Muhamad Sahlan

Sequential action of ATP-dependent subunit conformational change and interaction between helical protrusions in the closure of the built-in lid of group II chaperonins

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.

Thermodynamic Characterization of the Interaction between Prefoldin and Group II Chaperonin

Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1). In this study, we determined the crystal structure of the T. KS-1 PFDbeta2 subunit and characterized the interactions between T. KS-1 CPNs (CPNalpha and CPNbeta) and T. KS-1 PFDs (PFDalpha1-beta1 and PFDalpha2-beta2). As predicted from its amino acid sequence, the PFDbeta2 subunit conforms to a structure similar to those of the PFDbeta1 subunit and the Pyrococcus horikoshii OT3 PFDbeta subunit, with the exception of the tip of its coiled-coil domain, which is thought to be the CPN interaction site. The interactions between T. KS-1 CPNs and PFDs (CPNalpha and PFDalpha1-beta1; CPNalpha and PFDalpha2-beta2; CPNbeta and PFDalpha1-beta1; and CPNbeta and PFDalpha2-beta2) were analyzed using the Biacore T100 system at various temperatures ranging from 20 to 45 degrees C. The affinities between PFDs and CPNs increased with an increase in temperature. The thermodynamic parameters calculated from association constants showed that the interaction between PFD and CPN is entropy driven. Among the four combinations of PFD-CPN interactions, the entropy difference in binding between CPNbeta and PFDalpha2-beta2 was the largest, and affinity significantly increased at higher temperatures. Considering that expression of PFDalpha2-beta2 and CPNbeta subunit is induced upon heat shock, our results suggest that PFDalpha1-beta1 is a general PFD for T. KS-1 CPNs, whereas PFDalpha2-beta2 is specific for CPNbeta.

Contribution of the C-Terminal Region of a Group II Chaperonin to its Interaction with Prefoldin and Substrate Transfer.

Prefoldin is a molecular chaperone that captures an unfolded protein substrate and transfers it to a group II chaperonin. Previous studies have shown that the interaction sites for prefoldin are located in the helical protrusions of group II chaperonins. However, it does not exclude the possibility of the existence of other interaction sites. In this study, we constructed C-terminal truncation mutants of a group II chaperonin and examined the effects of these mutations on the chaperones function and interaction with prefoldin. Whereas the mutants with up to 6 aa truncation from the C-terminus retained more than 90% chaperone activities for protecting citrate synthase from thermal aggregation and refolding of green fluorescent protein and isopropylmalate dehydrogenase, the truncation mutants showed decreased affinities for prefoldin. Consequently, the truncation mutants showed reduced transfer efficiency of the denatured substrate protein from prefoldin and subsequent chaperonin-dependent refolding. The results clearly show that the C-terminal region of group II chaperonins contributes to their interactions with prefoldin, the transfer of the substrate protein from prefoldin and its refolding.

Hyperthermophilic archaeal prefoldin shows refolding activity at low temperature

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. Previous studies of archaeal prefoldins have shown that prefoldin only possesses holdase activity and is unable to fold unfolded proteins by itself. In this study, we have demonstrated for the first time that a prefoldin from hyperthermophilic archaeon, Pyrococcus horikoshii OT3 (PhPFD), exhibits refolding activity for denatured lysozyme at temperatures relatively lower than physiologically active temperatures. The interaction between PhPFD and denatured lysozyme was investigated by use of a surface plasmon resonance sensor at various temperatures. Although PhPFD showed strong affinity for denatured lysozyme at high temperature, it exhibited relatively weak interactions at lower temperature. The protein-folding seems to occur through binding and release from PhPFD by virtue of the weak affinity. Our results also imply that prefoldin might be able to contribute to the folding of some cellular proteins whose affinity with prefoldin is weak.

