Protein Quality Control by Molecular Chaperones in Neurodegeneration

Ciechanover, Aaron,Kwon, Yong Tae Frontiers Media S.A. 2017 Frontiers in neuroscience Vol.11 No.-

<P>Protein homeostasis (proteostasis) requires the timely degradation of misfolded proteins and their aggregates by protein quality control (PQC), of which molecular chaperones are an essential component. Compared with other cell types, PQC in neurons is particularly challenging because they have a unique cellular structure with long extensions. Making it worse, neurons are postmitotic, i.e., cannot dilute toxic substances by division, and, thus, are highly sensitive to misfolded proteins, especially as they age. Failure in PQC is often associated with neurodegenerative diseases, such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), and prion disease. In fact, many neurodegenerative diseases are considered to be protein misfolding disorders. To prevent the accumulation of disease-causing aggregates, neurons utilize a repertoire of chaperones that recognize misfolded proteins through exposed hydrophobic surfaces and assist their refolding. If such an effort fails, chaperones can facilitate the degradation of terminally misfolded proteins through either the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (hereafter autophagy). If soluble, the substrates associated with chaperones, such as Hsp70, are ubiquitinated by Ub ligases and degraded through the proteasome complex. Some misfolded proteins carrying the KFERQ motif are recognized by the chaperone Hsc70 and delivered to the lysosomal lumen through a process called, chaperone-mediated autophagy (CMA). Aggregation-prone misfolded proteins that remain unprocessed are directed to macroautophagy in which cargoes are collected by adaptors, such as p62/SQSTM-1/Sequestosome-1, and delivered to the autophagosome for lysosomal degradation. The aggregates that have survived all these refolding/degradative processes can still be directly dissolved, i.e., disaggregated by chaperones. Studies have shown that molecular chaperones alleviate the pathogenic symptoms by neurodegeneration-causing protein aggregates. Chaperone-inducing drugs and anti-aggregation drugs are actively exploited for beneficial effects on symptoms of disease. Here, we discuss how chaperones protect misfolded proteins from aggregation and mediate the degradation of terminally misfolded proteins in collaboration with cellular degradative machinery. The topics also include therapeutic approaches to improve the expression and turnover of molecular chaperones and to develop anti-aggregation drugs.</P>

Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation

LEE, JIN-HAENG,JEONG, JAEHO,JEONG, EUI MAN,CHO, SUNG-YUP,KANG, JEONG WOOK,LIM, JISUN,HEO, JINBEOM,KANG, HYUNSOOK,KIM, IN-GYU,SHIN, DONG-MYUNG UNKNOWN 2014 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Vol.33 No.4

<P>Aberrant activation of transglutaminase 2 (TGase2) contributes to a variety of protein conformational disorders such as neurodegenerative diseases and age-related cataracts. The accumulation of improperly folded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), which promotes either repair or degradation of the damaged proteins. Inadequate UPR results in protein aggregation that may contribute to the development of age-related degenerative diseases. TGase2 is a calcium-dependent enzyme that irreversibly modifies proteins by forming cross-linked protein aggregates. Intracellular TGase2 is activated by oxidative stress which generates large quantities of unfolded proteins. However, the relationship between TGase2 activity and UPR has not yet been established. In the present study, we demonstrated that ER stress activated TGase2 in various cell types. TGase2 activation was dependent on the ER stress-induced increase in the intracellular calcium ion concentration but not on the TGase2 protein expression level. Enzyme substrate analysis revealed that TGase2-mediated protein modification promoted protein aggregation concurrently with decreasing water solubility. Moreover, treatment with KCC009, a TGase2 inhibitor, abrogated ER stress-induced TGase2 activation and subsequent protein aggregation. However, TGase2 activation had no effect on ER stress-induced cell death. These results demonstrate that the accumulation of misfolded proteins activates TGase2, which further accelerates the formation of protein aggregates. Therefore, we suggest that inhibition of TGase2 may be a novel strategy by which to prevent the protein aggregation in age-related degenerative diseases.</P>

