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1.
Methods Protoc ; 6(3)2023 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-37218904

RESUMO

The detection and quantification of protein-protein interactions (PPIs) is a crucial technique that often involves the use of recombinant proteins with fusion protein tags, such as maltose-binding protein (MBP) and glutathione-S-transferase (GST). In this study, we improved the cohesive and sticky properties of gelatinized starch by supplementing it with agarose, resulting in a harder gel that could coat the bottom of a microtiter plate. The resulting gelatinized starch/agarose mixture allowed for the efficient immobilization of MBP-tagged proteins on the coated plates, enabling the use of indirect ELISA-like PPI assays. By using the enzymatic activity of GST as an indicator, we succeeded in determining the dissociation constants between MBP-tagged and GST-tagged proteins on 96-well microtiter plates and a microplate reader without any expensive specialized equipment.

3.
Curr Res Struct Biol ; 4: 158-166, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35663930

RESUMO

Human stomatin (hSTOM) is a component of the membrane skeleton of erythrocytes that maintains the membrane's shape and stiffness through interconnecting spectrin and actin. hSTOM is a member of the protein family that possesses a single stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of the molecule. Although SPFH domain proteins are widely distributed from archaea to mammals, the detailed function of the domain remains unclear. In this study, we first determined the solution structure of the SPFH domain of hSTOM (hSTOM(SPFH)) via NMR. The solution structure of hSTOM(SPFH) is essentially identical to the already reported crystal structure of the STOM SPFH domain (mSTOM(SPFH)) of mice, except for the existence of a small hydrophilic pocket on the surface. We identified this pocket as a phosphate-binding site by comparing its NMR spectra with and without phosphate ions. Meanwhile, during the conventional process of protein NMR analysis, we eventually discovered that hSTOM(SPFH) formed a unique solid material after lyophilization. This lyophilized hSTOM(SPFH) sample was moderately slowly dissolved in a physiological buffer. Interestingly, it was resistant to dissolution against the phosphate buffer. We then found that the lyophilized hSTOM(SPFH) formed a fibril-like assembly under electron microscopy. Finally, we succeeded in reproducing this fibril-like assembly of hSTOM(SPFH) using a centrifugal ultrafiltration device, thus demonstrating that the increased protein concentration may promote self-assembly of hSTOM(SPFH) into fibril forms. Our observations may help understand the molecular function of the SPFH domain and its involvement in protein oligomerization as a component of the membrane skeleton. (245 words).

4.
Front Mol Biosci ; 9: 869851, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35558561

RESUMO

The anti-oncogenic protein p53 is a transcription factor that prevents tumorigenesis by inducing gene repair proteins or apoptosis under DNA damage. Since the DNA-binding domain of p53 (p53C) is aggregation-prone, the anti-oncogenic function of p53 is often lost in cancer cells. This tendency is rather severe in some tumor-related p53 mutants, such as R175H. In this study, we examined the effect of salts, including KCl and sugars, on the aggregation of p53C by monitoring two distinct aggregates: amorphous-like and amyloid-like. The amorphous aggregates are detectable with 8-(phenylamino)-1-naphthalenesulfonic acid (ANS) fluorescence, whereas the amyloid aggregates are sensitive to thioflavin-T (ThT) fluorescence. We found that KCl inhibited the formation of amorphous aggregates but promoted the formation of amyloid aggregates in a p53C R175H mutant. The salts exhibited different effects against the wild-type and R175H mutants of p53C. However, the ratio of ANS/ThT fluorescence for the wild-type and R175H mutant remained constant. KCl also suppressed the structural transition and loss of the DNA-binding function of p53C. These observations indicate the existence of multiple steps of p53C aggregation, probably coupled with the dissociation of Zn. Notably, amorphous aggregates and amyloid aggregates have distinct properties that could be discriminated by various small additives upon aggregation.

5.
Biophys Rev ; 14(1): 267-275, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35340607

RESUMO

The tumor suppressor protein p53, a transcription product of the anti-oncogene TP53, is a critical factor in preventing cellular cancerization and killing cancer cells by inducing apoptosis. As a result, p53 is often referred to as the "guardian of the genome." Almost half of cancers possess genetic mutations in the TP53 gene, and most of these mutations result in the malfunction of p53, which promotes aggregation. In some cases, the product of the TP53 mutant allele shows higher aggregation propensity; the mutant co-aggregates with the normal (functional) p53 protein, thus losing cellular activity of the p53 guardian. Cancer might also progress because of the proteolytic degradation of p53 by activated E3 ubiquitination enzymes, MDM2 and MDM4. The inhibition of the specific interaction between MDM2 (MDM4) and p53 also results in increased p53 activity in cancer cells. Although the molecular targets of the drugs are different, two drug discovery strategies with a common goal, "rescuing p53 protein," have recently emerged. To conduct this approach, various biophysical methods of protein characterization were employed. In this review, we focus on these two independent strategies based on the unique biophysical features of the p53 protein.

