The pathology studies widely deal with many cellular and nuclear altered structures other than these one of the important and interesting features is the observation of various histopathological bodies. These inclusion bodies is an important diagnostic-aid in identifying the underlying disease. Therefore in this article present different inclusion bodies seen in various diseases.
H, Masthan K. Inclusion Bodies. Biomed Pharmacol J ;9 2. Inclusion bodies are nuclear or cytoplasmic aggregates which are stainable substances, usually proteins, and formed due to viral multiplication or genetic disorders in human beings these bodies are either intracellular or extracellular abnormalities and they are specific to certain diseases. When a foreign gene or the infectious agent is injected into a cell, the complementary DNA translated from a messenger RNA may code for a protein, which are fails to undergo further modification, transport, condensation of the cell, result in inclusion body.
In some diseased conditions, cells modified and may become pathognomonic for that particular disease. In Keratinized stratified squamous epithelium is shows membrane-coating granules called Odland bodies.
It is also called as lamellar bodies, keratinosomes. These are seen in the upper stratum spinosum and stratum granular cell layers which are rich in glycolipids. These lipids are discharged extracellularly to form a permeability barrier that prevent absorption of aqueous fluids. Interestingly, recent algorithms take into account not only the primary sequence of the polypeptide, but also experimental proteome data, including information about cellular localization, cytosolic, periplasmic and membrane proteins.
It is worth mentioning that other factors affect the aggregation of recombinant protein expression in bacteria such as temperature and growth rate, fusion to soluble protein tags, specific codon usage, tRNA availability, and general optimization of codons in the heterologous expressed sequence Cortazzo et al.
The formation and structure of IBs in E. Several excellent reviews summarize the current knowledge on IBs structure and formation De Groot et al. Here we present a concise summary and extend a little more on the most recent published data. IBs are normally observed in the cytoplasm of the producing bacteria as dense, large and apparently spherical or cylindrical particle, ranging from 0.
IBs may also contain other proteins, like small heat shock proteins IbpA and IbpA and chaperones like the DnaK system , phospholipids from membranes and nucleic acids and other background proteins that co-purify with aggregates Jurgen et al. Interestingly, the cellular composition of IBs evolves during cell growth so that cellular proteins are predominant during the first steps of formation, while heterologous proteins become predominant at the end. These changes occur in concert with the evolution of other parameters inherent to cell growth, such as division time and growth rate, leading to the idea that aging of bacterial population could be related to protein aggregation Lindner et al.
Notwithstanding, IBs may also contain variable amounts of natively folded proteins or partially folded proteins that can acquire their native conformation even if they are embedded in an aggregate Gonzalez-Montalban et al. In fact aggregates have been found to be composed of a wide spectrum of conformations, ranging from native conformation to misfolded aggregates Schrodel et al.
Moreover, aggregation and disaggregation have been shown to occur simultaneously in vivo in actively producing recombinant bacteria Carrio and Villaverde, The solubilized proteins can then reach their native state or alternatively suffer partial proteolytic degradation Corchero et al. The proportion of functional protein is characteristic of the target protein's sequence Upadhyay et al.
They propose that IBs present a sponge-like structure, where the PK resistant fibrils constitute a scaffold which confers mechanical stability to IBs, while the functional, PK sensitive fraction accumulate in the gaps of this scaffold. In accordance to this, Walther et al.
They propose that the solubilization process involves different layers in the IBs: a core, consisting of the IBs agglomerates, a reactive and a diffusion layer. The densely packed inner cores of protein shrink as the solubilized protein diffuses to the outer layers and subsequently through a porous barrier layer into free solution. The authors propose that this model correlates well with the IBs structure suggested by Cano-Garrido et al.
This approach is strongly supported by the concept that protein aggregation is part of a conserved cellular response. Three examples have been chosen to illustrate how IBs are employed as a model to study aggregation proteins involved in particular human diseases and as a useful screening approach for the search for aggregation inhibitors. Huntington disease is a neurodegenerative disorder that affects muscle coordination, followed by cognitive and psychiatric problems.
The disease is caused by mutations in the Huntingtin gene, in which expansion of the triplet CAG within the first exon of the gene produces a protein carrying stretches of repeated glutamines polyQ. When polyQ exceeds a critical length, huntingtin protein undergoes amyloid aggregation Orr and Zoghbi, This question was recently explored in E. For this purpose, the toxicity of three variants expressed in E. The authors showed that toxicity was correlated to the formation of soluble cytosolic oligomers, but not to peptide aggregation.
