Fluorescence Polarization of αs1, κ-Caseins and αs1-κ-Casein Complex

Conjugates of α^s1-,κ-caseins and α^s1-,κ-casein complex were prepared with dimethylaminonaphthalenesulfonate and pyrenebutyrate. Their fluorescence lifetimes and the rotational relaxation times were measured by single photon counting technique and fluorescence depolarization technique, respectively. Both dimethylaminonaphthalenesulfonate and pyrenebutyrate conjugates had more than two lifetimes and the longer lifetime of pyrenebutyrate conjugates was near 140 nsec.

The rotational relaxation time of pyrenebutyrate α^s1-,κ-casein complex was smaller than that of pyrenebutyrate κ-casein polymer, which suggested that the complex formation of α^s1- and κ-casein polymers led to dissociation of the κ-casein polymer.

Changes of the rotational relaxation time as a function of weight ratio of α^s1- and κ-casein polymers (α^s1/κ) showed the specific variation and it was suggested that 4 moles of α^s1-κ-casein complex were formed from one mole of κ-casein polymer.     

Aggregation-defective α-synuclein mutants inhibit the fibrillation of Parkinson’s disease-linked α-synuclein variants

α-Synuclein comprises the fibrillar core of Lewy bodies, which is one of the histologically defining lesions of Parkinson’s disease. Previously, we screened for α-synuclein substitution mutants that do not form fibrils. For preventative or therapeutic uses, it is essential to suppress the oligomerization/fibrillation of the wild-type and PD-linked α-synuclein proteins. Here we have examined the effects of fibrillation-retarded α-synuclein mutants on fibril formation by wild-type and PD-linked α-synuclein molecules. Six self-aggregation-defective α-synuclein mutants completely inhibit the fibrillation of both wild-type and Parkinson’s disease-linked α-synuclein variants. These results suggest future applications for gene therapy: the transplantation of a fibrillation-blocking mutant α-synuclein gene into individuals who carry an early-onset PD-associated α-synuclein allele. Short synthetic peptides derived from these mutant sequences may also serve as a lead compound for the development of therapeutics for Parkinson’s disease.     

Environmental toxins and α-synuclein in Parkinson’s disease

In recent years, environmental influences have been thought to play an important role in Parkinson’s disease (PD). Evidence from epidemiological investigations suggests that environmental factors might take part in the disease process. Intriguingly, most of environmental toxins share the common mechanism of causing mitochondria dysfunction by inhibiting complex I and promoting α-synuclein aggregation, a key factor in PD. Therefore, understanding the mechanism of interactions between α-synuclein and environmental factors could lead to new therapeutic approaches to PD.     

Antigenic Reactivity of Dephosphorylated αs1-Casein,Phosphopeptide from β-Casein and O-Phospho-l-serine towards the Antibody to Native αs1 -Casein

To study whether the phosphoserine residue is associated with the antigenicity of bovine α_s1- casein, we examined the antigenic reactivity of dephosphorylated α_s1-casein, peptide 1~25 from bovine β-casein and three chemical reagents with IgG antibody specific to native α_s1-casein by an enzyme-linked immunosorbent assay.

The reaction between native α_s1-casein and its IgG antibody was inhibited more strongly by native α_s1-casein than by dephosphorylated α_s1-casein. Peptide 1~25, having a phosphoserine residue-concentrated region from bovine β-casein, noticeably inhibited the reaction between native α_s1 -casein and its antibody. Furthermore, the O-phospho-l-serine residue inhibited the reaction of peptide 61~123 with anti-native α_s1-casein antibody, although l-serine and sodium phosphate showed no measurable inhibition.

These results suggest that the phosphoserine residue associated with part of an antigenic site in bovine α_sl-casein.     

Northern analysis of highly folded goat αs1 Casein mRNA

Northern blotting using glyoxal to denature a highly folded mRNA, such as goat α_s1-Casein E, can lead to the detection of multiple incompletely denatured forms. Formaldehyde appears to be the most suitable agent for Northern blotting due to its effective denaturing capacity and lower toxicity than methylmercuric hydroxide.     

