The alveoli of mammalian lungs are covered in a thin lipid film referred to as pulmonary surfactant. The primary purpose of pulmonary surfactant is to reduce the surface tension of the air/liquid interface allowing breathing with minimal effort required. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is inconsequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that for bothanionic DOPG and cationic DOTAP films which are in the fluid phase, amyloid-β interacts with the bilayer much quicker than in zwitterionic DPPC which is in the gel phase. Approaching 24 hours, we see small fibrils form on the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution. For fluid phase bilayer membrane, disruption is also observed. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (pulmonary surfactant BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is in consequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that in anionic DOPG films, amyloid-β inserts into the bilayer much quicker than in zwitterionic DPPC. Approaching 24 hours, we see small fibrils form in the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution.

The alveoli of mammalian lungs are covered in a thin lipid film referred to as pulmonary surfactant. The primary purpose of pulmonary surfactant is to reduce the surface tension of the air/liquid interface allowing breathing with minimal effort required. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is inconsequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that for bothanionic DOPG and cationic DOTAP films which are in the fluid phase, amyloid-β interacts with the bilayer much quicker than in zwitterionic DPPC which is in the gel phase. Approaching 24 hours, we see small fibrils form on the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution. For fluid phase bilayer membrane, disruption is also observed. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (pulmonary surfactant BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is in consequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that in anionic DOPG films, amyloid-β inserts into the bilayer much quicker than in zwitterionic DPPC. Approaching 24 hours, we see small fibrils form in the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution.

Atomic force microscopy is one of the powerful instruments used to explore the mechanical properties of nanoscale materials. It not only can produce high-resolution images and surface mechanical properties, but also can make use of its probe for surface etching. In this study, we first use atomic force microscopy to measure the Adhesion Map of insulin amyloid fibers, then conduct mechanical lithography on the surface with the probe. In the end, we discuss the effect on insulin amyloid fibrils due to exert different forces and different speeds with the probe. According to Nanoindentation theory and Hertzian model, we can derive the Young's modulus of insulin amyloid fibrils from force-indentation relations. Then we cut the Insulin amyloid fibers with probe. The results showed that when we applied 3.23 nN force by the probe, the insulin amyloid fibers began to break. When we applied 7.07 nN force, insulin amyloid fibers are cut off easily. Therefore, we can bite off insulin amyloid fibers of different lengths and sections, and arrange in the desired pattern by atomic force microscope.

Many proteins can easily attain a non-native fold and be of no use or even a detriment to the host. The host cell has a myriad of molecules dedicated to assisting nascent and existing proteins in folding properly and maintaining the native fold. Of these molecular chaperones, the small Heat Shock Proteins (sHSP’s) are an important group and worthy of study. The sHSP’s are a diverse group of proteins that have in common an a-crystallin domain and generally display a chaperone activity. A sHSP (HSP20) isolated from the cattle parasite Babesia bovis has similar activities, and limited sequence homology to other a-crystallins. The gene encoding HSP20 was cloned into an expression system where the gene product was induced and purified for study. It was shown that HSP20 inhibits thermally induced aggregation of alcohol dehydrogenase at equimolar ratios. HSP20 was also used to significantly reduce amyloid formation of the b-Amyloid (1-40) Peptide in vitro at the sub-stoichiometric ratio of 1:10. A study of the oligomeric forms of HSP20 using size exclusion chromatography and gel electrophoresis revealed a broad range of multimers present in solution. The distribution of oligomers was affected by altering the solution conditions and concentration of the protein. The domains responsible for multimerization of HSP20 were mapped via sequence homology with known a-crystallins. These regions correspond to 12 carboxy-terminal amino acids and 50 amino-terminal amino acids. Truncated versions of HSP20 lacking these proposed oligomerization domains were created using PCR of the original gene and cloning into an expression vector as before. Using size exclusion chromatography, gel electrophoresis and analytical centrifugation, we show that the deleted domains alter the multimeric population of the protein in solution. The carboxy-terminal domain has a slight effect on multimerization while the amino-terminal deletion results in a drastic reduction in any multimers above a dimer under the conditions tested. Despite this drastic change in the multimerization of HSP20, there were no changes in the activities observed when compared to the full-length form. From this we conclude that the regions responsible for multimerization play little role in the observed activities of HSP20.

