| 1. |
- Moparthi, Satish Babu, et al.
(författare)
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A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding
- 2009
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 18:2, s. 475-479
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Tidskriftsartikel (refereegranskat)abstract
- The protein folding process is often in vitro rate-limited by slow cis-trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active-site located Arg 55 is not required for catalysis of the rate-limiting prolyl cis-trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis-trans proline isomerization in HCA II occurs in the active-site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding-assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.
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| 2. |
- Moparthi, Satish Babu
(författare)
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Biophysical studies of protein folding upon interaction with molecular chaperones
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are biological macromolecules that serve all functions in cells. Every protein consists of a sequence of amino acids that is folded into a three‐dimensional structure to maintain the unique information it contains and to allow the protein to perform its specific actions. Improper folding caused by mutations in the amino acid sequence or environmental stress can lead to protein aggregation and ultimately to protein conformational disorders such as Parkinson’s disease and other dreadful diseases. Nature has developed special classes of protein guards called foldases and chaperones that can increase folding efficiency in the crowded intracellular milieu by preventing protein aggregation. The present research was aimed to elucidate how chaperones and foldases interact with their target proteins during folding. Special attention was focused on refolding kinetics and dynamic remodulation of site‐specific labeled cysteine variants of the protein human carbonic anhydrase (HCA II) upon interaction with the PPIase cyclophilin18 (Cyp18) and the chaperonin GroEL. Part of the work also compared properties of the group I chaperonin GroEL and the group II chaperonin TRiC, considering how they mediate structural alterations uponinteraction with the cytoskeletal target protein β‐actin. These interactions were studied by various fluorescence techniques, including fluorescence resonance energy transfer (FRET) and fluorescence anisotropy.Refolding of HCA II is an extremely complicated process that involves very fast and slow folding events, and research has shown that Cyp18 enhances the slow rate‐limiting cistrans proline isomerization steps during the refolding process. Furthermore, the active‐site mutant Cyp18R55A has been reported to posses only about 1% catalytic efficiency when acting on short chromogenic peptide substrates. However, we found that Cyp18R55A is as efficient as the wild‐type Cyp18 in accelerating HCA II refolding. We also noted that Cyp18 enhanced the final yield of the severely destabilized HCA IIH107N, and HCA IIH107F mutants by rescuing transient molten globule intermediates from misfolding as a result of condensation of hydrophobic patches at very early stages of the folding process. These findings led to the conclusion that Arg 55, located in the active site of Cyp18, is not required for prolyl cistrans isomerization of protein substrates, and that Cyp18 can function as both a folding catalyst and a chaperone during HCA II folding.Studies have demonstrated that sequestering of protein substrates by the chaperonin GroEL alone results in binding‐induced unfolding of aggregation‐prone molten globule intermediates. It was previously assumed that the co‐chaperonin GroES does not play an independent role in folding. However, based on FRET measurements, we found that GroEL alone stretches the protein substrate as an early event, and also that GroES alone can transiently remodulate the structure of the molten globule intermediate during the refolding process. In addition, GroES acts in i concert with GroEL to exert additive transient stretchng effects on the protein core, and it reverses the unfoldase activity of the GroEL termini, leading to compaction of the structure to attain the more constrained native state.Earlier investigations have shown that partially folded β‐actin binds to both GroEL and the TRiC chaperonin. However, only TRiC guides correct folding of β‐actin, whereas the GroEL–β‐actin interaction is non‐productive. Homo‐FRET measurements on β‐actin mutants labeled with fluorescein during interaction with GroEL and TRiC indicated that interplay with both the chaperonins lead to binding‐induced unfolding and dynamic remodulation of β‐actin. More specifically, the interaction with TRiC resulted in considerable expansion of the entrance of the ATP‐binding cleft of β‐actin by effecting specific modulation of the β‐actin sub‐domains followed by the formation of a compressed state (native‐like) during release from TriC. Conformational rearrangements of β‐actin by GroEL on the other and were ore modest. β‐actin remained rather compact in the complex and consequently did not lead to the native‐like state ven in the encapsulated cis‐cavity when capped by GroES.
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| 3. |
- Moparthi, Satish Babu, et al.