6.
BMC Biol ; 20(1): 42, 2022 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-35144600

RESUMO

BACKGROUND: Primary cilia are sensory organelles crucial for organ development. The pivotal structure of the primary cilia is a microtubule that is generated via tubulin polymerization reaction that occurs in the basal body. It remains to be elucidated how molecules with distinct physicochemical properties contribute to the formation of the primary cilia. RESULTS: Here we show that brain expressed X-linked 1 (Bex1) plays an essential role in tubulin polymerization and primary cilia formation. The Bex1 protein shows the physicochemical property of being an intrinsically disordered protein (IDP). Bex1 shows cell density-dependent accumulation as a condensate either in nucleoli at a low cell density or at the apical cell surface at a high cell density. The apical Bex1 localizes to the basal body. Bex1 knockout mice present ciliopathy phenotypes and exhibit ciliary defects in the retina and striatum. Bex1 recombinant protein shows binding capacity to guanosine triphosphate (GTP) and forms the condensate that facilitates tubulin polymerization in the reconstituted system. CONCLUSIONS: Our data reveals that Bex1 plays an essential role for the primary cilia formation through providing the reaction field for the tubulin polymerization.


Assuntos
Cílios , Proteínas do Tecido Nervoso/metabolismo , Tubulina (Proteína) , Animais , Condensados Biomoleculares , Cílios/metabolismo , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo
7.
Biophys Rev ; 14(1): 1-2, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35222732

RESUMO

On behalf of the Australian Society for Biophysics (ASB) and the Editors of this Special Issue, I would like to express our appreciation to Editor-in-Chief, Damien Hall, for arranging the publication of this Special Issue. The ASB is about five times smaller than our sister the Biophysical Society for Japan (BSJ) and tenfold smaller than the US Biophysical Society (USBS), but our meetings are notable because of the encouragement the Society gives to emerging biophysicists. It can be a terrifying experience for a PhD student to have to face a roomful of professors and senior academics, but invariably they appreciate the experience. Another feature of the ASB meetings is the inclusion of contributions from the Asian Pacific region. We now have formal ties with our New Zealand colleagues and our meetings with the BSJ contain joint sessions (see below). In 2020, despite the impact of COVID-19 (see Adam Hill's Commentary), there is a joint session with the University of California Davis. This Special Issue comprises 2 Editorials, 3 Commentaries, and 25 reviews.

8.
Food Funct ; 13(4): 1953-1964, 2022 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-35088789

RESUMO

Glycyrrhizin (GL) is known to exhibit a variety of useful pharmacological activities, including anti-inflammation, anti-hepatotoxicity, and enhancement of intestinal drug absorption. GL has been reported to modify the assembly of actin filaments, thereby modulating tight junction (TJ) integrity, but the detailed molecular mechanisms of this remain unclear. In this study, we first found that GL binds to the first PDZ domain of zonula occludens-1 (ZO-1(PDZ1)) through NMR experiments. The structure of the GL-ZO-1(PDZ1) complex was then constructed using HADDOCK with the transferred nuclear Overhauser effect-based inter-hydrogen distance constraints as well as restrictions on the interfacial residues identified from 1H-15N HSQC spectral changes. We identified the relevant interactions between the glucuronate-2 moiety of GL and the carboxylate binding loop of the ligand binding site of ZO-1(PDZ1). We further examined the interaction of ZO-1(PDZ1) with glycyrrhetinic acid and with GA-3-monoglucuronide and observed a much lower affinity for each than for that with GL, with good agreement with the model. The other contacts found in the model were examined by using an amino acid substitution mutant of ZO-1(PDZ1). Finally, we reproduced the experiments reported by Sakai et al. in which high-dose GL prolonged the TJ-opening mediated with sodium deoxycholate as indicated by reduced transepithelial electrical resistance.