Characterization of the Testis-specific Proteasome Subunit α4s in Mammals

The 26 S proteasome is responsible for regulated proteolysis in eukaryotic cells. It is composed of one 20 S core particle (CP) flanked by one or two 19 S regulatory particles. The CP is composed of seven different α-type subunits (α1-α7) and seven different β-type subunits, three of which are catalytic. Vertebrates encode four additional catalytic β subunits that are expressed predominantly in immune tissues and produce distinct subtypes of CPs particularly well suited for the acquired immune system. In contrast, the diversity of α subunits remains poorly understood. Recently, another α subunit, referred to as α4s, was reported. However, little is known about α4s. Here we provide a detailed characterization of α4s and the α4s-containing CP. α4s is exclusively expressed in germ cells that enter the meiotic prophase and is incorporated into the CP in place of α4. A comparison of structural models revealed that the differences in the primary sequences between α4 and α4s are located on the outer surface of the CP, suggesting that α4s interacts with specific molecules via these unique regions. α4s-containing CPs account for the majority of the CPs in mouse sperm. The catalytic β subunits in the α4s-containing CP are β1, β2, and β5, and immunosubunits are not included in the α4s-containing CP. α4s-containing CPs have a set of peptidase activities almost identical to those of α4-containing CPs. Our results provide a basis for understanding the role of α4s and male germ cell-specific proteasomes in mammals.     

High-level expression of human αs1-casein in Escherichia coli

Human _s1-casein was expressed efficiently in Escherichia coli. The overproduced recombinant human a _s1-casein was about 25% of the total cell protein. Two different vectors were constructed to express Met-_s1-casein and Met-_s1-casein with a His-affinity tag at the C-terminus. Recombinant Met-_s1-casein with a His-affinity tag was purified to homogeneity using Ni-nitrilotriacetic acid resin. N-terminal sequence of the first 10 amino acid residues of this purified protein was identical to that of mature human _s1-casein with an extra methionine residue at the N-terminus.     

The Central Theme of Parkinson’s Disease: α-Synuclein

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, defined by the presence of resting tremor, muscular rigidity, bradykinesia, and postural instability. PD is characterized by the progressive loss of dopaminergic neurons within the substantia nigra pars compacta of the midbrain. The neuropathological hallmark of the disease is the presence of intracytoplasmic inclusions, called Lewy bodies (LBs) and Lewy neurites (LNs), containing α-synuclein, a small protein which is widely expressed in the brain. The α-synuclein gene, SNCA, is located on chromosome 4q22.1; SNCA-linked PD shows an autosomal dominant inheritance pattern with a relatively early onset age, and it usually progresses rapidly. Three missense mutations, A53T, A30P, and E46K, in addition to gene multiplications of the SNCA have been described so far. Although it is clear that LBs and LNs contain mainly the α-synuclein protein, the mechanism(s) which leads α-synuclein to accumulate needs to be elucidated. The primary question in the molecular pathology of PD is how wild-type α-synuclein aggregates in PD, and which interacting partner(s) plays role(s) in the aggregation process. It is known that dopamine synthesis is a stressfull event, and α-synuclein expression somehow affects the dopamine synthesis. The aberrant interactions of α-synuclein with the proteins in the dopamine synthesis pathway may cause disturbances in cellular mechanisms. The normal physiological folding state of α-synuclein is also important for the understanding of pathological aggregates. Recent studies on the α-synuclein protein and genome-wide association studies of the α-synuclein gene show that PD has a strong genetic component, and both familial and idiopathic PD have a common denominator, α-synuclein, at the molecular level. It is clear that the disease process in Parkinson’s disease, as in other neurodegenerative disorders, is very complicated; there can be several different molecular pathways which are responsible for diverse and possibly also unrelated functions inside the neuron, playing roles in PD pathogenesis.     