Analysis of the Globular Nature of Proteins

정성훈,손현석 한국유전체학회 2011 Genomics & informatics Vol.9 No.2

Numerous restraints and simplifications have been developed for methods that anticipate protein structure to reduce the colossal magnitude of possible conformational states. In this study, we investigated if globularity is a general characteristic of proteins and whether they can be applied as a valid constraint in protein structure simulations with approximated measurements (Gb-index). Unexpectedly, most of the proteins showed strong structural globularity (i.e., mode of approximately 76%similarity to the perfect globe) with only a few percent of proteins being outliers. Small proteins tended to be significantly non-globular (R^2=0.79) and the minimum Gb-index showed a logarithmic increase with the increase in protein size (R^2=0.62), strongly implying that the non-globular characteristics might be more acceptable for smaller proteins than larger ones. The strong perfect globe-like character and the relationship between small size and the loss of globular structure of a protein may imply that living organisms have mechanisms to aid folding into the globular structure to reduce irreversible aggregation. This also implies the possible mechanisms of diseases caused by protein aggregation,including some forms of trinucleotide repeat expansion-mediated diseases.

Analysis of the Globular Nature of Proteins

Jung, Sung-Hoon,Son, Hyeon-Seok Korea Genome Organization 2011 Genomics & informatics Vol.9 No.2

Numerous restraints and simplifications have been developed for methods that anticipate protein structure to reduce the colossal magnitude of possible conformational states. In this study, we investigated if globularity is a general characteristic of proteins and whether they can be applied as a valid constraint in protein structure simulations with approximated measurements (Gb-index). Unexpectedly, most of the proteins showed strong structural globularity (i.e., mode of approximately 76% similarity to the perfect globe) with only a few percent of proteins being outliers. Small proteins tended to be significantly non-globular ($R^2$=0.79) and the minimum Gb-index showed a logarithmic increase with the increase in protein size ($R^2$=0.62), strongly implying that the non-globular characteristics might be more acceptable for smaller proteins than larger ones. The strong perfect globe-like character and the relationship between small size and the loss of globular structure of a protein may imply that living organisms have mechanisms to aid folding into the globular structure to reduce irreversible aggregation. This also implies the possible mechanisms of diseases caused by protein aggregation, including some forms of trinucleotide repeat expansion-mediated diseases.

혈소판응집과 단백인산화에 대한 PHOSPODIESTERASE-억제제의 효과

차진한 大韓成形外科學會 1995 Archives of Plastic Surgery Vol.22 No.3

There are many evidences suggesting that phosphodiesterase(PDE) inhibitors, amitriptyline(AM) and chlorpromazine(CP) inhibit the platelet aggregation. Therefore, the antiplatelet activities of several PDE inhibitors ; 3-isobutyl-1-methylxanthine(IBMX) and 4, 5-dihydro-6-(3-nitro-4-methoxyphenyl)-5-methyl-3-(2H)-pyridazinone ; KR30075(KR), sodium nitroprusside(SNP), amitriptyline(AM) and chlorpromazine(CP) were compared in terms of their inhibitory effects on aggregation and protein phosphoylation of the rabbit platelet in response to thrombin(0.25units/ml;TB), collagen(200mg/ml;CO), adenosine diphosphate(4.0×10-5M;ADP)and eqinephrine(4.0×10-6M;EP). The following results were obtained. 1. KR30075 inhibited the aggregation of platelets in response to EP>CO>ADP>TB in the order of its antiplatelet activities, showing IC50 of less than 1.7×10-7M. PDE inhibitors, showed the inhibitory effects on the platelet aggregation in response to several agonists in similar patterns. The antiplatelet activity of KR (IC50:less than 1.7×10-7M) was significantly greater than that of SNP(IC50:less than 1.57×10-6M). But the antiplatelet activity of other drugs used in this study was not so meaningful compared with those of KR and SNP. 2. The platelet aggregation induced by each of the agonists was accompanied with the increased phosphorylation of 41 kDa protein and the decreased 47 kDa and 20 kDa proteins. PDE inhibitors (KR, IBMX) and SNP significantly inhibited the increase of 41 kDa protein phosphorylation and produced somewhat of an attenuation of the decreases of 47 kDa and 20 kDa protein phosphorylations. AM and CP, however had little effect on the decrease of 47 kDa protein phosphorylation, but inhibited the decrease of 20 kDa protein phosphorylation. Even so KR, SNP and IBMX produced the increases of 43 kDa & 22 kDa protein phosphorylations in order of KR>SNP>IBMX. The antiplatelet activity of KR 30075 seems to correlate with the decrease of 41 kDa protein phosphorylation and the increases of 43 kDa and 22 kDa protein phosphorylations.

