Folding and Aggregation in Bacteria


Folding and Aggregation in Bacteria



Bacteria com a model de plegament i biotecnologia.

Resum del treball.

The field of protein misfolding and aggregation has become a widely active area of research in recent years, mainly because of the connection between the formation of insoluble protein deposits in human tissues and the development of dozens of conformational diseases. These protein deposits are constituted mainly by fibrillar structures known as amyloid aggregates that are characterized by a high protein organization in a cross-β structure. In addition, protein aggregation occurs during recombinant expression in prokaryotic systems as insoluble protein deposits named inclusion bodies (IBs) limiting the application of bacteria for protein production in biotechnology industry.

IBs formation was long regarded a dull mishap, although, gradually emerged the notion that IBs are more complex. In this way, the last studies of IBs formation and composition show a number of common features with the highly ordered amyloid fibril {Carrio, 2005 #16}. Both processes are nucleation driven, sequence specific and lead to the formation of -sheet rich aggregates. However, structural characterisation of this type of aggregates is extremely challenging and the degree that IBs resemble to amyloids is still unclear essentially. Recent studies provide compelling evidences of a direct relationship between these conformational diseases and the formation protein aggregates in bacterial cells {Morell, 2008 #26; Wang, 2008 #27}. These data suggest that inclusion bodies (IBs) could be considered as isolated reservoirs of amyloid aggregates. Moreover, IBs are influenced by the cellular environment resulting in a dynamic equilibrium between protein deposition and removal {Gonzalez-Montalban, 2006 #36; Carrio, 2001 #43; Carrio, 2002 #19; Carrio, 2003 #53}. Consequently, together with the β-sheet aggregates it could be found native-like protein and other different conformations of the over-expressed polypeptide {Gonzalez-Montalban, 2006 #36}. Overall, prokaryotic cells could provide a simple powerful system to study the mechanisms of in vivo amyloid aggregation opening up new horizons to the understanding of protein deposition related to conformation diseases. This exciting possibility suggests that the utilization of bacterial cells in order to perform a first cribbage of molecules with activity anti-prion or anti-aggregation could be, in the most next future, an election method for researchers and the pharmaceutical industry.

Protein synthesis in the ribosome

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The ribosome is the factory of the cellular proteins. The bacterial ribosome is composed of three RNA molecules (that represents about two third of the ribosome mass) and more than 50 proteins where its key components are highly conserved {Cech, 2000 #1; Yusupov, 2001 #10}. The bacterial 70S ribosome comprises two subunits: a large 50S subunit, and a small 30S subunit. The 30S subunit contains a 16S rRNA (ribosomal RNA) plus 20 proteins. The 50S subunit contains a 23S and a 5S rRNA plus over 30 proteins. The 23S rRNA of the large 50S subunit folds into six secondary structural domains (I to VI) containing over 130 RNA helices {Voisset, 2008 #2}. Nevertheless, exactly how all of the different ribosomal components contribute to protein synthesis and folding is still not clear {Cech, 2000 #1}.
Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct mRNA start site and the reading frame are selected when, with the help of initiation factors, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNA anticodon. This yields a 30S initiation complex that is an intermediate in the formation of the 70S initiation complex that occurs on joining of the 50S ribosomal subunit to the 30S and release of the initiation factors {Simonetti, 2009 #5; Simonetti, 2008 #3}. The atomic resolution of the two subunits of the large ribosome has allowed deduce that RNA components of the large subunit accomplish the key peptidyl transferase reaction. This chemical catalysis is simple -the joining of amino acids through amide (peptide) linkages- it performs the remarkable task of choosing the amino acids to be added to the growing polypeptide chain by reading successive messenger RNA (mRNA) codons {Cech, 2000 #1; Nissen, 2000 #6}; In the process, about 30 nucleotides of the mRNA are wrapped in a groove that encircles the neck of the 30S subunit. At the interface, only about eight nucleotides, centred on the junction between the A and P codons, are exposed, and bond almost exclusively to 16S rRNA {Yusupova, 2001 #9}. Thus, ribosomal RNA (rRNA) does not exist as a framework to organize catalytic proteins. Instead, the proteins are the structural units and they help to organize key ribozyme (catalytic RNA) elements {Ban, 2000 #7; Nissen, 2000 #6; Ban, 1999 #8}. In brief, the ribosomal proteins interact with the rRNA structures forming a specific and active structure.

Relationship between mRNA expression levels and protein solubility

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Regulació expression gènica. Operons. Micro-arrays.

