Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T

The persistence of high concentrations of beta-2-microglobulin (β2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of β2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length β2M (β2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6β2m and ΔN10β2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of β2M amyloid fibrils with affinity ~104 M−1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with β2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-β2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10β2m fibrils from other β2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between β2m and ΔN6β2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the β2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit β2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

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Molecular variants of β2-microglobulin in renal insufficiency

Biochemical Journal ◽

10.1042/bj2980181 ◽

1994 ◽

Vol 298 (1) ◽

pp. 181-187 ◽

Cited By ~ 15

Author(s):

C Vincent ◽

L Dennoroy ◽

J P Revillard

Keyword(s):

Amino Acid ◽

Renal Insufficiency ◽

Ascitic Fluid ◽

Amyloid Fibrils ◽

Biological Fluids ◽

Amino Acid Sequences ◽

Molecular Forms ◽

Molecular Variants ◽

Terminal Amino ◽

Beta 2 Microglobulin

Many patients with renal insufficiency treated by dialysis for more than 10 years have tissue deposits of amyloid material containing polymerized beta 2-microglobulin (beta 2m). The mechanisms of beta 2m polymerization and degradation remain unknown. In biological fluids (serum and urine) from haemodialysis patients and in dialysis fluids from patients treated by chronic ambulatory peritoneal dialysis (CAPD), we have characterized different molecular forms of beta 2m, including proteolytic split products. beta 2m isoforms of pI 5.7, 5.3 and 4.5-5.0 were isolated from urine and CAPD fluid. The pI 5.3 beta 2m, but not the other forms, was recovered both as monomers and as dimers. Such dimers were also detected in serum from patients but not from healthy controls. pI 5.3 and 5.7 beta 2m isoforms were found to be nearly identical by mass spectrometry and by their amino acid sequences. The amino acid sequence of the 43 N-terminal amino acids of beta 2m of pI 5.0 showed identity with the corresponding region of pI 5.7 beta 2m. Fragments recovered from CAPD fluid were similar to proteolytic fragments generated from pure pI 5.7 beta 2m by incubation in mouse ascitic fluid at acidic pH. Furthermore, pure pI 5.7 beta 2m was converted into more acidic forms of 12 kDa upon incubation in mouse ascitic fluid at acid pH. beta 2m dimers found in serum may represent a precursor of amyloid fibrils.

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beta 2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils

European Journal of Biochemistry ◽

10.1046/j.1432-1327.1998.2580061.x ◽

1998 ◽

Vol 258 (1) ◽

pp. 61-67 ◽

Cited By ~ 84

Author(s):

Vittorio Bellotti ◽

Monica Stoppini ◽

Palma Mangione ◽

Margaret Sunde ◽

Carol Robinson ◽

...

Keyword(s):

Amyloid Fibrils ◽

Ex Vivo ◽

Native State ◽

Beta 2 ◽

Beta 2 Microglobulin

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Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6663 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6663-6669 ◽

Cited By ~ 50

Author(s):

L Trieschmann ◽

Y V Postnikov ◽

A Rickers ◽

M Bustin

Keyword(s):

Amino Acids ◽

Structural Features ◽

Binding Domain ◽

Full Length ◽

Modular Structure ◽

Chromosomal Proteins ◽

Nucleosome Core ◽

Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

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Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

Journal of Virology ◽

10.1128/jvi.75.13.6095-6106.2001 ◽

2001 ◽

Vol 75 (13) ◽

pp. 6095-6106 ◽

Cited By ~ 212

Author(s):

Stephen J. Polyak ◽

Khalid S. A. Khabar ◽

Denise M. Paschal ◽

Heather J. Ezelle ◽

Gilles Duverlie ◽

...