Proteins need fold to perform their biological function. Thus, understanding how proteins fold could be the key to understanding life. In the first study, the stability and structure of several !-hairpin peptide variants derived from the C-terminus of the B1 domain of protein G (PGB1) were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N-terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N-terminal charge-charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the !-turn conformation. Our results indicate that the structure and stability of this !?hairpin peptide can be modulated in numerous ways and thus contributes towards a more complete understanding of this important model !-hairpin as well as to the folding and stability of larger peptides and proteins. The second study revealed that PGB1 and its variants can form amyloid fibrils in vitro under certain conditions and these fibrils resemble those from other proteins that have been implicated in diseases. To gain a further understanding of molecular mechanism of PGB1 amyloid formation, we designed a set of variants with mutations that change the local secondary structure propensity in PGB1, but have similar global conformational stability. The kinetics of amyloid formation of all these variants have been studied and compared. Our results show that different locations of even a single mutation can have a dramatic effect on PGB1 amyloid formation, which is in sharp contrast with a previous report. Our results also suggest that the "-helix in PGB1 plays an important role in the amyloid formation process of PGB1. In the final study, we investigate the forces that contribute to protein stability in a very general manner. Based on what we have learned about the major forces that contribute to the stability of globular proteins, protein stability should increase as the size of the protein increases. This is not observed: the conformational stability of globular proteins is independent of protein size. In an effort to understand why large proteins are not more stable than small proteins, twenty single-domain globular proteins ranging in size from 35 to 470 residues have been analyzed. Our study shows that nature buries more charged groups and more non-hydrogen-bonded polar groups to destabilize large proteins.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 121-158).

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder, characterized by global cognitive decline and progressive memory loss. As many other neurological disorders characterized by “proteinopathy”, pathology of AD includes beta-amyloid plaques and tau neurofibrillary tangles, which imply a crucial role of the cellular degradation systems in maintaining homeostasis of protein turnover. This is especially important for post-mitotic neuronal cells since aggravating protein crisis cannot be alleviated by cell division. Autophagy is a cellular degradation process that removes or recycles long-lived proteins and damaged organelles, with its enhancement being remarkably implicated during the progression of Alzheimer’s disease (AD). The majority of studies have hitherto focused on the mechanism of how oligomeric Ah, as one of the potent toxic species in AD, activates autophagy. However, how autophagy is activated remains to be elucidated. The goal of this study is to reveal the underlying mechanisms of autophagy and the subsequent events. Using imaging and biochemical analysis in primary cultures of rat hippocampal neurons, I found that oligomeric An-induced autophagy was initiated by aggregation of the endoplasmic reticulum (ER), in an mTOR-independent pathway. Ao-triggered autophagosomes were derived from omegasomes, starting from the ER aggregation sites. Aggregation of the ER facilitated the clustering of Atg14L to propel the recruitment of Beclin1 and Vps34, which contributes to generation of omegasomes. I further found that p62 targeted to ER aggregates possibly through the enhanced ubiquitinated ER chaperones trapped at ER aggregation sites, implicating the underlying mechanism for how p62 are recruited to autophagosome formation sites (omegasomes). Herein, I report key steps for activation of AH-triggered autophagy, whereby a mechanistic link between ER aggregation, autophagic activation and recruitment of p62 to autophagosome formation sites is revealed. First, Ao-induced ER aggregation triggers autophagy, via the recruitment of Beclin 1 and Vps34 to Atg14L clusters, which is a promoting factor for omegasome formation at the ER aggregation site. Second, the recruitment of p62 to omegasomes is likely mediated by the attraction of the underlying accumulation of ubiquitinated ER chaperones at the ER aggregation site. Up-regulation of autophagy is an early sign of AD. The activation of autophagy without tightly manipulation may contribute to neuronal damage in AD. In addition, how the autophagic substrates can be efficiently incorporated into the autophagic pathway is important for understanding the sustainability of autophagy. Therefore, my study on elucidating how ER aggregation initiates autophagy and the autophagic substrate/cargo receptor p62 are loaded onto autophagosome formation sites may help us to identify a potential therapeutic strategy or target for AD patients. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

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