Instead, interestingly, the aggregates appeared to be protective against cell toxicity Invernizzi et al. Prions are protein aggregates with self-perpetuating ability and thus infectious reviewed in Villar-Pique and Ventura, Prions are involved in transmissible spongiform encephalopathies TSEs , a family of rare progressive neurodegenerative disorders that affect both humans and animals. Bacterial IBs have been exploited as a tool for the study of the structural and functional characteristics of prions.
Het-s, from the fungus Podospora anserina , was the first prion protein whose bacterial IBs were shown to display amyloid-like properties Sabate et al.
These E. A similar observation was reported in the case of the yeast prion Sup These results highlight the fact that the infectivity rate can be easily modulated by tuning the environmental conditions during the formation of IBs Radchenko et al. There is an increasing interest in developing methods to identify cellular factors that trigger the aggregation of proteins inside the organism as well as to discover drugs able to interfere with these factors.
Villar-Pique et al. The authors further propose that, as many proteins form IBs when expressed in bacteria De Groot et al. Considering IBs as a source of almost pure proteins, one possible way is to attain the dissolution of aggregates in order to obtain native-folded, active protein. The challenge is then to solubilize and refold as much aggregated protein as possible and obtain a stable, functional product. The cost of the whole process must be taken into consideration if the aim is to produce a large-scale manufactured product.
The rate and yield of the solubilization process seem to be influenced by the conditions used, like chaotrope addition, concentration, temperature, pressure, etc. Solubilization and refolding are the most critical steps in the procedure and successful conditions still depend mostly on trial-and-error strategies Burgess, There are very good recent reviews that compile the different methods employed in the different steps of the recovery process.
In accordance to the above mentioned findings on the structure and solubilization mechanisms of IBs, the tendency is to employ milder extraction conditions, avoiding strong denaturation and refolding conditions. In Table 1 we listed the most recent approaches reported in the last 3 years.
A recent report showed that peptide hormones of the pituitary gland are stored intracellularly as amyloid aggregates within the secretion granules. A somewhat similar situation occurs with bacterial IBs.
In fact, IBs are composed by amyloid-like aggregates from which substantial amounts of functional recombinant protein can be released in vivo as well as under mild non-denaturing conditions in vitro.
As a proof of concept, Vazquez et al. Also, dihydrofolate reductase IBs complemented the intrinsic cell deficiency of this enzyme. Furthermore, IBs are spontaneously internalized by cultured cells, as was directly demonstrated with green fluorescent protein IBs Villaverde et al.
Following a similar approach, Liovic et al. The common view of IBs as undesirable by-products in recombinant protein production have been lastly reconsidered, in view of the potential of these structures for different purposes.
On the other hand, IBs provide to constitute an easy to handle model for the study of the molecular basis of conformational diseases. On the other hand, IBs provide a source of almost pure polypeptides and are a potentially useful source of ready-to-use protein.
In this sense, the aim is then to obtain IBs containing as much folded, functional protein as possible. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Basu, A. Refolding of proteins from inclusion bodies: rational design and recipes. Burgess, R. Refolding solubilized inclusion body proteins. Methods Enzymol. Cano-Garrido, O. Supramolecular organization of protein-releasing functional amyloids solved in bacterial inclusion bodies.
Acta Biomater. Carrio, M. Proteolytic digestion of bacterial inclusion body proteins during dynamic transition between soluble and insoluble forms. Acta , — Construction and deconstruction of bacterial inclusion bodies.
Chow, M. Nucleic Acids Res. Protein Expr. Conchillo-Sole, O. BMC Bioinformatics Corchero, J. Limited in vivo proteolysis of aggregated proteins. Cortazzo, P. Silent mutations affect in vivo protein folding in Escherichia coli. Cubarsi, R. In situ proteolytic digestion of inclusion body polypeptides occurs as a cascade process.
Datta, I. Microwave assisted solubilization of inclusion bodies. CrossRef Full Text. De Groot, N. Methods Mol. Studies on bacterial inclusion bodies.
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A novel protein refolding system using lauroyl-l-glutamate as a solubilizing detergent and arginine as a folding assisting agent.
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