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Response ◽

Full Length ◽

Interleukin 8 ◽

Antiviral Resistance ◽

Ns5a Protein ◽

Terminal Amino

ABSTRACT Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-ΔN110), 222 (NS5A-ΔN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-ΔN110 and NS5A-ΔN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-κB and AP-1 were important in NS5A-ΔN222 transactivation in the presence of tumor necrosis factor alpha and that NF–IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

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The C-Terminal of MYH11 Is Required for Cbfb-MYH11 Activity During Embryonic Hematopoiesis and Leukemogenesis

Blood ◽

10.1182/blood.v118.21.2472.2472 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2472-2472

Author(s):

R. Katherine Hyde ◽

Yasuhiko Kamikubo ◽

Ling Zhao ◽

Lemlem Alemu ◽

Lisa Garrett ◽

...

Keyword(s):

Gene Expression ◽

Amino Acids ◽

Transcription Factor ◽

Null Allele ◽

Fusion Gene ◽

Full Length ◽

Primitive Hematopoiesis ◽

Single Allele ◽

Terminal Amino ◽

Definitive Hematopoiesis

Abstract Abstract 2472 RKH, YK, and LZ all contributed equally to this work Inv(16) is found in nearly all patients with acute myeloid leukemia (AML) subtype M4Eo. Inv(16) results in the fusion of the transcription factor gene CBFB, and the MYH11 gene, which encodes Smooth Muscle Myosin Heavy Chain (SMMHC). This results in the fusion gene CBFB-MYH11, which encodes CBFβ-SMMHC. Previously we showed that knock-in mice with a single allele of Cbfb-MYH11 (Cbfb+/MYH11) have severe differentiation defects in primitive hematopoiesis and a total block in definitive hematopoiesis. In addition, chimeric mice generated from Cbfb+/MYH11 ES cells consistently developed leukemia within a few months after treatment with the mutagen N-ethyl-N-nitrosourea (ENU). It is currently not clear which functional domains of CBFβ-SMMHC are responsible for its activity in differentiation and leukemogenesis. In vitro experiments have indicated that CBFβ-SMMHC can form multimeric complexes via the C terminal domain. It has been postulated that this multimerization may be important for the function of CBFβ-SMMHC by resulting in large macromolecular complexes and/or sequestration of its binding partner, the transcription factor RUNX1. To determine the importance of this domain in vivo, we generated knock-in mice expressing a mutant Cbfb-MYH11 allele with a deletion of the 95 C-terminal amino acids (Cbfb+/MYH11ΔC95). In analysis of primitive hematopoiesis, we found that Cbfb+/MYH11ΔC95 and CbfbMYH11ΔC95/MYH11ΔC95 mice had no or very mild differentiation defects, statistically significantly less severe (p<.05) than seen in embryos expressing full-length Cbfb-MYH11. During definitive hematopoesis, there were no observable defects in Cbfb+/MYH11ΔC95 mice, but CbfbMYH11ΔC95/MYH11ΔC95 embryos showed a complete block in definitive hematopoiesis, as seen in mice expressing a single allele of full length Cbfb-MYH11. This indicates that Cbfb-MYH11ΔC95 is less effective in blocking differentiation than the full length fusion gene. Interestingly, both the primitive and definitive embryonic blood phenotypes of the CbfbMYH11ΔC95/MYH11ΔC95 were similar to that observed in embryos lacking functional Cbfb (Cbfb−/−), implying that Cbfb-MYH11ΔC95 may act as a null allele. To test this possibility we used gene expression microarrays to compare gene expression profiles in the peripheral blood from embryonic day 12 CbfbMYH11ΔC95/MYH11ΔC95, Cbfb−/−, and Cbfb+/MYH11, as well as their Cbfb+/+ littermates. Surprisingly, CbfbMYH11ΔC95/MYH11ΔC95 embryos showed deregulated expression of a distinct gene set as compared to both Cbfb−/− and Cbfb+/MYH11 embryos. This implies that Cbfb-MYH11ΔC95 is not a null allele of Cbfb, and likely retains some, but not all, of the neomorph properties of full length Cbfb-MYH11. Consistent with this finding, we observe the accumulation of abnormal myeloid cells in some adult Cbfb+/MYH11ΔC95 mice after ENU treatment, which has not been reported in Cbfb+/− mice. However, we found that Cbfb-MYH11ΔC95 has not retained the most critical of the fusion gene's activities: the ability to induce leukemogenesis. Importantly, none of the Cbfb+/MYH11ΔC95 mice developed leukemia after treatment with ENU. This is in contrast to mice expressing full length Cbfb-MYH11, which all develop leukemia under these conditions. Together, these results indicate that the 95 C-terminal amino acids of CBFβ-SMMHC are required for both embryonic hematopoietic defects and leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Domain assembly of the GLUT1 glucose transporter

