The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.
FKBP22 of a psychrophilic bacterium, Shewanella sp. SIB1 (SIB1 FKBP22), is a member of peptidyl-prolyl cis-trans isomerase (PPIase) and consists of N- and C-domains responsible for chaperone-like and PPIase catalytic activities, respectively. The chaperone-like activity of SIB1 FKBP22 was previously evidenced by its ability to prevent dithiothreitol (DTT)-induced insulin aggregation. Nevertheless, the mechanism by which this protein inhibits the aggregation remains unclear. To address this, the binding affinity of SIB1 FKBP22 to the native or reduced states of insulin was examined using surface plasmon resonance (SPR). The native and reduced states refer to insulin in the absence or DTT presence, respectively. The SPR sensorgram showed that SIB1 FKBP22 binds specifically to the reduced state of insulin, with a KD value of 37.31 ± 3.20 μM. This binding was facilitated by the N-domain, as indicated by the comparable KD values of the N-domain and SIB1 FKBP22. Meanwhile, the reduced state of insulin was found to have no affinity towards the C-domain. The KD value of SIB1 FKBP22 was slightly decreased by NaCl but was not severely affected by FK506, a specific FKBP inhibitor. Similarly, the prevention of DTT-induced aggregation by SIB1 FKBP22 was also modulated by the N-domain and was not affected by FK506. Further, the reduced and native states of insulin had no effect on the catalytic efficiency (kcat/KM) of SIB1 FKBP22 towards a peptide substrate. Nevertheless, the reduced state of insulin slightly reduced the catalytic efficiency towards refolding RNase T1, at up to 1.5-fold lower than in the absence of insulin. These results suggested that the binding event was mainly facilitated by hydrophobic interaction and was independent from its PPIase activity. Altogether, a possible mechanism by which SIB1 FKBP22 prevents DTT-induced insulin aggregation was proposed.
Leigh syndrome (LS) is a common neurodegenerative disease affecting neonates with devastating sequences. One of the characteristic features for LS is the phenotypic polymorphism, which-in part-can be dedicated to variety of genetic causes. A strong correlation with mitochondrial dysfunction has been assumed as the main cause of LS. This was based on the fact that most genetic causes are related to mitochondrial complex I genome. The first animal LS model was designed based on NDUFS4 knockdown. Interestingly, however, this one or others could not recapitulate the whole spectrum of manifestations encountered in different cases of LS. We show in this chapter a new animal model for LS based on silencing of one gene that is reported previously in clinical cases, FOXRED1. The new model carries some differences from previous models in the fact that more histopathological degeneration in dopaminergic system is seen and more behavioral changes can be recognized. FOXRED1 is an interesting gene that is related to complex I assembly, hence, plays important role in different neurodegenerative disorders leading to different clinical manifestations.
The ability of eukaryotes to adapt to an extreme range of temperatures is critically important for survival. Although adaptation to extreme high temperatures is well understood, reflecting the action of molecular chaperones, it is unclear whether these molecules play a role in survival at extremely low temperatures. The recent genome sequencing of the yeast Glaciozyma antarctica, isolated from Antarctic sea ice near Casey Station, provides an opportunity to investigate the role of molecular chaperones in adaptation to cold temperatures. We isolated a G. antarctica homologue of small heat shock protein 20 (HSP20), GaSGT1, and observed that the GaSGT1 mRNA expression in G. antarctica was markedly increased following culture exposure at low temperatures. Additionally, we demonstrated that GaSGT1 overexpression in Escherichia coli protected these bacteria from exposure to both high and low temperatures, which are lethal for growth. The recombinant GaSGT1 retained up to 60 % of its native luciferase activity after exposure to luciferase-denaturing temperatures. These results suggest that GaSGT1 promotes cell thermotolerance and employs molecular chaperone-like activity toward temperature assaults.
Thalassemia is known as a diverse single gene disorder, which is prevalent worldwide. The molecular chaperones are set of proteins that help in two important processes while protein synthesis and degradation include folding or unfolding and assembly or disassembly, thereby helping in cell homeostasis. This review recaps current knowledge regarding the role of molecular chaperones in thalassemia, with a focus on beta thalassemia.