Alternative Nonallelic Deletion Is Constitutive of Ruminant αs1-Casein

Multiple forms of α_s1-casein were identified in the four major ruminant species by structural characterization of the protein fraction. While α_s1-casein phenotypes were constituted by a mixture of at least seven molecular forms in ovine and caprine species, there were only two forms in bovine and water buffalo species. In ovine and caprine forms the main component corresponded to the 199-residue-long form, and the deleted proteins differed from the complete one by the absence of peptides 141–148, 110–117, or Gln78, or a combination of such deletions. The deleted segments corresponded to the sequence regions encoded by exons 13 and 16, and by the first triplet of exon 11 (CAG), suggesting that the occurrence of the short protein forms is due to alternative skipping, as previously demonstrated for some caprine and ovine phenotypes. The alternative deletion of Gln78 in α_s1-casein, the only form common to the milk of all the species examined and located in a sequence region joining the polar phosphorylation cluster and the hydrophobic C-terminal domain of the protein, may play a functional role in the stabilization of the milk micelle structure.     

Studies on the Interaction between αs1- and β-Caseins

The interaction of α_s1-casein with β-, dephosphorylated β-,γ- and R-caseins was studied. It was proved by the sedimentation velocity experiments that α_s1-casein formed a complex with each of these components at 25±C in the presence of 3 mm CaCl₂.

In the presence of 10 mm CaCl₂, β- and dephosphorylated β-casein prevented the precipitation of α_s1-casein and gave micelle-like turbid solutions. However, γ- and R-caseins, fragments of β-casein, did not stabilize α_s1-casein. It was concluded from these results that α-casein interacted with α_s1-casein through its hydropholic region corresponding to R-casein and that hydrophilic region of β-casein was responsible for the stabilization of α_s1-casein.     

Thermodynamics of the Interaction between αs1- and κ-Caseins

Pyrenebutyrate-conjugated α_s1-casein was prepared and the complex formation between α_s1- and κ-casein polymers was investigated by fluorescence polarization. The complex formation was also investigated by a microcalorimetric technique. The positive enthalpy and entropy changes and endothermic nature suggested the hydrophobic interaction between α_s1- and κ-casein polymers.

The degree of polarization of κ-casein polymer decreased with the addition of 1-anilino-8-naphthalenesulfonate (ANS), while that of α_s1-casein polymer and α_s1-κ-casein complex was invariant. Moreover the reaction of κ-casein polymer and ANS was exothermic. These facts suggested that the intermolecular hydrophobic regions in κ-casein polymer were disrupted by the adsorption of ANS. The rotational relaxation time of pyrenebutyrate conjugated complex between cyanoethyl-κ-casein and α_s1-casein polymer was smaller than that of cyanoethyl-κ-casein alone. From these results, it was postulated that the dissociation of κ-casein polymer by the complex formation with α_s1-casein polymer might be caused by the disruption of the intermolecular hydrophobic bonds in κ-casein polymer.     

A novel angiotensin-I-converting enzyme inhibitory peptide from human αs1-casein

We have previously reported (Kim et al., J. Dairy Res., 1999, in press) three novel angiotensin-I-converting enzyme (ACE) inhibitory peptides, Tyr-Pro-Glu-Arg, Tyr-Tyr-Pro-Gln-Ile-Met-Gln-Tyr and Asn-Asn-Val-Met-Leu-Gln-Trp, isolated from the tryptic digest of recombinant human _s1-casein. A series of C-terminal di- and tripeptides from these three peptides have now been synthesized and their ACE inhibitory activities have been measured. Among the peptides tested, the tripeptide Leu-Gln-Trp had the highest ACE inhibitory activity (IC₅₀=3.8 mol l^–1).     

Synthesis of hydroquinone-α-glucoside by α-glucosidasefrom baker’s yeast

Hydroquinone-α-glucoside was synthesised from hydroquinone and maltose as glucosyl donor by transglucosylation in a water system with α-glucosidase from baker’s yeast. Only one phenolic –OH group was α-anomer-selectively glucosylated. The optimum conditions for transglucosylation reaction were at 30 °C for 20 h with 50 mM hydroquinone and 1.5 M maltose in 100 mM sodium citrate/phosphate buffer at pH 5.5. The glucoside was obtained at 0.6 mg/ml with a 4.6% molar yield with respect to hydroquinone.     

Variants within the 5′-flanking regions of bovine milk-protein-encoding genes. III. Genes encoding the Ca-sensitive caseins αs1, αs2 and β

The 5-flanking regions of the Ca-sensitive casein-encoding gene family were analysed for DNA variants by automated DNA sequencing of 13 cows belonging to seven breeds. About 1 kbp of each 5-flanking region, including non-coding exon I, was amplified by PCR and sequenced bidirectionally. A total number of 34 variable sites (17 for the _s1, 10 for the _s2, and 7 for the casein encoding gene) was identified. Variants were computer-analysed for location in putative regulatory sites in order to predict potential influences on gene expression.     

