuropathies which fall into two main groups: demyelinating CMT1 with reduced nerve conduction velocity and axonal CMT2 with normal nerve conduction velocity. The neuropathological features correspond in most cases to this classification. Four genes were recently identified to cause autosomal dominant CMT2, including the neurofilament light gene. Thus far, only few mutations have been reported in neurofilament light involving eight amino acids of the gene. We identified a novel mutation, Glu397Lys, in a conserved motive signaling the end of the rod domain. The affected family members from three generations showed strikingly different clinical phenotypes, including weakness of the lower extremities, foot deformities, and deafness. The mutation was associated with nerve conduction velocities ranging from 27 m/s in a 25-year-old female to 43 m/s in an 82-year-old male in the lower extremity motor nerves. Sural nerve biopsies of two affected subjects were analyzed by light and electron microscopy. The pathological changes consisted of a reduction of predominantly large myelinated nerve fibers and various stages of onion bulb formation as typically seen in CMT1. This correlative study further confirms that neurofilament light gene mutations cause a wide clinical spectrum. Thus, analysis of the neurofilament light gene should not be restricted to pure axonal neuropathies.

ENAM mutations with incomplete penetrance.

Amelogenesis imperfecta (AI) is a genetic disease affecting tooth enamel formation. AI can be an isolated entity or a phenotype of syndromes. To date, more than 10 genes have been associated with various forms of AI. We have identified 2 unrelated Turkish families with hypoplastic AI and performed mutational analysis. Whole-exome sequencing identified 2 novel heterozygous nonsense mutations in the ENAM gene (c.454G>T p.Glu152* in family 1, c.358C>T p.Gln120* in family 2) in the probands. Affected individuals were heterozygous for the mutation in each family. Segregation analysis within each family revealed individuals with incomplete penetrance or extremely mild enamel phenotype, in spite of having the same mutation with the other affected individuals. We believe that these findings will broaden our understanding of the clinical phenotype of AI caused by ENAM mutations.

Cleft lip in oculodentodigital dysplasia suggests novel roles for connexin43.

Oculodentodigital Dysplasia (ODDD) is a rare syndrome involving anomalies in eye, tooth, and digit formation, caused by mutations in CX43/GJA1. In addition to classic dental features, ODDD includes oral and craniofacial accessory symptoms such as characteristic facial appearance and cleft palate. However, there have been no reports of ODDD accompanied by cleft lip. Herein we report, for the first time, a male, sporadic, Asian proband presenting bilateral cleft lip. By direct sequence analysis, our proband was diagnosed as having ODDD with a heterozygous mutation, codon 142 G>A in GJA1 and CX43E48K. We excluded the possibility of pathogenic mutations in B3GALTL, BMP4, TFAP2A, PVRL1, IRF6, and MSX1. To address how CX43/GJA1 is related to cleft lip, we performed immunohistochemistry using mouse and human mid-facial tissue. CX43 expression was detected in the nasal compartment and nasal and maxillary processes at murine developmental stage E12.5. Furthermore, CX43 expression was found in the epithelial tissue inside the human subepithelial cleft lip that completes epithelial fusion. Therefore, we suggest that CX43/GJA1 is involved in lip formation. Our case report of ODDD with a bilateral cleft lip suggests that CX43/GJA1 might be a novel candidate gene for syndromic cleft lip.

Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin.

BACKGROUND: Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem. METHODS: A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr(-) cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized. RESULTS: The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement. DISCUSSION: This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.

Clinical, pathological and genetic evaluations of Chinese patients with autosomal-dominant hypophosphatasia.

OBJECTIVES: Hypophosphatasia (HPP) is an inherited disorder characterised by defective bone and tooth mineralisation and deficient serum and bone alkaline phosphatase activity, and it results from mutations in alkaline phosphatase (ALPL) encoding tissue-nonspecific alkaline phosphatase (TNAP). The objective of the present work was to explore the correlations between genotype and phenotype in a Chinese family affected by autosomal-dominant HPP. DESIGN: We examined all individuals of a HPP family by clinical and radiographic examinations as well as laboratory assays. Furthermore, a prematurely exfoliated tooth was observed histopathologically. Based on the clinical and pathological manifestations, the causative gene ALPL was selected for further analysis and screened for mutations. RESULTS: The proband presented the characteristic clinical features of childhood HPP such as rachitic skeletal changes, early loss of primary teeth, and short root anomalies of the permanent teeth. Histopathological evaluation of a tooth revealed a "shell" structure, severe mineralisation defects of dentin, and an absence of cementum. The patient's mother and grandfather were clinically diagnosed with adult HPP. The family showed autosomal-dominant moderate hypophosphatasia. DNA sequencing and analysis revealed a novel missense mutation (c.251A>T) in exon4 of ALPL. This mutation (p.E84V) is located in the secondary structure of TNAP's homodimer interface, and it was predicted to have a dominant negative effect. CONCLUSION: Our findings suggest the missense transversion (c.251A>T, p.E84V) should be responsible for the HPP phenotype in this Chinese family.