Biochemical Journal ◽

10.1042/bj3000291 ◽

1994 ◽

Vol 300 (2) ◽

pp. 291-294 ◽

Cited By ~ 30

Author(s):

D L Cope ◽

G D Holman ◽

S A Baldwin ◽

A J Wolstenholme

Keyword(s):

Amino Acids ◽

Binding Sites ◽

Glucose Transporter ◽

Cytochalasin B ◽

Insect Cells ◽

Full Length ◽

Stable Complex ◽

C And N ◽

Terminal Amino ◽

Glut1 Glucose Transporter

A full-length construct of the glucose transporter isoform GLUT1 has been expressed in Sf9 (Spodoptera frugiperida Clone 9) insect cells, and a photolabelling approach has been used to show that the expressed protein binds the bismannose compound 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine (ATB-BMPA) and cytochalasin B at its exofacial and endofacial binding sites respectively. Constructs of GLUT1 which produce either the N-terminal (amino acids 1-272) or C-terminal (amino acids 254-492) halves are expressed at levels in the plasma membrane which are similar to that of the full-length GLUT1 (approximately 200 pmol/mg of membrane protein), but do not bind either ATB-BMPA or cytochalasin B. When Sf9 cells are doubly infected with virus constructs producing both the C- and N-terminal halves of GLUT1, then the ligand labelling is restored. Only the C-terminal half is labelled, and, therefore, the labelling of this domain is dependent on the presence of the N-terminal half of the protein. These results suggest that the two halves of GLUT1 can assemble to form a stable complex and support the concept of a bilobular structure for the intact glucose transporters in which separate C- and N-domain halves pack together to produce a ligand-binding conformation.

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The Fluorescent Dye 1,6-Diphenyl-1,3,5-Hexatriene Binds to Amyloid Fibrils Formed by Human Amylin and Provides a New Probe of Amylin Amyloid Kinetics

10.1101/2021.05.10.443442 ◽

2021 ◽

Author(s):

Ming-Hao Li ◽

Lakshan Manathunga ◽

Erwin London ◽

Daniel Raleigh

Keyword(s):

Time Course ◽

Amyloid Fibrils ◽

Fold Increase ◽

Fluorescent Dye ◽

Thioflavin T ◽

Lag Phase ◽

Amyloid Formation ◽

Islet Amyloid ◽

Human Amylin ◽

Mutant Forms

The fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is widely used as a probe of membrane order. We show that DPH also interacts with amyloid fibrils formed by human amylin (also known as islet amyloid polypeptide) in solution and this results in a 100-fold increase in DPH fluorescence for a sample of microM human amylin and 0.25 microM DPH. No increase in DPH fluorescence is observed with the non-amyloidogenic rat amylin or with freshly dissolved, non-fibrillar human amylin. The time course of amyloid formation by amylin was followed by monitoring the fluorescence of added DPH as a function of time and was similar to that monitored by the standard fluorescent probe thioflavin-T. The inclusion of DPH in the buffer did not perturb the time course of amyloid formation under the conditions examined and the time course was independent of the range of DPH concentrations tested (0.25 to 5 microM). Maximum final fluorescence intensity is observed at substoichiometric ratios of DPH to amylin. No significant increase in fluorescence was observed during the lag phase of amyloid formation, and the implications for the structure of amylin pre-fibril oligomers are discussed. Human amylin contains three aromatic residues. A triple aromatic to leucine mutant forms amyloid and DPH binds to the resulting fibrils, indicating that interactions with aromatic side chains are not required for DPH amylin amyloid interactions. DPH may be especially useful for studies on mutant amylins and other polypeptides in which changes in charged residues might complicate interpretation of thioflavin-T fluorescence.

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