The ribosome-borne protein folding activity
Chaperone activity in the cytosol medium

Composition and characterization of inclusion bodies

Usually recombinant protein expression in bacteria results in the accumulation of the main polypeptide in IBs. The observation of one of this protein deposits by electron microscopy reveals an apparently amorphous electro dens mass. Deeper analysis show that they are high dense (1.3mg•mL-1), porous and refractive particles from 0.5 to 1.3 m diameter that could be found in cytoplasm or periplasm {Carrio, 1998 #12; Bowden, 1991 #13; Arie, 2006 #14}. Its composition is constituted mainly by the recombinant protein (>90% in overexpressed proteins) which could be presented at different conformations from unfolded to native {Arie, 2006 #14; Garcia-Fruitos, 2005 #15; Carrio, 2005 #16; {Carrio, 2005 #17; {Villaverde, 2003 #18; Carrio, 2002 #19; Clark, 2001 #20; Schrodel, 2005 #21; Garcia-Fruitos, 2007 #22}; At early stages of deposition, host proteins might represent up to 50% of the IB composition {Carrio, 2000 #23}. The analysis of purified IBs, simultaneously with the recombinant polypeptide reveals proteolytic fragments of this protein, ribosomal components, traces of membrane proteins, phospholipids and nuclei acids, some of these probably were retained during IBs treatment {Rinas, 2007 #24}. In addition, together with the IBs components are present polypeptide quality control molecules, as small heat-shock proteins IbpA and IbpB or chaperones as DnaK and GroEL {Carrio, 2005 #17}, this data reveals the close relation of this machinery with the IBs formation.

Aggregation in bacteria: Inclusion bodies formation

The over-expression of amyloidogenic proteins in prokaryotic systems usually involve in the formation of IBs {West, 1999 #49}. More and more, recombinant protein production is an essential tool used to obtain amyloid proteins and their mutated partners for studying the polypeptide deposition process principally due to its relation with a large number of human conformational diseases. The accumulation of polypeptides as insoluble deposits has been also observed after the over-expression of proteins with high aggregation propensity. The compilation of all these data suggests a connection between the propensity of a protein to form amyloid deposits and their ability to develop IBs in bacterial cell.

The recombinant expression of unstructured proteins widely related with the development of amyloid aggregates, as amyloid -peptide, PrP or HET-s(218-289), commonly results in the establishment of intracellular aggregation associated with the IBs development. A similar process can be observed with the recombinant expression of globular proteins with high aggregation propensity. As follows, globular proteins related to amyloid disorders as transthyretin, β2-microglobulin, insulin or lysozyme implicated diverse fatal diseases have a high chance to aggregate as IBs. Moreover, recently it has been reported that the recombinant polypetide distribution between the soluble and insoluble cellular fractions is related with the protein aggregation propensity. In the amyloid-β-peptide case, a recent study realized using a set of mutants reveals that those variants predicted to possess the higher predisposition to aggregate are mainly detected in the insoluble fraction whereas those with the lower deposition tendency are in the soluble one {de Groot, 2006 #50; de Groot, 2006 #51; de Groot, 2006 #52}.

PNAS Taddei!!!

Specificity during inclusion bodies formation

At biological conditions the protein globular structure is stabilized by several weak contacts that cooperate to support the native form. This conformation is preserved by a delicate thermodynamic balance between different parameters as hydrogen bonds, hydrophobicity and electrostatic interactions. This equilibrium prevents the transition into unfolded and misfolded forms and, as a result, avoids protein aggregation. In contrast, under recombinant expression conditions the elevate tax of protein can entail a balance disequilibrium and entail the apparition of unfolded or partially unfolded protein with exposed hydrophobic regions able to develop intermolecular contacts and start protein aggregation into IBs. This event has been observed during the overexpression of different proteins that do not share any similarity between sequence, structure, size or origin however it is not a simple process driven by non-specific interactions as has been purposed short time ago. Moreover, recently it has been reported that the molecular contacts related with the IB assembly are sequence-specific {Carrio, 2005 #16}. In this sense, later works have presented interesting evidences of this specificity analysing the aggregation of different proteins in vitro. These results showed protein aggregation as concentration dependent event developed via homologous and specific contacts between the polypeptide chains {London, 1974 #54; Speed, 1996 #55}.