Mechanistic aspects of Parkinson’s disease: α-synuclein and the biomembrane

A key feature in Parkinson’s disease is the deposition of Lewy bodies. The major protein component of these intracellular deposits is the 140-amino acid protein α-synuclein that is widely distributed throughout the brain. α-synuclein was identified in presynaptic terminals and in synaptosomal preparations. The protein is remarkable for its structural variability. It is almost unstructured as a monomer in aqueous solution. Self-aggregation leads to a variety of β-structures, while membrane association may result in the formation of an amphipathic helical structure. The present article strives to give an overview of what is currently known on the interaction of α-synuclein with lipid membranes, including synthetic lipid bilayers, membraneous cell fractions, synaptic vesicles and intact cells. Manifestations of a functional relevance of the α-synuclein–lipid interaction will be discussed and the potential pathogenicity of oligomeric α-synuclein aggregates will be briefly reviewed.     

SDS Induced Structural Changes in α-Crystallin and It’s Effect on Refolding

-Crystallin, a major eye lens protein and a key member of the small heat shock protein family, acts like a chaperone by preventing aggregation of substrate proteins. One of the hallmarks of most small heat shock proteins is their existence as a large oligomer, the role of which in its function is not understood at present. We have studied the role of the oligomer in the stability of its structure against SDS induced destabilization by CD measurements. -Crystallin from bovine source as well as recombinant preparation was used for this purpose. As SDS concentration was gradually increased, the -sheet structure was diminished followed by concomitant increase in the -helical structure. The quaternary structural changes in presence of SDS were also monitored by light scattering, polarization and anisotropy measurements. It was found that the breakdown of the oligomeric structure was nearly complete above 1 mM SDS concentration. The results were compared with that of a monomeric -crystallin, which is also a major -sheet protein like -crystallin. When -crystallin was first converted into monomeric random coil structure in presence of 6 M urea and allowed to refold in SDS solution, amount of -helix was more than that incubated directly in the same concentration of SDS. The results show that -crystallin attains extra structural stability against external stress due to its oligomeric structure. The implication for the extra stability is discussed in reference to its function as molecular chaperone.     

Synthesis of new α-heterocyclic α-aminoesters

alpha-Heterocyclic alpha-aminoesters were obtained in good yields by reaction of a glycine cation equivalent and different heterocyclic nucleophiles; diastereoselectivity using a carbohydrate (galactopyranose) as N-protecting group was modest.     

TOM40 Mediates Mitochondrial Dysfunction Induced by α-Synuclein Accumulation in Parkinson’s Disease

Alpha-synuclein (α-Syn) accumulation/aggregation and mitochondrial dysfunction play prominent roles in the pathology of Parkinson’s disease. We have previously shown that postmortem human dopaminergic neurons from PD brains accumulate high levels of mitochondrial DNA (mtDNA) deletions. We now addressed the question, whether alterations in a component of the mitochondrial import machinery -TOM40- might contribute to the mitochondrial dysfunction and damage in PD. For this purpose, we studied levels of TOM40, mtDNA deletions, oxidative damage, energy production, and complexes of the respiratory chain in brain homogenates as well as in single neurons, using laser-capture-microdissection in transgenic mice overexpressing human wildtype α-Syn. Additionally, we used lentivirus-mediated stereotactic delivery of a component of this import machinery into mouse brain as a novel therapeutic strategy. We report here that TOM40 is significantly reduced in the brain of PD patients and in α-Syn transgenic mice. TOM40 deficits were associated with increased mtDNA deletions and oxidative DNA damage, and with decreased energy production and altered levels of complex I proteins in α-Syn transgenic mice. Lentiviral-mediated overexpression of Tom40 in α-Syn-transgenic mice brains ameliorated energy deficits as well as oxidative burden. Our results suggest that alterations in the mitochondrial protein transport machinery might contribute to mitochondrial impairment in α-Synucleinopathies.