Scientific Challenges - Literature review Example

Scientific challenges Introduction There are several neurodegenerative disorders that are associated with themisfolding of some proteins. The disorders include the Parkinson disease where the protein misfolds into toxic oligomers that are soluble in nature and also stable cross-β fibers. Amyloidogenesis is also another problem encountered with by the researchers; it involves the purification of the recombinant protein that originates from diverse systems such as animal cells and bacteria. In this process, the proteins combine to form inclusions and take the amyloid form. As a result, the amyloid hinders the manufacture of therapeutic proteins by pharmacists. It also frustrates functionally as well as structural studies (Martin et al 2005, p.1115). Scientists as well as researchers do not understand how Hsp104, which is a hexameric AAA+ ATPase obtained from yeast, disaggregates various structures that include aggregates induced by stress, the prions and also the α-synuclein conformers are related with the Parkinson disease. The researcher aims in establishing the Hsp104 hexamers undergo various mechanisms of collaborating intersubunitly to disaggregate the aggregates that are induced by stress versus the amyloid. So as to resolve the aggregates that are disordered the Hsp104 subunits team up in a non-cooperating manner by the employment of probabilistic substrate binding and also the ATP hydrolysis. The solutions to these problems enable humanity understand the reverse function played by cells in amyloid formation (Barnhart & Chapman 2006, p. 140). Discussion Paper 1 (Martin et al., 2005, Nature, 437, 1115-20) In making efforts to optimize and also reduce the side effects of therapy, the Hsp104 gets engineered and also potentiated to be able to dissolve specific aggregates. The aggregates should be related to the Parkinson disease. The disaggregase activity of Hsp104can is tailored and also enhanced for any protein. Therefore, Hsp104 variant that have been optimized by a substrate increases solubility of proteins. It also enhances the purification of proteins in the various settings. Nevertheless, the limited understanding of the structure and mechanics of Hsp104 hexamers hinders the research. The individual subunits of the Hsp104 hexamer are not understood how they coordinate the translocation of substrates. It is also not understood how ATP hydrolysis to solubilize proteins that are unrelated and have been trapped in the aggregates (Martin et al 2005, p.1111). The major question is how the single subunits work together to promote remodeling of the substrate. The experiments involve all NTP-fueled, hexameric translocases and not only for Hsp104. There are several proposals based on different intersubunit collaboration made to aid in finding solutions. One of the proposals is the probabilistic models. In this model, the functioning of the individual subunits is independently and also non-co-operatively. Models of sub-global are the second models. In this model the subunits co-operate. The third model is a model of global cooperativity, in this method subunits co-operate. They do so in sequence and also in concert. The three models centre their arguments on the event coordination by the NTPase. The models have not given any attention to the individual subunits that are within the hexamer and their contribution to the translocation and also binding of the substrates (Tucker and Sallai 2007, p. 12). It remains unclear on whether cooperative activities of the ATPase must get unified so as to substrate handling cooperatively. The aspect that requires be resolved is to determine whether a ring-translocase can get various modes of intersubunit unification so as to remodel the substrates that force various mechanical demands. In the method, it is made clear that Hsp104 unify using the various available different mechanisms to disaggregate the disordered aggregates against amyloids. The findings indicated that the E. coli that are a homolog of Hsp104, ClpB coordinated the collaboration of the subunits on different ground as Hsp104. It is irrespective of the fact that Hsp104 and ClpB function using a similar mechanism (Ogura et al. 2008, p. 34). The Hsp104 has operational plasticity that is similar to the adaptable disaggregates activities that are intended to the demands of the yeast proteome. It also includes the prion disaggregation. On the other hand, ClpB is placed for optimal disordered aggregate dissolution. It also has little ability in dissolving the amyloid. The strategy used in the determination of the individual subunits employs the use of mutant doping. It is towards making efforts in protein disaggregation and makes the definition of the mechanochemical coupling of the of Hsp104 hexamers. Therefore, mutant subunits that are defective in ATP hydrolysis, substrate binding can also get mixed with wild subunits and yield heterohexamer with different ratios of mutant protein and WT. The strategy has resulted to major insights to other NTP ring-translocases. However, the strategies are dependent on the mixing of WT and mutant subunits in the level of monomers (Martin et al 2005, p.1119). The use of Hsp104DPL is employed to indicate how Hsp104 subunits move towards the binding of the substrates. The Hsp104DPl has the Y662A and also the Y257A mutations. They incorporate the WT hexamers and have little effects on the activities of ATPase. The dilution of Hsp104 by the usage of buffers has minimal effects. On the other hand, the addition of Hsp104DPL resulted to a linear decrease in the activities of disaggregates. The same data was obtained with the heat-denatured