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Interestingly, amyloid aggregation in vitro is also a sequence specific process which undergoes the formation of homologous aggregates in fibrillar form where cross-seeding and heterologous coaggregation is very rare {Chiti, 2006 #25}. In this way, it has been described that the development of these deposits is a nucleation-polymerization process that depends on the initial protein concentration. This process can be mathematically analyzed as an autocatalytic reaction that can be accelerated by addition of homologous preaggregated protein acting as “aggregation seeds” {Harper, 1997 #56; Jarrett, 1993 #57; Sabate, 2003 #58}. This property has been also observed in vitro after the addition of purified IBs into a solution of soluble recombinant protein. This study shows that the IBs seeding is specific to dose and sequence but also to protein conformation since an IB can act as an effective aggregation seed for the deposition of homologous but not heterologous polypeptides and attracts the fully or partially unfolded protein but not the native structure counterpart {Carrio, 2005 #16; Morell, 2008 #26}. Furthermore, the study in vivo of two high aggregation prone proteins coexpressed in the same prokaryotic cell using a plasmid encoding both genes permits to observe two extreme situations: the formation of two different types of IBs enriched in one type of recombinant protein {Hart, 1990 #59} or the formation of one IB with different aggregation zones enriched in one type of recombinant protein {Morell, 2008 #26}. In the second situation, the kinetic of protein folding and aggregation influences on the coaggregation since after the expression of the protein with higher aggregation propensity is the first to start the deposition and as a result it is contained in the most buried zone whereas the other one is localized at the IB surface {de Groot, 2009 #60}.

The importance of polypeptide sequence and structure

IBs in the cell: Quality and dynamics

To control protein quality and prevent misfolding and aggregation evolution provide cells with an accurate machinery system {Vendruscolo, 2003 #28}. The fail of this regulatory system in human is associated to the occurrence of protein deposition diseases as Alzheimer {Fonte, 2002 #29} or Parkinson disease {McNaught, 2002 #30; Tanaka, 2004 #31; Gao, 2008 #32}. Moreover, the different living systems share similarities between the quality control elements and the deposits formation {Doyle, 2008 #33; Vendruscolo, 2003 #28; Morell, 2008 #26}. Therefore bacteria could be a simple model to study the different factors that influence in the development of protein deposit in vivo. In addition, members of this quality system have been found physically associated with amyloid deposits however the reason of this connection remains unknown {Wilson, 2008 #34}.

The cellular milieu where the IBs are building on is a key factor for their final structure and composition. As follows, IBs are associated to a dynamic equilibrium between protein deposition and removal involving a constant traffic of soluble and insoluble polypeptide forms {Carrio, 1999 #35}. This exchange of protein is actively regulated by the cell quality control system which is stimulate under stress states and operates disaggregating, unfolding and activating the proteins {Gonzalez-Montalban, 2006 #36; Mogk, 2004 #37; Mogk, 2003 #40; Mogk, 2003 #41; Mogk, 2003 #42; Weibezahn, 2004 #38; Dougan, 2002 #39}. In this way, it has been reported that few hours after stopping the polypeptide expression the control machinery is able to decrease the IBs mass and almost eliminate them from the cellular inner {Carrio, 2001 #43}. According with this, the IB appears to play a role of protein reservoir {Carrio, 2002 #19; Carrio, 2001 #43; Gonzalez-Montalban, 2006 #36} where the polypeptide can be stored and extracted {Carrio, 1999 #35; Vera, 2005 #44; Corchero, 1997 #45}. The constantly activity of this control machinery modifies actively the organization the IBs components. This effect has been observed studying the consequence of the removal of misfolding protein from the IBs surfaced {Schlieker, 2004 #46; Schlieker, 2004 #47} which produce a gradually increment of the amount of native-like and active protein in the IBs inner {Garcia-Fruitos, 2007 #22}. In this way, Villaverde and coworkers has used bacterial knockouts to study the effect of deleting different chaperones upon the protein solubility distribution {Garcia-Fruitos, 2007 #48}. On the whole, it has been detected a general increasing of β-sheet compactness inside de IBs, a decrease of the proteolytic activity and an unexpected increment of the protein activity in both soluble and insoluble fractions. These results show that chaperones lead protein digestion even against active polypeptide conformations resulting in the improvement of solubility but also the sacrifice of protein functionality.

Throughout, the cellular inner is populated by a huge variety of protein conformations that could by located in the soluble or in the insoluble fractions. As follows, it is possible to find incorrect folded polypeptides as soluble aggregates or native like polypeptide inside the IBs. Moreover, the active protein is not an exclusive feature of the soluble fraction since there could be present several non functional structures and the IBs inner could contain a quantity of active conformations. The complexity of this mixture is increased by the fact that this variety of structures coexist in a constant fluctuation between partially folded states and native states. Despite the intellectual input accumulated during the last years, it is still unknown how could coexist of misfolded prone regions enclosed in the β-sheet architecture of IBs and native-like folded domains.

