Defining the mechanism of PDI interaction with disulfide-free amyloidogenic proteins: Implications for exogenous protein expression and neurodegenerative disease

Yan, Pingyu; Zou, Zhiyuan; Zhang, Shiyao; Wang, Rui; Niu, Tingting; Zhang, Xia; Liu, Defu; Zhou, Xuejie; Chang, Alan K.; Milton, Nathaniel G.N.; Jones, Gary W.; He, Jing

doi:10.1016/j.ijbiomac.2021.01.172

Cited by 5 publications

(3 citation statements)

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“…Molecular chaperones are another important binding partner of the N-terminal region of α-syn in mammalian cells 38 . PDI interacts with the hydrophobic region of the N-terminal region, inhibiting aggregation 43,52,53 . To investigate the impact of the C-terminal truncation on the interaction with PDI, we acquired 1 H- 15 N HSQC spectra of uniformly 15 N-enriched FL-and CT-αsyn in the presence of PDI at a mole ratio of 2:1, which enabled us to assess intermolecular interactions at the residue level via chemical shift perturbations (CSPs) and intensity changes.…”

Section: Resultsmentioning

confidence: 99%

C-terminal truncation modulates α-Synuclein’s cytotoxicity and aggregation by promoting the interactions with membrane and chaperone

et al. 2022

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Abstractα-Synuclein (α-syn) is the main protein component of Lewy bodies, the major pathological hallmarks of Parkinson’s disease (PD). C-terminally truncated α-syn is found in the brain of PD patients, reduces cell viability and tends to form fibrils. Nevertheless, little is known about the mechanisms underlying the role of C-terminal truncation on the cytotoxicity and aggregation of α-syn. Here, we use nuclear magnetic resonance spectroscopy to show that the truncation alters α-syn conformation, resulting in an attractive interaction of the N-terminus with membranes and molecular chaperone, protein disulfide isomerase (PDI). The truncated protein is more toxic to mitochondria than full-length protein and diminishes the effect of PDI on α-syn fibrillation. Our findings reveal a modulatory role for the C-terminus in the cytotoxicity and aggregation of α-syn by interfering with the N-terminus binding to membranes and chaperone, and provide a molecular basis for the pathological role of C-terminal truncation in PD pathogenesis.

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Section: Resultsmentioning

confidence: 99%

C-terminal truncation modulates α-Synuclein’s cytotoxicity and aggregation by promoting the interactions with membrane and chaperone

et al. 2022

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“…Our results showed that co-expression of PDI with wild-type cC or mutated cC (I66Q or ΔW) could improve the secretion of cC to different extents. However, the distinct client-recruiting system of molecular chaperones may have a limit in improving the secretion of foreign proteins that may not be properly folded ( Yan et al, 2021 ). This limitation could prevent P. pastoris from becoming an ideal protein production platform that can accommodate a variety of production requirements.…”

Section: Discussionmentioning

confidence: 99%

Comparative transcriptomic analysis-based identification of the regulation of foreign proteins with different stabilities expressed in Pichia pastoris

Niu

Cui

et al. 2022

Front. Microbiol.

