• Biotechnology and Bioprocess Engineering:BBE
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• 제6권4호
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• pp.237-243
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• 2001

Substantial progress has been made towards understanding the folding mechanisms of proteins in virto and in vivo even though the general rules governing such folding events remain unknown. This paper reviews current folding models along with experimental approaches used to elucidate the folding pathways. Protein misfolding is discussed in relation to disease states, such as amyloidosis, and the recent findings on the mechanism of converting normally soluble proteins into amyloid fibrils through the formation of intermediates provide an insight into understanding the pathogenesis of amyloid formation and possible cules for the development of therapeutic treatments. Finally, some commonly adopted refolding strategies developed over the part decade are summarized.

• 생명과학회지
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• 제17권8호통권88호
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• pp.1129-1134
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• 2007

bPDI가 ER내 misfolding 단백질의 생성을 제한함으로써 곤충변역과 관계하는지를 해석하기 위하여 bPDI가 과발현(overexpression)되는 곤충세포주와 이와 반대로 bPDI가 억제발현(knock-down)되는 곤충세포주를 제작하여 bPDI가 곤충면역에 관련하는지를 해석하였다. bPDI가 과발현되는 세포주 (Sf9-bPDI)는 정상세포주(Sf9)나 pIZT/V5-His 벡터만 도입된 세포주(Sf9-pIZT)에 비하여 생존율이 30% 이상 높았지만, bPDI의 전사체 발현이 억제된 세포주(Sf9-bPDI-dsRNA)는 오히려 정상세포주나 pIZT/V5-His 벡터만 도입된 세포주에 비하여 생존율이 약 15%낮았다. 이와 같은 결과로써, bPDI는 ER내 misfolding 단백질의 생성을 제한함으로써 곤충의 ERSE과 밀접하게 관련할 것이라 추정할 수 있었다.

• BMB Reports
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• 제39권1호
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• pp.55-60
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• 2006

We describe a phage display strategy, based on the differential resistance of proteins to denaturant-induced unfolding, that can be used to select protein variants with improved conformational stability. To test the efficiency of this strategy, wild-type and two stable variants of ${\alpha}_1$-antitrypsin (${\alpha}_1AT$) were fused to the gene III protein of M13 phage. These phages were incubated in unfolding solution containing denaturant (urea or guanidinium chloride), and then subjected to an unfavorable refolding procedure (dialysis at $37^{\circ}C$). Once the ${\alpha}_1AT$ moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint ($C_m$) of the ${\alpha}_1AT$ variant, around 20% of the phage retained binding affinity to anti-${\alpha}_1AT$ antibody due to a low refolding efficiency. Moreover, this affinity reduced to less than 5% when 10 mg/mL skimmed milk (a misfolding-promoting additive) was included during the unfolding/refolding procedure. In contrast, most binding affinity (>95%) remained if the ${\alpha}_1AT$ variant was stable enough to resist unfolding. Because this selection procedure does not affect the infectivity of M13, the method is expected to be generally applicable to the high-throughput screening of stable protein variants, when activity-based screening is not possible.

• BMB Reports
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• 제50권1호
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• pp.37-42
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• 2017

The tubby protein (Tub), a putative transcription factor, plays important roles in the maintenance and function of neuronal cells. A splicing defect-causing mutation in the 3'-end of the tubby gene, which is predicted to disrupt the carboxy-terminal region of the Tub protein, causes maturity-onset obesity, blindness, and deafness in mice. Although this pathological Tub mutation leads to a loss of function, the precise mechanism has not yet been investigated. Here, we found that the mutant Tub proteins were mostly localized to puncta found in the perinuclear region and that the C-terminus was important for its solubility. Immunocytochemical analysis revealed that puncta of mutant Tub co-localized with the aggresome. Moreover, whereas wild-type Tub was translocated to the nucleus by extracellular signaling, the mutant forms failed to undergo such translocation. Taken together, our results suggest that the malfunctions of the Tub mutant are caused by its misfolding and subsequent localization to aggresomes.

• BMB Reports
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• 제38권3호
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• pp.275-280
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• 2005

For most of proteins to be active, they need well-defined three-dimensional structures alone or in complex. Folding is a process through which newly synthesized proteins get to the native state. Protein folding inside cells is assisted by various chaperones and folding factors, and misfolded proteins are eliminated by the ubiquitin-proteasome degradation system to ensure high fidelity of protein expression. Under certain circumstances, misfolded proteins escape the degradation process, yielding to deposit of protein aggregates such as loop-sheet polymer and amyloid fibril. Diseases characterized by insoluble deposits of proteins have been recognized for long time and are grouped as conformational diseases. Study of protein folding mechanism is required for better understanding of the molecular pathway of such conformational diseases.