Salt-Induced Protein Precipitation in Aqueous Solution: Single and Binary Protein Systems

Kim, Sang-Gon,Bae, Young-Chan The Polymer Society of Korea 2003 Macromolecular Research Vol.11 No.1

A molecular-thermodynamic model is developed for the salt-induced protein precipitation. The protein molecules interact through four intermolecular potentials. An equation of state is derived based on the statistical mechanical perturbation theory with the modified Chiew's equation for the fluid phase, Young's equation for the solid phase as the reference system and a perturbation based on the protein-protein effective two body potential. The equation of state provides an expression for the chemical potential of the protein. In a single protein system, the phase separation is represented by fluid-fluid equilibria. The precipitation behaviors are simulated with the partition coefficient at various salt concentrations and degree of pre-aggregation effect for the protein particles. In a binary protein system, we regard the system as a fluid-solid phase equilibrium. At equilibrium, we compute the reduced osmotic pressure-composition diagram in the diverse protein size difference and salt concentrations.

대장균 트립토판 중합효소 α 소단위체의 응집 형성에 미치는 잔기 173 치환체의 억제 효과

정재갑,박후휘,임운기 한국생명과학회 2022 생명과학회지 Vol.32 No.9

Aggregation of normally soluble proteins can cause disease-related problems. Tryptophan synthase α-subunit (αTS) in E. coli adopts one of most popular structural scaffolds, the TIM barrel fold. Previous mutagenesis of the αTS gene resulted in many aggregation-prone mutant proteins. Here, Y173F (Tyr at residue 173 to Phe) substitution, which imparts increased stability, was tested for its ability to suppress aggregation of aggregation-prone mutant proteins (Y4C, S33L, P28L, P28S, G44S, D46N, P96L, and P96S). Aggregation was suppressed in all eight severe aggregate-forming mutants (all differing in their mutation positions), by the Y173F replacement. P28L αTS, which was available in pure form, was further analyzed and showed reduced secondary structure content, lower stability, and a looser structure with more exposed hydrophobic surface compared to the wild type protein. A double mutant P28L/Y173F protein showed almost no indication of these changes compared to the wild type protein. We hypothesized that Tyr at position 173 in αTS is positioned at the hydrophobic core and may serve to suppress the aggregation of this protein caused by other residues. Important residue (s) could be working widely in the prevention/suppression of protein aggregation.

Zinc boosts EGCG's hIAPP amyloid Inhibition both in solution and membrane

Lee, Young-Ho,Lin, Yuxi,Cox, Sarah J.,Kinoshita, Misaki,Sahoo, Bikash R.,Ivanova, Magdalena,Ramamoorthy, Ayyalusamy Elsevier 2019 Biochimica et biophysica acta. Proteins and proteo Vol.1867 No.5

