Bacterial inclusion bodies contain amyloid-like structure

Protein aggregation is a process in which identical proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as amorphous, lacking any long-range order, or highly ordered fibrils. Protein fibrils can be composed of native globular molecules, such as the hemoglobin molecules in sickle-cell fibrils, or can be reorganized beta-sheet-rich aggregates, termed amyloid-like fibrils. Amyloid fibrils are associated with several pathological conditions in humans, including Alzheimer disease and diabetes type II. We studied the structure of bacterial inclusion bodies, which have been believed to belong to the amorphous class of aggregates. We demonstrate that all three in vivo-derived inclusion bodies studied are amyloid-like and comprised of amino-acid sequence-specific cross-beta structure. These findings suggest that inclusion bodies are structured, that amyloid formation is an omnipresent process both in eukaryotes and prokaryotes, and that amino acid sequences evolve to avoid the amyloid conformation.     

Inclusion bodies: a new concept

In the last decades, the understanding of inclusion body biology and consequently, of their properties and potential biotechnological applications have dramatically changed. Therefore, the development of new purification protocols aimed to preserve those properties is becoming a pushing demand.     

Inclusion bodies in Plesiomonas shigelloides

Inclusion bodies were discovered in seven environmental isolates of Plesiomonas shigelloides and the P. shigelloides control (ATCC 14029). Differential staining indicated that the inclusion bodies may be composed of polyphosphates, and developmental stages of the bodies may occur. The inclusion bodies may be useful for rapid presumptive identification of this organism.     

Inclusion bodies in Plesiomonas shigelloides.

Inclusion bodies were discovered in seven environmental isolates of Plesiomonas shigelloides and the P. shigelloides control (ATCC 14029). Differential staining indicated that the inclusion bodies may be composed of polyphosphates, and developmental stages of the bodies may occur. The inclusion bodies may be useful for rapid presumptive identification of this organism.     

Tryptophanyl substitutions in apomyoglobin determine protein aggregation and amyloid-like fibril formation at physiological pH

Myoglobin is an alpha-helical globular protein that contains two highly conserved tryptophan residues located at positions 7 and 14 in the N-terminal region of the protein. Replacement of both indole residues with phenylalanine residues, i.e. W7F/W14F, results in the expression of an unstable, not correctly folded protein that does not bind the prosthetic group. Here we report data (Congo red and thioflavine T binding assay, birefringence, and electron microscopy) showing that the double Trp/Phe replacements render apomyoglobin molecules highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions in which most of the wild-type protein is in the native state. In refolding experiments, like the wild-type protein, the W7F/W14F apomyoglobin mutant formed a soluble, partially folded helical state between pH 2.0 and pH 4.0. A pH increase from 4.0 to 7.0 restored the native structure only in the case of the wild-type protein and determined aggregation of W7F/W14F. The circular dichroism spectrum recorded immediately after neutralization showed that the polypeptide consists mainly of beta-structures. In conclusion, under physiological pH conditions, some mutations that affect folding may cause protein aggregation and the formation of amyloid-like fibrils.     

PDZ domains and their binding partners: structure, specificity, and modification

PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins. They regulate multiple biological processes such as transport, ion channel signaling, and other signal transduction systems. This review discusses the structural characterization of PDZ domains and the use of recently emerging technologies such as proteomic arrays and peptide libraries to study the binding properties of PDZ-mediated interactions. Regulatory mechanisms responsible for PDZ-mediated interactions, such as phosphorylation in the PDZ ligands or PDZ domains, are also discussed. A better understanding of PDZ protein-protein interaction networks and regulatory mechanisms will improve our knowledge of many cellular and biological processes.     

Aggresomes, inclusion bodies and protein aggregation

Intracellular and extracellular accumulation of aggregated protein are linked to many diseases, including ageing-related neurodegeneration and systemic amyloidosis. Cells avoid accumulating potentially toxic aggregates by mechanisms including the suppression of aggregate formation by molecular chaperones and the degradation of misfolded proteins by proteasomes. Once formed, aggregates tend to be refractory to proteolysis and to accumulate in inclusion bodies. This accumulation has been assumed to be a diffusion-limited process, but recent studies suggest that, in animal cells, aggregated proteins are specifically delivered to inclusion bodies by dynein-dependent retrograde transport on microtubules. This microtubule-dependent inclusion body is called an aggresome.     

Inclusion bodies in loggerhead erythrocytes are associated with unstable hemoglobin and resemble human Heinz bodies

The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grünwald-Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species.     

Preparation of amyloid-like fibrils containing magnetic iron oxide nanoparticles: effect of protein aggregation on proton relaxivity

A method to prepare amyloid-like fibrils functionalized with magnetic nanoparticles has been developed. The amyloid-like fibrils are prepared in a two step procedure, where insulin and magnetic nanoparticles are mixed simply by grinding in the solid state, resulting in a water soluble hybrid material. When the hybrid material is heated in aqueous acid, the insulin/nanoparticle hybrid material self assembles to form amyloid-like fibrils incorporating the magnetic nanoparticles. This results in magnetically labeled amyloid-like fibrils which has been characterized by Transmission Electron Microscopy (TEM) and electron tomography. The influence of the aggregation process on proton relaxivity is investigated. The prepared materials have potential uses in a range of bio-imaging applications.     

Dynamics of in vivo protein aggregation: building inclusion bodies in recombinant bacteria

Time-dependent aggregation of a plasmid-encoded β-galactosidase fusion protein, VP1LAC, has been carefully monitored during its high-rate synthesis in Escherichia coli. Immediately after recombinant gene induction, the full-length form of the protein steadily accumulates into rapidly growing cytoplasmic inclusion bodies. Their volume increases during at least 5 h at a rate of 0.4 μm³ h⁻¹, while the average density remains constant. Protein VP1LAC accounts for about 90% of the aggregated protein throughout the building process. Minor components, such as DnaK and GroEL chaperones, have been identified in variable, but low concentrations. The homogeneous distribution of inclusion bodies among the cell population and the coexistence of large, still growing bodies with newly appearing aggregates indicate that the aggregation cores are mutually exclusive, this fact being a main determinant of the in vivo dynamics of protein aggregation.     

Relation between the structure of alliin analogues and their inhibitory effect on platelet aggregation

Alliin analogues have been synthesized and tested for their inhibitory activity on platelet aggregation. It is found that only allicin, the S-oxodiallyl disulphide, has a strong inhibitory effect, comparable to that of alliin, while all the other tested compounds do not show any inhibitory effect even at concentrations of 10^?3 M.