We have analyzed unfractionated sera of newly diagnosed patients (n=10) with breast carcinoma (BC), prior to treatment, and patients (n=5) with fibrocystic disease of the breast (FDB) by two-dimensional gel electrophoresis (2-DE) and silver staining. The patients' 2-DE serum protein profiles obtained were then subjected to image analysis and compared to similar data generated from sera of normal healthy female controls (n=10) of the same range of age. The relative expression of alpha1-antichymotrypsin (ACT), clusterin, and complement factor B was significantly higher in all BC patients as compared to normal controls. However, the expression of alpha1-antitrypsin (AAT) in BC patients was apparently lower than that of the controls. Similar differential expression of ACT was detected in the FDB patients. The aberrant expression of the serum acute-phase proteins of patients with BC and FDB was confirmed by competitive enzyme-linked immunosorbent assay (ELISA). Similar altered proteins expression was also observed from immunohistochemical studies of malignant (n=5) and benign (n=5) breast lesions of the respective patients performed using antisera to the aberrantly expressed proteins. However, the malignant breast lesions were instead positively stained for AAT. The differential expression of the serum proteins was apparently abrogated when a six-month follow-up study was performed on nine of the BC patients subsequent to treatment.
Eukaryotic ribosomes are composed of rRNAs and ribosomal proteins. Ribosomal proteins are translated in the cytoplasm and imported into the nucleus for assembly with the rRNAs. It has been shown that chaperones or karyopherins responsible for import can maintain the stability of ribosomal proteins by neutralizing unfavorable positive charges and thus facilitate their transports. Among 79 ribosomal proteins in yeast, only a few are identified with specific chaperones. Besides the classic role in maintaining protein stability, chaperones have additional roles in transport, chaperoning the assembly site, and dissociation of ribosomal proteins from karyopherins. Bcp1 has been shown to be necessary for the export of Mss4, a phosphatidylinositol 4-phosphate 5-kinase, and required for ribosome biogenesis. However, its specific function in ribosome biogenesis has not been described. Here, we show that Bcp1 dissociates Rpl23 from the karyopherins and associates with Rpl23 afterward. Loss of Bcp1 causes instability of Rpl23 and deficiency of 60S subunits. In summary, Bcp1 is a novel 60S biogenesis factor via chaperoning Rpl23 in the nucleus.
Human diploid fibroblasts (HDFs) cultured in vitro have limited capacity to proliferate after population doubling is repeated several times, and they enter into a state known as replicative senescence or cellular senescence. This study aimed to investigate the effect of Chlorella vulgaris on the replicative senescence of HDFs by determining the expression of senescence-associated genes. Young and senescent HDFs were divided into untreated control and C. vulgaris-treated groups. A senescence-associated gene transcription analysis was carried out with qRT-PCR. Treatment of young HDFs with C. vulgaris reduced the expression of SOD1, CAT and CCS (p
We describe four families with affected siblings showing unique clinical features: early-onset (before 1 year of age) progressive diffuse brain atrophy with regression, postnatal microcephaly, postnatal growth retardation, muscle weakness/atrophy, and respiratory failure. By whole-exome sequencing, we identified biallelic TBCD mutations in eight affected individuals from the four families. TBCD encodes TBCD (tubulin folding co-factor D), which is one of five tubulin-specific chaperones playing a pivotal role in microtubule assembly in all cells. A total of seven mutations were found: five missense mutations, one nonsense, and one splice site mutation resulting in a frameshift. In vitro cell experiments revealed the impaired binding between most mutant TBCD proteins and ARL2, TBCE, and β-tubulin. The in vivo experiments using olfactory projection neurons in Drosophila melanogaster indicated that the TBCD mutations caused loss of function. The wide range of clinical severity seen in this neurodegenerative encephalopathy may result from the residual function of mutant TBCD proteins. Furthermore, the autopsied brain from one deceased individual showed characteristic neurodegenerative findings: cactus and somatic sprout formations in the residual Purkinje cells in the cerebellum, which are also seen in some diseases associated with mitochondrial impairment. Defects of microtubule formation caused by TBCD mutations may underlie the pathomechanism of this neurodegenerative encephalopathy.