GFP aggregates and also the heat-denatured citrate synthase aggregates. The tolerance of the Hsp104 hexamers to the Hsp104DPL subunits indicates that for the disordered aggregates substrate in a manner that is probabilistic. The research has, therefore, been useful in providing answers to the scientists (Tsonis 2003). Paper 2 (Joly and Buck, 2011, J Biol Chem, 286, 12734-42) The authors used various strategies in addressing their problems that entailed majority of the AAA+ proteins. The authors indicated that most of the AAA+ is as active as homo-oligomers. However, the knowledge of individual’s contribution subunits to the activities of the hexamers is not complete or often absent. Lack of this knowledge hence puts the validity of the methods employed by the authors to address this problem into question. Several analyses from the methods employed gave six virtually identical subunits. Their precise functionality and organization in the hexamer tends to be very difficult to probe. A major challenge that is presented in the method used by the authors is the fixation of the hexamer’s organization via linking the subunits together (Barnhart & Chapman 2006, p.142). Chemical linking is one of the methods that were used in addressing the indicated problem. It played a role the role of tracking the amount of subunits that were present in the oligimer. However, it fails to give a good tool of addressing the subunits’ geometrical distribution for functionality. The authors also employed two additional methods in their attempts of exhaustively addressing the problem. One of the methods involved mixing experiments that were based on the hetero-oligimers’ reconstitution that possessed various variants. The second method is a single chain strategy on the basis of the engineering of a polypeptide that is covalently linking two or more oligomer’s subunits. The method was aimed at allowing the subunit organization’s differentiation in the final oligomer (Tucker and Sallai 2007, p. 13). As a method of identifying a solution to this problem, the authors combined these two methods and made use of the model AAA+ activator protein that is specialized in σ54 that is dependent on transcription activation and the bacterial enhancer that binds proteins. After hexamerization had taken place, bEBPs would use the mechanochemical energy originating from the ATP hydrolysis. The energy would remodel the RNA polymerase that was inactive to become transcriptionally active. The use of the nucleotide analog in these methods highlighted the objective of conserved PspF in an interaction that was direct with σ54 factors present in RPc prior to the ATP hydrolysis. However, the bEBP hexamer’s intrinsic organization raised questions of the number of GAFTGA motifs that were required both interact with RPc and at the same time activate the transcription (Ogura et al. 2008, p. 34). The use of a single chain polypeptide method reveals the novel characteristics of the initial interaction of the PspF-σ54. The interactions suggest an asymmetry as well as possible ring discontinuity in conjunction with the obligations of the sole subunits within the hexamer on the basis of substrate remodeling activity. After establishing the single chain forms of the trimer and dimer the methods also helped to determine whether all the hexamer’s subunits were necessary for σ54 RPo formation as well as binding interactions. Through the investigation of the impact caused by the defective subunit’s relative location within the hexamer, the methods by the authors of this article finally revealed that for a given number of T and W subunits, the activity’s level can differ on the basis of certain hexamer organization (Joly and Buck 2011, p.12738). The authors also confirmed that the requirement of the two adjacent W subunits for binding the optimal σ54. The results demonstrated that there exist several arrangements of the hexamer that supports σ54 binding. The arrangement can also be subdivided in either productive or unproductive RPo formation. The other result that was achieved is that possible interactions between the σ54 and PspF hexamer can occur. The position of the subunits determines the final activities of the hexamer. The validity of the research is satisfactory as it provides answers that help researchers understand the relationship between the disorders that include the Parkinson disease and the proteins misfolding into toxic oligomers that are soluble in nature and also stable cross-β fibers (Joly and Buck 2011, p.12741). References List Barnhart, M. M . & Chapman, M. R. (2006). Curli biogenesis and function. Annu Rev Microbiol. 60, pp131–147 Bosl, B., Grimminger, V. & Walter, S. (2005). Substrate Binding to the Molecular Chaperone Hsp104 and Its Regulation by Nucleotides. Journal of Biological Chemistry. 280, pp38170–38176. Davidson, E. H. (2006). The Regulatory Genome: Gene Regulatory Networks In Development And Evolution. Burlington, Elsevier. Joly, N. and Buck, M. (2011). Gene Regulation: Single Chain Forms of the Enhancer Binding Protein PspF Provide Insights into Geometric Requirements for Gene Activation. Journal of Biological Chemistry 286, pp12734-12742. Martin, A., Baker, T. A. & Sauer, R. T. (2005). Rebuilt AAA1 motors reveal operating principles for ATP-fuelled machines. Nature 437, pp.1115-1119. Ogura, T., Matsushita-Ishiodori,Y., Johjima, A., Nishizono, M., Nishikori, S., Esaki, M., & Yamanaka, K. (2008). Biochem. Soc. Trans. 36, 68–71  Tucker, P. A., & Sallai, L. (2007). Current Opinion. Struct. Biol. 17, 641–652. Tsonis, P. A. (2003). Anatomy of gene regulation: A three-dimensional structural analysis. New York, Cambridge University Press. Read More