Novel inhibitor for prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis and details of substrate-recognition mechanism.

A new inhibitor, H-Ala-Ile-pyrrolidin-2-yl boronic acid, was developed as an inhibitor against prolyl tripeptidyl aminopeptidase with a K(i) value of 88.1 nM. The structure of the prolyl tripeptidyl aminopeptidase complexed with the inhibitor (enzyme-inhibitor complex) was determined at 2.2 A resolution. The inhibitor was bound to the active site through a covalent bond between Ser603 and the boron atom of the inhibitor. This structure should closely mimic the structure of the reaction intermediate between the enzyme and substrate. We previously proposed that two glutamate residues, Glu205 and Glu636, are involved in the recognition of substrates. In order to clarify the function of these glutamate residues in substrate recognition, three mutant enzymes, E205A, E205Q, and E636A were generated by site-directed mutagenesis. The E205A mutant was expressed as an inclusion body. The E205Q mutant was expressed in soluble form, but no activity was detected. Here, the structures of the E636A mutant and its complex with the inhibitor were determined. The inhibitor was located at almost the same position as in the wild-type enzyme-inhibitor complex. The amino group of the inhibitor interacted with Glu205 and the main-chain carbonyl group of Gln203. In addition, a water molecule in the place of Glu636 of the wild-type enzyme interacted with the amino group of the inhibitor. This water molecule was located near the position of Glu636 in the wild-type and formed a hydrogen bond with Gln203. The k(cat)/K(M) values of the E636A mutant toward the two substrates used were smaller than those of the wild-type by two orders of magnitude. The K(i) value of our inhibitor for the E636A mutant was 48.8 microM, which was 554-fold higher than that against the wild-type enzyme. Consequently, it was concluded that Glu205 and Glu636 are significant residues for the N-terminal recognition of a substrate.

N-Terminal domain of annexin 2 regulates Ca(2+)-dependent membrane aggregation by the core domain: a site directed mutagenesis study.

Annexin 2 binds and aggregates biological membranes in a Ca(2+)-dependent manner. This protein exists as a monomer (p36) or as a heterotetramer (p90) in which two p36 chains are associated with a dimer of p11, a member of the S100 protein family. Protein kinase C phosphorylates the protein at the level of the N-terminal tail on serines 11 and 25, thereby modifying its oligomeric structure and its properties of membrane aggregation. To analyze these effects, the properties of a series of mutants in which serines 11 and 25 were replaced by alanine and/or glutamic acid were investigated. The affinity for p11 light chain was decreased in the S11E mutants. Glutamic acid residues in positions 11 or 25 did not change membrane binding, either in the tetrameric or in the monomeric form. On the other hand, these mutations affected the aggregation properties of the two forms. For the tetramer, the aggregation efficiency was decreased but not the Ca(2+) sensitivity, whereas the latter was affected in the case of the monomer. The effects were stronger in the S11E mutants, and they were cumulative in the double mutant. They suggest a different conformation of the N-terminal domain in the mutants (and in the phosphorylated protein), a hypothesis which is supported by proteolysis experiments. This conformational change would affect aggregation by the monomer through a dimerization step.

Ser787 in the proline-rich region of human MAP4 is a critical phosphorylation site that reduces its activity to promote tubulin polymerization.

p34cdc2 kinase-phosphorylation sites in the microtubule (MT)-binding region of MAP4 were determined by peptide sequence of phosphorylated MTB3, a fragment containing the carboxy-terminal half of human MAP4. In addition to two phosphopeptides containing Ser696 and Ser787 which were previously indicated to be in vivo phosphorylation sites, two novel phosphopeptides, containing Thr892 or Thr901 and Thr917 as possible phosphorylation sites, were isolated, though only in in vitro phosphorylation. The role of phosphorylation at Ser696 and Ser787, which were differently phosphorylated during the cell cycle (Ookata et al., (1997). Biochemistry, 36: 15873-15883), was investigated in MT-polymerization, using MAP4 Ser to Glu mutants, which mimic phosphorylation at each site. Mutation of Ser787 to Glu strikingly reduced the MAP4's MT-polymerization activity, while Glu-mutation at Ser696 did not. These results suggest that Ser787 could be the critical phosphorylation site causing MTs to be dynamic at mitosis.