<P><B>Abstract</B></P> <P>Amyloid aggregation of human islet amyloid polypeptide (hIAPP) is linked to insulin-producing islet cell death in type II diabetes. Previous studies have shown that zinc (Zn(II)) and insulin, co-secreted with hIAPP, have an inhibition effect on hIAPP aggregation. Lipid membranes have also been shown to significantly influence the aggregation kinetics of hIAPP. An increasing number of studies report the importance of developing small molecule inhibitors to suppress the hIAPP's aggregation and subsequent toxicity. The ability of epigallocatechin-gallate (EGCG) to inhibit aggregation of a variety of amyloid peptide/proteins initiated numerous studies as well as the development of derivative compounds to potentially treat amyloid diseases. In this study, a combination of Thioflavin-T fluorescence kinetics, transmission electron microscopy, isothermal titration calorimetery, circular dicrosim and nucelar magnetic resonance experiments were used to demonstrate a significant enhancement in EGCG's efficiency when complexed with Zn(II). We demonstrate that the Zn-EGCG complex is able to significantly suppress hIAPP's amyloid aggregation both in presence and absence of lipid membrane. Circular dichroism experiments indicate the formation and stabilization of a helical structure of hIAPP in presence of the EGCG:Zn(II) complex. Our results also reveal the ability of EGCG or EGCG:Zn(II) to efficiently suppress hIAPP's cellular toxicity. We believe that the reported results could be useful to develop strategies to trap hIAPP intermediates for further biophysical and structural studies, and also to devise approaches to abolish amyloid aggregation and cellular toxicity.</P> <P><B>Highlights</B></P> <P> <UL> <LI> EGCG:Zn(II) inhibits amyloid aggregation in solution as well as in the presence of membrane. </LI> <LI> EGCG:Zn(II) complex efficiently suppresses human-IAPP's toxicity to pancreatic β-cells. </LI> <LI> EGCG suppresses Zinc's toxicity to pancreatic β-cells. </LI> <LI> Human-IAPP forms a helical structure in presence of EGCG:Zn(II) in membrane. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Enhanced intranasal insulin delivery by formulations and tumor protein-derived protein transduction domain as an absorption enhancer

Kim, Nam Ah,Thapa, Ritu,Jeong, Seong Hoon,Bae, Hae-duck,Maeng, Jeehye,Lee, Kyunglim,Park, Kinam Elsevier 2019 Journal of controlled release Vol.294 No.-

<P><B>Abstract</B></P> <P>One of the key factors for successful development of an intranasal insulin formulation is an absorption enhancer that would deliver insulin efficiently across nasal membranes without causing damage to mucosa or inducing protein aggregation under physiological conditions. In the present study, a protein transduction domain (PTD1) and its L-form with the double substitution A6L and I8A (PTD4), derived from human translationally controlled tumor protein, were used as absorption enhancers. PTD4 exhibited higher compatibility with insulin in terms of biophysical properties analyzed using μDSC, DLS, and CD. In addition, thermodynamic properties indicated stable complex formation but higher propensity of protein aggregation. Arginine hydrochloride (ArgHCl) was used to suppress protein aggregation and carbohydrates (i.e., mannitol, sucrose, and glycerin) were used as osmolytes in the formulation. The relative bioavailability of insulin co-administered intranasally using PTD4, 16 mg/mL glycerin and 100 mM ArgHCl was 58% and that using PTD4, 1 <I>w</I>/<I>v</I>% sucrose, and 25 mM ArgHCl was 53% of the bioavailability obtained via the subcutaneous route. These values represented a remarkable increase in bioavailability of intranasal insulin, causing a significant decrease in blood glucose levels within one hour. The pharmacokinetic properties of intranasal absorption were dependent on the concentration of carbohydrates used. These results suggest that the newly designed formulations with PTD represent a useful platform for intranasal delivery of insulin and other biomolecules.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Dimerization, Oligomerization, and Aggregation of Human Amyotrophic Lateral Sclerosis Copper/Zinc Superoxide Dismutase 1 Protein Mutant Forms in Live Cells

Kim, Jiho,Lee, Honggun,Lee, Joo Hyun,Kwon, Do-yoon,Genovesio, Auguste,Fenistein, Denis,Ogier, Arnaud,Brondani, Vincent,Grailhe, Regis American Society for Biochemistry and Molecular Bi 2014 The Journal of biological chemistry Vol.289 No.21

<P>More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. <I>In vitro</I> studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and <I>in vivo</I> studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.</P>