Assuntos
Ácido Glicirrízico/farmacologia , Domínios PDZ , Junções Íntimas , Proteína da Zônula de Oclusão-1 , Células CACO-2 , Humanos , Junções Íntimas/química , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/antagonistas & inibidores , Proteína da Zônula de Oclusão-1/metabolismo
9.
Biophys Rev ; 13(4): 485-486, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34188719

RESUMO

This Commentary describes a call for submissions for the upcoming Special Issue focused on the research topics presented at the Australian Society of Biophysics (ASB) in 2020 and 2021. Submissions from past and present ASB members who could not attend these meetings are also welcome as contributions to this special issue.

10.
J Alzheimers Dis ; 80(1): 159-174, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33492290

RESUMO

BACKGROUND: Brain amyloid-ß (Aß) peptide is released into the interstitial fluid (ISF) in a neuronal activity-dependent manner, and Aß deposition in Alzheimer's disease (AD) is linked to baseline neuronal activity. Although the intrinsic mechanism for Aß generation remains to be elucidated, interleukin-like epithelial-mesenchymal transition inducer (ILEI) is a candidate for an endogenous Aß suppressor. OBJECTIVE: This study aimed to access the mechanism underlying ILEI secretion and its effect on Aß production in the brain. METHODS: ILEI and Aß levels in the cerebral cortex were monitored using a newly developed ILEI-specific ELISA and in vivo microdialysis in mutant human Aß precursor protein-knockin mice. ILEI levels in autopsied brains and cerebrospinal fluid (CSF) were measured using ELISA. RESULTS: Extracellular release of ILEI and Aß was dependent on neuronal activation and specifically on tetanus toxin-sensitive exocytosis of synaptic vesicles. However, simultaneous monitoring of extracellular ILEI and Aß revealed that a spontaneous fluctuation of ILEI levels appeared to inversely mirror that of Aß levels. Selective activation and inhibition of synaptic receptors differentially altered these levels. The evoked activation of AMPA-type receptors resulted in opposing changes to ILEI and Aß levels. Brain ILEI levels were selectively decreased in AD. CSF ILEI concentration correlated with that of Aß and were reduced in AD and mild cognitive impairment. CONCLUSION: ILEI and Aß are released from distinct subpopulations of synaptic terminals in an activity-dependent manner, and ILEI negatively regulates Aß production in specific synapse types. CSF ILEI might represent a surrogate marker for the accumulation of brain Aß.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Citocinas/genética , Citocinas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Sinapses , Idoso , Idoso de 80 Anos ou mais , Precursor de Proteína beta-Amiloide/genética , Animais , Autopsia , Córtex Cerebral/metabolismo , Citocinas/líquido cefalorraquidiano , Espaço Extracelular/metabolismo , Feminino , Técnicas de Introdução de Genes , Genes Supressores , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microdiálise , Atividade Motora , Proteínas de Neoplasias/líquido cefalorraquidiano , Receptores de AMPA/metabolismo , Receptores de GABA/metabolismo
11.
Biophys Physicobiol ; 17: 86-93, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33194509

RESUMO

An increasing number of proteins, which have neither regular secondary nor well-defined tertiary structures, have been found to be present in cells. The structure of these proteins is highly flexible and disordered under physiological (native) conditions, and they are called "intrinsically disordered" proteins (IDPs). Many of the IDPs are involved in interactions with other biomolecules such as DNA, RNA, carbohydrates, and proteins. While these IDPs are largely unstructured by themselves, marked conformational changes often occur upon binding to an interacting partner, which is known as the "coupled folding and binding mechanism", which enable them to change the conformation to become compatible with the shape of the multiple target biomolecules. We have studied the structure and interaction of eukaryotic transcription factors Sp1 and TAF4, and found that both of them have long intrinsically disordered regions (IDRs). One of the IDRs in Sp1 exhibited homo-oligomer formation. In addition, the same region was used for the interaction with another IDR found in the TAF4 molecule. In both cases, we have not detected any significant conformational change in that region, suggesting a prominent and novel binding mode for IDPs/IDRs, which are not categorized by the well-accepted concept of the coupled folding and binding mechanism.

12.
Biochem Biophys Rep ; 20: 100704, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31867447

RESUMO

Perturbation of the homeostasis of brain membrane lipids has been implicated in the pathomechanism of Alzheimer's disease (AD). The ε4 allele of the apolipoprotein E gene (APOE) confers an increased risk, in a dosage-dependent manner, for brain amyloid-ß accumulation and the development of sporadic AD. An effect of the APOE genotype on brain lipid homeostasis may underlie the AD risk associated with the ε4 allele. In this research, we examined an effect of APOE ε4 on the lipid class composition of crude membranes and raft-enriched fractions of brains. We applied enzymatic reaction-based methods for the quantification of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and sphingomyelin. Our results indicate that brain lipid class composition was neither significantly altered in AD subjects nor affected by the presence of the APOE ε4 allele.