Construction and characterization of the hetero-oligomer of the group II chaperonin from the hyperthermophilic archaeon, Thermococcus sp. strain KS-1

The hyperthermophilic archaeon Thermococcus sp. strain KS-1 (T. KS-1) expresses two different chaperonin subunits, α and β, for the folding of its proteins. The composition of the subunits in the hexadecameric double ring changes with temperature. The content of the β subunit significantly increases according to the increase in temperature. The homo-oligomer of the β subunit, Cpnβ, is more thermostable than that of the α subunit, Cpnα. Since Cpnα and Cpnβ also have different protein folding activities and interactions with prefoldin, the hetero-oligomer is thought to exhibit different characteristics according to the content of subunits. The hetero-oligomer of the T. KS-1 chaperonin has not been studied, however, because the α and β subunits form hetero-oligomers of varying compositions when they are expressed simultaneously. In this study, we characterized the T. KS-1 chaperonin hetero-oligomer, Cpnαβ, containing both α and β in the alternate order, which was constructed by the expression of α and β subunits in a coordinated fashion and protease digestion. Cpnαβ protected citrate synthase from thermal aggregation, promoted the folding of acid-denatured GFP in an ATP-dependent manner, and exhibited an ATP-dependent conformational change. The yield of refolded GFP generated by Cpnαβ was almost equivalent to that generated by Cpnβ but lower than that generated by Cpnα. In contrast, Cpnαβ exhibited almost the same level of thermal stability as Cpnα, which was lower than that of Cpnβ. The affinity of Cpnαβ to prefoldin was found to be between those of Cpnα and Cpnβ, as expected.

Solid State Fermentation using Agroindustrial Wastes to Produce Aspergillus Niger Lipase as a Biocatalyst Immobilized by an Adsorption-crosslinking Method for Biodiesel Synthesis

Although technological advances have fueled the rising demand for lipase as a biocatalyst, commercial availability remains limited and costs prohibitive. To meet this need, an extracellular lipase enzyme from Aspergillus niger can be produced through solid state fermentation (SSF) using agroindustrial wastes including tofu dregs, coconut dregs, and corn bran. These agroindustrial residues still contain nutrients, especially lipids/triglycerides, making them a potential fermentation medium to produce lipase. Lipase with the highest activity level (8.48 U/mL) was obtained using a tofu dreg substrate, 4% inducer concentration, and 9-day fermentation period. This crude lipase extract was then dried with a spray drier and immobilized in a macroporous anion resin using the adsorption-crosslinking method. The immobilized lipase’s activity was assayed by a biodiesel synthesis reaction; it showed 48.3% yield. The immobilized enzymes stability was also tested through four cycles of biodiesel synthesis; in the fourth cycle, the enzyme maintained 84% of its initial activity.

Prefoldin, a jellyfish-like molecular chaperone: functional cooperation with a group II chaperonin and beyond

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits and has the appearance of a jellyfish: its body consists of a double beta-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. The prefoldin-group II chaperonin system is thought to be important for the folding of newly synthesized proteins and for their maintenance, or proteostasis, in the cytosol. Based on structural information of archaeal prefoldins, the mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the role and mechanism of eukaryotic PFDs remain unknown. Recent studies have shown that prefoldin plays an important role in proteostasis and is involved in various diseases. In this paper, we review a series of studies on the molecular mechanisms of archaeal prefoldins and introduce recent findings about eukaryotic prefoldin.

Molecular Chaperones in Thermophilic Eubacteria and Archaea

Thermophilic organisms tolerate or adapt to high temperatures by making their proteins thermostable or thermophilic. Even though, thermophilic bacteria have their own optimal growth temperatures, and heat-shock responses are still induced at the temperatures higher than optimal temperatures. The molecular chaperone systems of thermophilic eubacteria are very similar to those of mesophilic eubacteria. On the contrary, the molecular chaperone system of hyperthermophilic archaea is much simpler than those of other organisms. Within the hyperthermophilic archaea, only the following six kinds of chaperones have so far been identified: group II chaperonins, prefoldin, small heat-shock proteins, peptidyl-prolyl cis-trans isomerases, AAA proteins, and NAC. These archaea lack the Hsp70 chaperone system as well as Hsp90 and Hsp100, though these are thought to be indispensable chaperones in all other organisms. Since group II chaperonins are highly induced at elevated temperatures and related to the stress response of hyperthermophilic archaea, this manuscript focuses on the group II chaperonin and its cofactor, prefoldin, in addition to sHsps that are ubiquitous in thermophilic eubacteria and archaea. The limited number of molecular chaperones in hyperthermophilic archaea might be due to the relatively high stability of their proteins. The molecular chaperones in hyperthermophilic archaea might contribute to the protection of only a limited number of relatively unstable proteins.