Chaperone activity in the aggregated protein

Amyloid protein structure in bacterial IBs

Amyloid fibrils are thread-like protein aggregates with a cross- structure, which is composed by -strands stacked perpendicular to the fibril axis, responsible for a number of fatal human protein deposition diseases {Chiti, 2006 #25}. Recently, different groups have converged to demonstrate unequivocally the existence of amyloid structures inside bacterial IBs {Morell, 2008 #4; Wang, 2008 #27}. As mentioned above, protein aggregation into IBs is a process that comprises proteins self-associate into imperfectly ordered macroscopic entities. Despite the IBs’ amorphous appearance, they are not only clusters of misfolded proteins stuck to each other through nonspecific hydrophobic contacts {Wang, 2008 #27} but also, as amyloid aggregates, they are often enriched by -sheet structure detectable by X-ray diffraction, Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD). Like this, they are able to bind amyloid-tropic dyes as Thioflavin-T (fluorescence spectroscopy) or Congo Red (UV/Vis spectroscopy and birefringence), present proteinase K digestion resistance and fibrillar structures are detectable by transmission electronic microscopy (TEM) or atomic forces microscopy (AFM). Moreover, as mention before, they have a seeding capacity reminiscent of amyloids, form homogeneous aggregates without cross-aggregation, and display aggregation propensities strongly affected by mutations.

Recently, two independent research groups have thoroughly analyzed the IBs fine structure of different recombinant proteins obtaining closed conclusions. Riek et al analyse the conformation of three inclusion bodies constituted by three proteins without disulfide bridges and with distinctive native foldings in order to cover the folding universe (-helix, -helix/-sheet and -sheet). In this work, the authors confirm by X-ray diffraction and CD spectroscopy that the studied IBs are organized -sheet-rich aggregates. In addition, they show that these deposits contain fibrillar structure observable by TEM and bind specific amyloid dyes. Moreover, an exhaustive analysis by measuring quenched hydrogen/deuterium (H/D) exchange with nuclear magnetic resonance (NMR) have been realised with the aim of identify the solvent-protected backbone amide protons involved in IBs hydrogen bonds,. The obtained results confirm that the IBs of these three proteins contain sequence-specific positions of regular secondary structure that exhibits cross-β signal {Wang, 2008 #27}. In this sense, Morell et al employed a complete battery of analytical techniques to determine the structure of A42 and Vp1 IBs demonstrating the existence of rich cross- aggregates by FTIR and CD as well as the presence of fibrillar material by TEM and AFM. Like this, the results obtained studying the binding of specific ThT amyloid dye, the seeding capability and the proteinase K resistance show that these IBs have typical amyloid characteristics {Morell, 2008 #26}.

In conclusion, as propose by Riek and co-workers in their excellent work: “bacterial inclusion bodies contain amyloid-like structure”. Furthermore, 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 are evolutionary selected to avoid the amyloid conformation {Wang, 2008 #27}.

Amyloid transmissibility: Inclusion bodies as an infectious agent

Prions represent a particular subclass of amyloids for which the aggregation process becomes self-perpetuating in vivo and thus infectious. In this sense, it could be very interesting to verify if the prion proteins deposited inside IBs are able to acquire the typical fibrillar structure and also display evident prion properties. Recently, Wasmer et al have analyzed purified IBs of HET-s(218-289), C-terminal region of HET-s spanning residue 218 to 289 as the most essential domain for amyloid formation and prion propagation of the filamentous fungus Podospora anserina. HET-s IBs were used to infect prion-free P. anserina strains using a protein transfection method. While insoluble extracts from a control strain containing only the empty vector did not induce the development of the [Het-s] prion form, strains transfected with HET-s(218-289) IBs acquired the [Het-s] prion aggregation at a high frequency comparable to the one obtained with HET-s(218-289) amyloids assembled in vitro, confirming that HET-s(218-289) IBs display [Het-s] prion infectivity {Wasmer, 2009 #11}. This results indicates that the HET-s(218-289) acquires an infectious prion fold in E. coli IBs. Then, the structural characterization of E. coli IBs of the fragment 218-289 from the fungal prion protein HET-s was realized {Wasmer, 2009 #11}. The HET-s(218-289) protein in IBs shows the highly ordered amyloid structure previously characterized for prion fibrils assembled in vitro. Solid state NMR confirms that the structure of HET-s(218-289) in IBs is similar to the fibrils obtained in vitro, suggesting that the amyloid fibril and IB formation processes are related {Wasmer, 2009 #11}.

Incloure informació dels Microbiol Cell Factories!!!

Functional amyloids in bacteria
Curli, Chaplins

Future goals