13.
Protein Sci ; 26(11): 2280-2290, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28857320

RESUMO

The expression of eukaryotic genes is precisely controlled by interactions between general transcriptional factors and promoter-specific transcriptional activators. The fourth element of TATA-box binding protein-associated factor (TAF4), an essential subunit of the general transcription factor TFIID, serves as a coactivator for various promoter-specific transcriptional regulators. Interactions between TAF4 and site-specific transcriptional activators, such as Sp1, are important for regulating the expression levels of genes of interest. However, only limited information is available on the molecular mechanisms underlying the interactions between these transcriptional regulatory proteins. We herein analyzed the interaction between the transcriptional factors Sp1 and TAF4 using high-resolution solution nuclear magnetic resonance spectroscopy. We found that four glutamine-rich (Q-rich) regions in TAF4 were largely disordered under nearly physiological conditions. Among them, the first Q-rich region in TAF4 was essential for the interaction with another Q-rich region in the Sp1 molecule, most of which was largely disordered. The residues responsible for this interaction were specific and highly localized in a defined region within a range of 20-30 residues. Nevertheless, a detailed analysis of 13 C-chemical shift values suggested that no significant conformational change occurred upon binding. These results indicate a prominent and exceptional binding mode for intrinsically disordered proteins other than the well-accepted concept of "coupled folding and binding."


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Fator de Transcrição Sp1/química , Fatores Associados à Proteína de Ligação a TATA/química , Fator de Transcrição TFIID/química , Motivos de Aminoácidos , Sítios de Ligação , Isótopos de Carbono , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fator de Transcrição Sp1/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Transcrição Gênica
14.
Protein Sci ; 25(11): 2006-2017, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27515574

RESUMO

The expression of eukaryotic genes is precisely controlled by specific interactions between general transcription initiation factors and gene-specific transcriptional activators. The general transcription factor TFIID, which plays an essential role in mediating transcriptional activation, is a multisubunit complex comprising the TATA box-binding protein (TBP) and multiple TBP-associated factors (TAFs). On the other hand, biochemical and genetic approaches have shown that the promoter-specific transcriptional activator Sp1 has the ability to interact with one of the components of TFIID, the TBP-associated factor TAF4. We herein report the structural details of the glutamine-rich domains (Q-domains) of Sp1 and TAF4 using circular dichroism (CD) and heteronuclear magnetic resonance (NMR) spectroscopy. We found that the two Q-domains of Sp1 and four Q-domains of TAF4 were disordered under physiological conditions. We also quantitatively analyzed the interaction between the Q-domains of Sp1 and TAF4 by NMR and surface plasmon resonance, and detected a weak but specific association between them. Nevertheless, a detailed analysis of CD spectra suggested that any significant conformational change did not occur concomitantly with this association, at least at the level of the overall secondary structure. These results may represent a prominent and exceptional binding mode for the IDPs, which are not categorized in a well-accepted concept of "coupled folding and binding."


Assuntos
Fator de Transcrição Sp1/química , Fatores Associados à Proteína de Ligação a TATA/química , Fator de Transcrição TFIID/química , Dicroísmo Circular , Humanos , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estrutura Secundária de Proteína , Fator de Transcrição Sp1/metabolismo , Ressonância de Plasmônio de Superfície , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo
15.
Protein Sci ; 21(10): 1481-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22855260