Antiviral activity of Acanthaster planci phospholipase A2 against human immunodeficiency virus

Aim: Investigation of antiviral activity of Acanthaster planci phospholipase A2 (AP-PLA2) from moluccas to human immunodeficiency virus (HIV). Materials and Methods: Crude venom (CV) and F20 (PLA2 with 20% fractioned by ammonium sulfate) as a sample of PLA 2 obtained from A. planci’s extract were used. Enzymatic activity of PLA2 was determined using the degradation of phosphatidylcholine (PC). Activity test was performed using in vitro method using coculture of phytohemagglutinin-stimulated peripheral blood mononuclear cell (PBMC) from a blood donor and PBMC from HIV patient. Toxicity test of AP-PLA2 was done using lethal concentration required to kill 50% of the population (LC50). Results: AP-PLA2 F20 had activity and purity by 15.66 times bigger than CV. The test showed that the LC50 of AP-PLA2 is 1.638 mg/ml. Antiviral analysis of AP-PLA2 in vitro showed the inhibition of HIV infection to PBMC. HIV culture with AP-PLA2 and without AP-PLA2 has shown the number of infected PBMC (0.299±0.212% and 9.718±0.802%). Subsequently, RNA amplification of HIV using reverse transcriptase-polymerase chain reaction resulted in the decrease of band intensity in gag gene of HIV. Conclusion: This research suggests that AP-PLA2 has the potential to develop as an antiviral agent because in vitro experiment showed its ability to decrease HIV infection in PBMC and the number of HIV ribonucleic acid in culture.

The effect of propolis honey candy on Streptococcus mutans prevalence in caries and caries-free subjects

This study was to evaluate the effect of Propolis Honey candy on Streptococcus mutans prevalence in caries and caries-free subject. The subject of this research was caries and caries-free subjects. The Streptococcus mutans colony was counted in saliva samples before and after a 7-day period of consuming Propolis Honey candy, Honey candy, and “X” candy. The Streptococcus mutans was proliferated in a TYS20B gelatin medium for 48 hours. The number of Streptococcus mutans colonies was expressed in CFU/ml. Compared with the pre-treatment group, the number of Streptococcus mutans colonies in the treatment group tends to show a statistically significant reduction (p<0.05). The amount of Streptococcus mutans after consuming Propolis honey candy were lower (5.8×106 CFU/ml) than before (2.4×1010 CFU/ml) in caries-free subject. In caries subject, the result of Propolis honey candy were also lower (2.2×107 CFU/ml) than before (5.8×109 CFU/ml). The study showed a decrease in the number of Streptococcus mutans colonies from caries and caries-free subjects after propolis honey candy consumption.This study was to evaluate the effect of Propolis Honey candy on Streptococcus mutans prevalence in caries and caries-free subject. The subject of this research was caries and caries-free subjects. The Streptococcus mutans colony was counted in saliva samples before and after a 7-day period of consuming Propolis Honey candy, Honey candy, and “X” candy. The Streptococcus mutans was proliferated in a TYS20B gelatin medium for 48 hours. The number of Streptococcus mutans colonies was expressed in CFU/ml. Compared with the pre-treatment group, the number of Streptococcus mutans colonies in the treatment group tends to show a statistically significant reduction (p<0.05). The amount of Streptococcus mutans after consuming Propolis honey candy were lower (5.8×106 CFU/ml) than before (2.4×1010 CFU/ml) in caries-free subject. In caries subject, the result of Propolis honey candy were also lower (2.2×107 CFU/ml) than before (5.8×109 CFU/ml). The study showed a decrease in the number of Streptococcus mutans colonies...