Mycobacterium leprae HSP18 is a small heat shock protein (sHSP). It is a major immunodominant antigen of M. leprae pathogen. Previously, we have reported the existence of two M. leprae HSP18 variants in various leprotic patients. One of the variants has serine at position 52, whereas the other one has proline at the same position. We have also reported that HSP18 having proline at position 52 ( HSP18P 52 ) is a nonameric protein and exhibits chaperone function. However, the structural and functional characterization of wild-type HSP18 having serine at position 52 ( HSP18S 52 ) is yet to be explored. Furthermore, the implications of the S52P mutation on the structure and chaperone function of HSP18 are not well understood. Therefore, we cloned and purified these two HSP18 variants. We found that HSP18S 52 is also a molecular chaperone and an oligomeric protein. Intrinsic tryptophan fluorescence and far-UV CD measurements revealed that the S52P mutation altered the tertiary and secondary structure of HSP18. This point mutation also reduced the oligomeric assembly and decreased the surface hydrophobicity of HSP18, as revealed by HPLC and 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid binding studies, respectively. Mutant protein was less stable against thermal and chemical denaturation and was more susceptible towards tryptic cleavage than wild-type HSP18. HSP18P 52 had lower chaperone function and was less effective in protecting thermal killing of Escherichia coli than HSP18S 52. Taken together, our data suggest that serine 52 is important for the larger oligomerization and chaperone function of HSP18. Because both variants differ in stability and function, they may have different roles in the survival of M. leprae in infected hosts. Abbreviations ACD, a-crystallin domain; bis-ANS, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt; CFU, colony-forming unit; FRET, F€ orster resonance energy transfer; Gu-HCl, guanidine hydrochloride; IPTG, isopropyl thio-b-D-galactoside; MDH, malate dehydrogenase; sHSP, small heat shock protein. Structured digital abstract

Adenosine-5’-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of “HSP18-ATP” interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.

αB-Crystallin is a chaperone and an anti-apoptotic protein that is highly expressed in many tissues, including the lens, retina, heart and kidney. In the human lens, several lysine residues in αB-crystallin are acetylated. We have previously shown that such acetylation is predominant at lysine92 (K92) and K166. We have investigated the effect of lysine acetylation on the structure and functions of αB-crystallin by the specific introduction of an Nε-acetyllysine (AcK) mimic at K92. The introduction of AcK slightly altered the secondary and tertiary structures of the protein. AcK introduction also resulted in an increase in the molar mass and hydrodynamic radius of the protein, and the protein became structurally more open and more stable than the native protein. The acetyl protein acquired higher surface hydrophobicity and exhibited 25-55% higher chaperone activity than the native protein. The acetyl protein had higher client protein binding per subunit of the protein and higher binding affinity relative to the native protein. The acetyl protein was at least 20% more effective in inhibiting chemically induced apoptosis than the native protein. Molecular modeling suggests that acetylation of K92 makes the ‘α-crystallin domain’ more hydrophobic. Together, our results reveal that the acetylation of a single lysine residue in αB-crystallin makes the protein structurally more stable and improves its chaperone and anti-apoptotic activities. Our findings suggest that lysine acetylation of αB-crystallin is an important chemical modification to enhance αB-crystallin’s protective functions in the eye.

Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31–43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25–43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min-1. Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18.

Acylation of lysine residues is a common post-translational modification of cellular proteins. Here, we show that lysine succinylation, a type of acylation, occurs in human lens proteins. All of the major crystallins exhibited Nε-succinyllysine (SuccK) residues. Quantification of SuccK in human lens proteins (from donors between the ages of 20 and 73 years) by LC–MS/MS showed a range between 1.2 and 14.3 pmol/mg lens protein. The total SuccK levels were slightly reduced in aged lenses (age > 60 years) relative to young lenses (age < 30 years). Immunohistochemical analyses revealed that SuccK was present in epithelium and fiber cells. Western blotting and immunoprecipitation experiments revealed that SuccK is particularly prominent in αB-crystallin, and succinylation in vitro revealed that αB-crystallin is more prone to succinylation than αA-crystallin. Mass spectrometric analyses showed succinylation at K72, K90, K92, K166, K175, and potentially K174 in human lens aB-crystallin. We detected succinylation at K72, K82, K90, K92, K103, K121, K150, K166, K175, and potentially K174 by mass spectrometry in mildly succinylated αB-crystallin. Mild succinylation improved the chaperone activity of αB-crystallin along with minor perturbation in tertiary and quaternary structure of the protein. These observations imply that succinylation is beneficial to αB-crystallin by improving its chaperone activity with only mild conformational alterations.

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The human lens contains three major protein families: α-, β-, and γ-crystallin. Among the several variants of γ-crystallin in the human lens, γD-crystallin is a major form. γD-Crystallin is primarily present in the nuclear region of the lens and contains a single lysine residue at the second position (K2). In this study, we investigated the acetylation of K2 in γD-crystallin in aging and cataractous human lenses. Our results indicated that K2 is acetylated at an early age and that the amount of K2-acetylated γD-crystallin increased with age. Mass spectrometric analysis revealed that in addition to K2, glycine 1 (G1) was acetylated in γD-crystallin from human lenses and in γD-crystallin acetylated in vitro. The chaperone ability of α-crystallin for acetylated γD-crystallin was lower than that for the nonacetylated protein. The tertiary structure and the microenvironment of the cysteine residues were significantly altered by acetylation. The acetylated protein exhibited higher surface hydrophobicity, was unstable against thermal and chemical denaturation, and exhibited a higher propensity to aggregate at 80 °C in comparison to the nonacetylated protein. Acetylation enhanced the GdnHCl-induced unfolding and slowed the subsequent refolding of γD-crystallin. Theoretical analysis indicated that the acetylation of K2 and G1 reduced the structural stability of the protein and brought the distal cysteine residues (C18 and C78) into close proximity. Collectively, these results indicate that the acetylation of G1 and K2 residues in γD-crystallin likely induced a molten globule-like structure, predisposing it to aggregation, which may account for the high content of aggregated proteins in the nucleus of aged and cataractous human lenses.

Axonal degeneration and death of retinal ganglion cells (RGCs) are the primary causes of vision loss in glaucoma. In this study, we evaluated the efficacy of a peptide (peptain-1) that exhibits robust chaperone and anti-apoptotic activities against RGC loss in two rodent models and in cultured RGCs. In cultures of rat primary RGCs and in rat retinal explants peptain-1 significantly decreased hypoxia-induced RGC loss when compared to a scrambled peptide. Intraperitoneally (i.p.) injected peptain-1 (conjugated to a Cy7 fluorophore) was detected in the retina indicative of its ability to cross the blood-retinal barrier. Peptain-1 treatment inhibited RGC loss in the retina of mice subjected to ischemia/reperfusion (I/R) injury. A reduction in anterograde axonal transport was also ameliorated by peptain-1 treatment in the retina of I/R injured mice. Furthermore, i.p. injections of peptain-1 significantly reduced RGC death and axonal loss and partially restored retinal mitochondrial cytochrome c oxidase subunit 6b2 (COX 6b2) levels in rats subjected to five weeks of elevated intraocular pressure. We conclude that i.p. injected peptain-1 gains access to the retina and protects both RGC somas and axons against the injury caused by I/R and ocular hypertension. Based on these findings, peptain-1 has the potential to be developed as an efficacious neuroprotective agent for the treatment of glaucoma.