RESUMO

The promoter-specific transcription factor Sp1 is expressed ubiquitously, and plays a primary role in the regulation of the expression of many genes. Domains A and B located in the N-terminal half of the protein are characterized by glutamine-rich (Q-rich) sequences. These Q-rich domains have been shown to be involved in the interaction between Sp1 and different classes of nuclear proteins, such as TATA-binding protein associated factors. Furthermore, the self-association of Sp1 via Q-rich domains is also important for the regulation of transcriptional activity. It has been considered that an Sp1 molecule bound to a "distal" GC-box synergistically interacts with another Sp1 molecule at a "proximal" binding site. Although the formation of multimers via Q-rich domains seems functionally important for Sp1, little is known about the structural and physicochemical nature of the interaction between Q-rich domains. We analyzed the structural details of isolated glutamine-rich B (QB) domains of Sp1 by circular dichroism (CD), analytical ultracentrifugation, and heteronuclear magnetic resonance spectroscopy (NMR). We found the isolated QB domains to be disordered under all conditions examined. Nevertheless, a detailed analysis of NMR spectra clearly indicated interaction between the domains. In particular, the C-terminal half was responsible for the self-association. Furthermore, analytical ultracentrifugation demonstrated weak but significant interaction between isolated QB domains. The self-association between QB domains would be responsible, at least in part, for the formation of multimers by full-length Sp1 molecules that has been proposed to occur during transcriptional activation.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Fator de Transcrição Sp1/química , Fator de Transcrição Sp1/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Glutamina/química , Humanos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Ultracentrifugação
16.
Primates ; 49(2): 139-42, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18204817

RESUMO

A variable number of tandem repeat (VNTR) polymorphism based on a 16 or 17-bp unit has been reported in the third intron of the human serotonin transporter gene (5-HTT). VNTRs have been shown to affect the transcriptional activity of genes, and VNTR polymorphisms possibly influence human personality and several psychoneurological disorders. To estimate the changes that occurred in the VNTRs during primate evolution, we amplified and sequenced the regions that corresponded to the human VNTRs in various primate species, including apes, Old World monkeys, and New World monkeys. The VNTR sequences were polymorphic in all the ape species examined, and alleles with repeat numbers of 18, 19, 23, and 24 in chimpanzees, 33, 35, 36, 38, and 40 in gorillas, 4 and 6 in orangutans, and 11, 13, 14, and 15 in gibbons were found. On the other hand, only a 5-repeat allele was detected in Old World monkeys such as the Japanese macaque and patas monkey. In this study we demonstrated for the first time that a repeat structure was not present in the corresponding regions in the New World monkeys examined, and only one unit sequence was found in them. These results suggested that the duplication of a unit in the VNTR region occurred in the Cercopithecidae species following the divergence of the Old World and New World monkeys, and various long repeated alleles were generated in humans and apes, except orangutans.


Assuntos
Variação Genética , Repetições Minissatélites/genética , Primatas/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/química , Evolução Molecular , Frequência do Gene , Humanos , Íntrons/genética , Dados de Sequência Molecular , Filogenia , Primatas/classificação , Alinhamento de Sequência , Especificidade da Espécie
17.
Primates ; 47(3): 248-54, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16467955

RESUMO

Two polymorphic trinucleotide repeats of human androgen receptor gene (hAR), CAG and GGN which encode glutamine and glycine, have been shown to be associated with human diseases. The number of repeats ranges from 8 to 35 for the CAG and from 10 to 30 for the GGN in human populations. Longer CAG repeats are associated with reduced hAR transcriptional activity, spinal bulbar muscular atrophy and lower cognitive function in older men, whereas shorter CAG repeats are associated with increased risk of prostate cancer and infertility in men. The functional roles of the CAG and GGN repeats have not been clarified. In order to compare the sequence of the CAG and GGN regions in apes, we analyzed 57 chimpanzees, 18 gorillas, 20 orangutans, 16 agile gibbons, and 17 siamangs by PCR and electrophoresis. Two bonobos and one long-tailed macaque were also sequenced and the sequences of all species were aligned, respectively, with one human registered sequence. Seventeen different alleles (4, 7, 8, 9, 12, 14, 15, and 17-26 repeats) and 11 alleles (11-14 and 16-22 repeats) were detected at the CAG and the GGN loci, respectively. Although the repeat tract was conserved among apes, chimpanzees had alleles with a wide range of repeat lengths: (CAG)(14-26) and (GGN)(14-22). Gorillas were less polymorphic with the (CAG)(8) and (GGN)(19) alleles being most common, and orangutans exhibited monomorphic (CAG)(11) and (GGN)(22) alleles. On the other hand, agile gibbons and siamangs had the shortest (CAG)(4) allele, but showed variable length of GGN repeats (11-13 in agile gibbons and 16-21 in siamangs). In chimpanzees, frequent haplotypes consisting of short CAG repeats and long GGN repeats or vice versa was observed as in humans.


Assuntos
Hominidae/genética , Polimorfismo Genético/genética , Receptores Androgênicos/genética , Repetições de Trinucleotídeos/genética , Animais , Feminino , Humanos , Masculino , Dados de Sequência Molecular
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