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IntroductionThe industrial yeast Pichia pastoris is widely used as a cell factory to produce proteins, chemicals and advanced biofuels. We have previously constructed P. pastoris strains that overexpress protein disulfide isomerase (PDI), which is a kind of molecular chaperone that can improve the expression of an exogenous protein when they are co-expressed. Chicken cystatin (cC) is a highly thermostable cysteine protease inhibitor and a homologous protein of human cystatin C (HCC). Wild-type cC and the two mutants, I66Q and ΔW (a truncated cC lacking the á-helix 2) represent proteins with different degrees of stability.MethodsWild-type cC, I66Q and ΔW were each overexpressed in P. pastoris without and with the coexpression of PDI and their extracellular levels were determined and compared. Transcriptomic profiling was performed to compare the changes in the main signaling pathways and cell components (other than endoplasmic reticulum quality control system represented by molecular chaperones) in P. pastoris in response to intracellular folding stress caused by the expression of exogenous proteins with different stabilities. Finally, hub genes hunting was also performed.Results and discussionThe coexpression of PDI was able to increase the extracellular levels of both wild-type cC and the two mutants, indicating that overexpression of PDI could prevent the misfolding of unstable proteins or promote the degradation of the misfolded proteins to some extent. For P. pastoris cells that expressed the I66Q or ΔW mutant, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of the common DEGs in these cells revealed a significant upregulation of the genes involved in protein processing, but a significant downregulation of the genes enriched in the Ribosome, TCA and Glycolysis/Gluconeogenesis pathways. Hub genes hunting indicated that the most downregulated ribosome protein, C4QXU7 in this case, might be an important target protein that could be manipulated to increase the expression of foreign proteins, especially proteins with a certain degree of instability.ConclusionThese findings should shed new light on our understanding of the regulatory mechanism in yeast cells that responds to intracellular folding stress, providing valuable information for the development of a convenient platform that could improve the efficiency of heterologous protein expression in P. pastoris.

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“…In the yeast K. phaffii , the overexpression of PDI is reported to amplify the secretion of both recombinant proteins rich in disulfide bonds [ 154 , 155 , 156 ] and proteins lacking thereof [ 157 ]. This evidence confirms the finding that protein disulfide isomerase PDI is an important molecular chaperone as well [ 158 ], whose catalytic activity is most pronounced toward partially unfolded and misfolded proteins [ 153 ].…”

Section: Specific Features Of K Phaffii As a Produ...mentioning

confidence: 99%

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

Khlebodarova,

Bogacheva,

Zadorozhny

et al. 2024

Microorganisms

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In the 1980s, Escherichia coli was the preferred host for heterologous protein expression owing to its capacity for rapid growth in complex media; well-studied genetics; rapid and direct transformation with foreign DNA; and easily scalable fermentation. Despite the relative ease of use of E. coli for achieving the high expression of many recombinant proteins, for some proteins, e.g., membrane proteins or proteins of eukaryotic origin, this approach can be rather ineffective. Another microorganism long-used and popular as an expression system is baker’s yeast, Saccharomyces cerevisiae. In spite of a number of obvious advantages of these yeasts as host cells, there are some limitations on their use as expression systems, for example, inefficient secretion, misfolding, hyperglycosylation, and aberrant proteolytic processing of proteins. Over the past decade, nontraditional yeast species have been adapted to the role of alternative hosts for the production of recombinant proteins, e.g., Komagataella phaffii, Yarrowia lipolytica, and Schizosaccharomyces pombe. These yeast species’ several physiological characteristics (that are different from those of S. cerevisiae), such as faster growth on cheap carbon sources and higher secretion capacity, make them practical alternative hosts for biotechnological purposes. Currently, the K. phaffii-based expression system is one of the most popular for the production of heterologous proteins. Along with the low secretion of endogenous proteins, K. phaffii efficiently produces and secretes heterologous proteins in high yields, thereby reducing the cost of purifying the latter. This review will discuss practical approaches and technological solutions for the efficient expression of recombinant proteins in K. phaffii, mainly based on the example of enzymes used for the feed industry.

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Defining the mechanism of PDI interaction with disulfide-free amyloidogenic proteins: Implications for exogenous protein expression and neurodegenerative disease

Cited by 5 publications

References 41 publications

C-terminal truncation modulates α-Synuclein’s cytotoxicity and aggregation by promoting the interactions with membrane and chaperone

C-terminal truncation modulates α-Synuclein’s cytotoxicity and aggregation by promoting the interactions with membrane and chaperone

Comparative transcriptomic analysis-based identification of the regulation of foreign proteins with different stabilities expressed in Pichia pastoris

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

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