• Molecules and Cells
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• 제23권2호
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• pp.123-131
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• 2007

Extracellular stresses induce heat shock response and render cells resistant to lethal stresses. Heat shock response involves induction of heat shock proteins (Hsps). Recently the roles of Hsps in neurodegenerative diseases and cancer are attracting increasing attention and have accelerated the study of heat shock response mechanism. This review focuses on the stress sensing steps, molecules involved in Hsps production, diseases related to Hsp malfunctions, and the potential of proteomics as a tool for understanding the complex signaling pathways relevant to these events.

• BMB Reports
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• 제38권3호
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• pp.259-265
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• 2005

Proteins must fold into their correct three-dimensional conformation in order to attain their biological function. Conversely, protein aggregation and misfolding are primary contributors to many devastating human diseases, such as prion-mediated infections, Alzheimer's disease, type II diabetes and cystic fibrosis. While the native conformation of a polypeptide is encoded within its primary amino acid sequence and is sufficient for protein folding in vitro, the situation in vivo is more complex. Inside the cell, proteins are synthesized or folded continuously; a process that is greatly assisted by molecular chaperones. Molecular chaperones re a group of structurally diverse and mechanistically distinct proteins that either promote folding or prevent the aggregation of other proteins. With our increasing understanding of the proteome, it is becoming clear that the number of proteins that can be classified as molecular chaperones is increasing steadily. Many of these proteins have novel but essential cellular functions that differ from that of more 'conventional' chaperones, such as Hsp70 and the GroE system. This review focuses on the emerging role of molecular chaperones in protein quality control, i.e. the mechanism that rids the cell of misfolded or incompletely synthesized polypeptides that otherwise would interfere with normal cellular function.

• 대한의생명과학회지
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• 제16권1호
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• pp.65-67
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• 2010

The endoplasmic reticulum (ER) is a multifunctional intercellular organelle in which several posttranslational modification steps occurred such as protein folding, lipid biosynthesis, calcium storage and release. Perturbations that disrupt ER homeostasis lead to the misfolding of proteins in the ER lumen and up-regulation of ER signaling pathway called the unfolded protein response (UPR). Here, we have demonstrated that ageing changes the expression of ER chaperone and associated ER membrane kinases of IRE1, ATF6 and PERK.

• 한국자기공명학회논문지
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• 제23권1호
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• pp.26-32
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• 2019

${\alpha}B$-crystallin (${\alpha}BC$) is a member of a small heat-shock protein (sHSP) superfamily and plays a predominant role in cellular protein homeostasis network by rescuing misfolded proteins from irreversible aggregation. ${\alpha}BC$ assembles into dynamic and polydisperse high molecular weight complexes containing 12 to 48 monomers; this variable stereochemistry of ${\alpha}BC$ has been linked to quaternary subunit exchange and its chaperone activity. The chaperone activity of ${\alpha}BC$ poses great potential as therapeutic agents for various neurodegenerative diseases. In this mini-review, we briefly outline the recent advancement in structural characterization of ${\alpha}BCs$ and its potential role to inhibit protein misfolding and aggregation in various human diseases. In particular, nuclear magnetic resonance (NMR) spectroscopy and its complimentary techniques have contributed much to elucidate highly-dynamic nature of ${\alpha}BCs$, among which notable advancements are discussed in detail. We highlight the importance of resolving the structural details of various ${\alpha}BC$ oligomers, their quaternary dynamics, and structural heterogeneity.

• Journal of Microbiology and Biotechnology
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• 제28권6호
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• pp.987-996
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• 2018

Bacterial programmed cell death is regulated by the toxin-antitoxin (TA) system. YhaV (toxin) and Pr1F (antitoxin) have been recently identified as a type II TA system in Escherichia coli. YhaV homologs have conserved active residues within the C-terminus, and to characterize the function of this region, we purified native YhaV protein (without denaturing) and constructed YhaV proteins of varying lengths. Here, we report a new low-temperature method of purifying native YhaV, which is notable given the existing challenges of purifying this highly toxic protein. The secondary structures and thermostability of the purified native protein were characterized and no significant structural destruction was observed, suggesting that the observed inhibition of cell growth in vivo was not the result of structural protein damage. However, it has been reported that excessive levels of protein expression may result in protein misfolding and changes in cell growth and mRNA stability. To exclude this possibility, we used an [$^{35}S$]-methionine prokaryotic cell-free protein synthesis system in vitro in the presence of purified YhaV, and two C-terminal truncated forms of this protein (YhaV-L and YhaV-S). Our results suggest that the YhaV C-terminal region is essential for mRNA interferase activity, and the W143 or H154 residues may play an analogous role to Y87 of RelE.