(författare)
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pRb2/p130 protein in relation to clinicopathological and biological variables in rectal cancers with a clinical trial of preoperative radiotherapy
- 2009
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Ingår i: INTERNATIONAL JOURNAL OF COLORECTAL DISEASE. - : Springer Science and Business Media LLC. - 0179-1958 .- 1432-1262. ; 24:11, s. 1303-1310
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Tidskriftsartikel (refereegranskat)abstract
- pRb2/p130 plays a key role in cell proliferation and is a considerable progress about expression patterns of pRb2/p130 in number of malignancies. However, pRb2/p130 expression and its significance in rectal cancer remain unknown. The purpose of the present study was to investigate pRb2/p130 protein patterns and their correlations with clinicopathological and biological factors in rectal cancer patients with or without preoperative radiotherapy (RT). pRb2/p130 protein was examined by immunohistochemistry in 130 primary tumors, along with the corresponding 61 distant normal mucosa specimens, 85 adjacent normal mucosa specimens, 34 lymph node metastases, and 93 primary tumor biopsies from rectal cancer patients who participated in a Swedish clinical trial of preoperative RT. The pRb2/p130 protein was mainly localized in the cytoplasm of tumor cells. In nonradiated cases, the lack of pRb2/p130 was related to advanced tumor-node-metastases stage, poorer differentiation, weak fibrosis, less inflammatory infiltration, higher Ki-67, and positive Cox-2 expression (p andlt; 0.05). In radiated cases, the lack of pRb2/p130 was related to nonstaining of Cox-2 and survivin (p andlt; 0.05). pRb2/p130 protein in primary tumors tended to be increased after RT (27% vs 16%, p = 0.07). pRb2/p130 was mainly localized in the cytoplasm rather than in the nucleus in rectal cancer. After RT, pRb2/p130 protein seems to be increased in primary tumors, and further the relationship of the pRb2/p130 with the clinicopathological and biological variables changed compared to the nonradiated cases. However, we did not find that the pRb2/p130 was directly related to RT, tumor recurrence, and patients survival.
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| 4. |
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| 5. |
- Villebeck, Laila, et al.
(författare)
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Domain-specific chaperone-induced expansion is required for ß-actin folding : a comparison of ß-actin conformations upon interactions with GroEL and tail-less complex polypeptide 1 ring complex (TRiC)
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:44, s. 12639-12647
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Tidskriftsartikel (refereegranskat)abstract
- Actin, an abundant cytosolic protein in eukaryotic cells, is dependent on the interaction with the chaperonin tail-less complex polypeptide 1 ring complex (TRiC) to fold to the native state. The prokaryotic chaperonin GroEL also binds non-native ß-actin, but is unable to guide ß-actin toward the native state. In this study we identify conformational rearrangements in ß-actin, by observing similarities and differences in the action of the two chaperonins. A cooperative collapse of ß-actin from the denatured state to an aggregation-prone intermediate is observed, and insoluble aggregates are formed in the absence of chaperonin. In the presence of GroEL, however, >90% of the aggregation-prone actin intermediate is kept in solution, which shows that the binding of non-native actin to GroEL is effective. The action of GroEL on bound flourescein-labeled ß-actin was characterized, and the structural rearrangement was compared to the case of the ß-actin-TRiC complex, employing the homo fluorescence resonance energy transfer methodology previously used [Villebeck, L., Persson, M., Luan, S.-L., Hammarström, P., Lindgren, M., and Jonsson, B.-H. (2007) Biochemistry 46 (17), 5083-93]. The results suggest that the actin structure is rearranged by a "binding-induced expansion" mechanism in both TRiC and GroEL, but that binding to TRiC, in addition, causes a large and specific separation of two subdomains in the ß-actin molecule, leading to a distinct expansion of its ATP-binding cleft. Moreover, the binding of ATP and GroES has less effect on the GroEL-bound ß-actin molecule than the ATP binding to TRiC, where it leads to a major compaction of the ß-actin molecule. It can be concluded that the specific and directed rearrangement of the ß-actin structure, seen in the natural ß-actin-TRiC system, is vital for guiding ß-actin to the native state. © 2007 American Chemical Society.
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