ABSTRACT
Investigation of the molecular mechanisms underlying amyloid-related human diseases attracts close attention. These diseases, the number of which currently is above 40, are characterized by formation of peptide or protein aggregates containing a cross-ß structure. Most of the amyloidogenesis mechanisms described so far are based on experimental studies of aggregation of short peptides, intrinsically disordered proteins, or proteins under denaturing conditions, and studies of amyloid aggregate formations by structured globular proteins under conditions close to physiological ones are still in the initial stage. We investigated the effect of amino acid substitutions on propensity of the completely helical protein sperm whale apomyoglobin (sw ApoMb) for amyloid formation from its structured state in the absence of denaturing agents. Stability and aggregation of mutated sw ApoMb were studied using circular dichroism, Fourier transform infrared spectroscopy, x-ray diffraction, native electrophoresis, and electron microscopy techniques. Here, we demonstrate that stability of the protein native state determines both protein aggregation propensity and structural peculiarities of formed aggregates. Specifically, structurally stable mutants show low aggregation propensity and moderately destabilized sw ApoMb variants form amyloids, whereas their strongly destabilized mutants form both amyloids and nonamyloid aggregates.
Subject(s)
Apoproteins/metabolism , Myoglobin/metabolism , Protein Aggregation, Pathological/metabolism , Amino Acid Sequence , Animals , Apoproteins/chemistry , Apoproteins/genetics , Calorimetry, Differential Scanning , Circular Dichroism , Electrophoresis , Escherichia coli , Microscopy, Electron , Mutation , Myoglobin/chemistry , Myoglobin/genetics , Protein Aggregation, Pathological/genetics , Protein Folding , Protein Stability , Protein Structure, Secondary , Spectroscopy, Fourier Transform Infrared , Sperm Whale , X-Ray DiffractionABSTRACT
BACKGROUND: YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. RESULTS: We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. CONCLUSION: These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.
Subject(s)
DNA-Binding Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/ultrastructure , Nuclear Proteins/metabolism , RNA-Binding Proteins/metabolism , Tubulin/metabolism , Animals , Chromatography, Affinity , Humans , Microscopy, Atomic Force , Microtubules/metabolism , Peptide Fragments/metabolism , RNA, Messenger/metabolism , Rabbits , Ribonucleoproteins/metabolism , Tissue Extracts , Tubulin/isolation & purification , Tubulin/ultrastructure , Y-Box-Binding Protein 1ABSTRACT
The time course of polysome formation was studied in a long-term wheat germ cell-free translation system using sedimentation and electron microscopy techniques. The polysomes were formed on uncapped luciferase mRNA with translation-enhancing 5' and 3' UTRs. The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases. First, short linear polysomes containing no more than six ribosomes were formed. Next, folding of these polysomes into short double-row clusters occurred. Subsequent gradual elongation of the clusters gave rise to heavy-loaded double-row strings containing up to 30-40 ribosomes. The formation of the double-row polysomes was considered to be equivalent to circularization of polysomes, with antiparallel halves of the circle being laterally stuck together by ribosome interactions. A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5' UTR, while de novo initiation including 5' UTR scanning proceeds at a much slower rate. Removal or replacements of 5' and 3' UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.
Subject(s)
Polyribosomes/metabolism , Polyribosomes/ultrastructure , Protein Biosynthesis , 3' Untranslated Regions/chemistry , 5' Untranslated Regions/chemistry , Cell-Free System , Centrifugation, Density Gradient , Kinetics , Luciferases/genetics , Luminescent Proteins/genetics , RNA, Messenger/metabolism , Ribosomes/metabolism , Tobacco Mosaic Virus/genetics , Triticum/geneticsABSTRACT
Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase.
Subject(s)
DNA, Bacterial/biosynthesis , DNA-Directed DNA Polymerase/metabolism , Deoxyribonuclease I/physiology , Escherichia coli Proteins/physiology , Base Sequence , Deoxyribonucleases, Type II Site-Specific/metabolism , Geobacillus stearothermophilus/enzymology , Microsatellite Repeats/physiologyABSTRACT
YB-1 is a universal major protein of cytoplasmic mRNPs, a member of the family of multifunctional cold shock domain proteins (CSD proteins). Depending on its amount on mRNA, YB-1 stimulates or inhibits mRNA translation. In this study, we have analyzed complexes formed in vitro at various YB-1 to mRNA ratios, including those typical for polysomal (translatable) and free (untranslatable) mRNPs. We have shown that at mRNA saturation with YB-1, this protein alone is sufficient to form mRNPs with the protein/RNA ratio and the sedimentation coefficient typical for natural mRNPs. These complexes are dynamic structures in which the protein can easily migrate from one mRNA molecule to another. Biochemical studies combined with atomic force microscopy and electron microscopy showed that mRNA-YB-1 complexes with a low YB-1/mRNA ratio typical for polysomal mRNPs are incompact; there, YB-1 binds to mRNA as a monomer with its both RNA-binding domains. At a high YB-1/mRNA ratio typical for untranslatable mRNPs, mRNA-bound YB-1 forms multimeric protein complexes where YB-1 binds to mRNA predominantly with its N-terminal part. A multimeric YB-1 comprises about twenty monomeric subunits; its molecular mass is about 700 kDa, and it packs a 600-700 nt mRNA segment on its surface.
