Disaggregation and reactivation of aggregated proteins by chaperones is well established. However, little is known regarding such kind of function of single-domain small cyclophilins (CyPs). Here we demonstrate that, with increasing concentrations, fully active adenosine kinase (AdK) of Leishmania donovani tends to form soluble aggregates, resulting in inactivation. Using this inactive enzyme as the substrate, it is shown that a CyP from L. donovani (LdCyP) alone can cause complete disaggregation, leading to reactivation of the enzyme. The reactivating ability of LdCyP remains unaffected even in the presence of cyclosporin A and macromolecular crowding agents. The reactivation occurs noncatalytically and is reversible. A truncated LdCyP, devoid of 88 amino acids from the N terminus, is found to be required in near stoichiometric proportion to reactivate AdK, suggesting essentiality of the C-terminal region. Gel filtration and light-scattering experiments together with protein cross-linking studies revealed that both full-length LdCyP and the truncated form directly interact with AdK and convert oligomeric forms of the enzyme to monomeric state. Homology modeling studies suggest that the exposed hydrophobic residues of LdCyP, by interacting with solvent-accessible hydrophobic surface of AdK, pull apart its aggregated inactive oligomers to functional monomers. Clearly, the results are consistent with the interpretation that the higher efficiency of the truncated LdCyP is most likely due to increased exposure of the hydrophobic residues on its surface. These observations, besides establishing L. donovani AdK as one of the model enzymes to study aggregation-disaggregation of proteins, raise the possibility that single-domain small CyPs, under physiological conditions, may regulate the activity of aggregation-prone proteins by ensuring their disaggregation.

Dithiocarbamates have emerged as potent carbonic anhydrase (CA) inhibitors in recent years. Given that CAs are important players in cellular metabolism, the objective of this work was to exploit the CA-inhibitory property of dithiocarbamates as a chemotherapeutic weapon against the Leishmania parasite. We report here strong antileishmanial activity of three hitherto unexplored metal dithiocarbamates, maneb, zineb, and propineb. They inhibited CA activity in Leishmania major promastigotes at submicromolar concentrations and resulted in a dose-dependent inhibition of parasite growth. Treatment with maneb, zineb, and propineb caused morphological deformities of the parasite and Leishmania cell death with 50% lethal dose (LD 50 ) values of 0.56 M, 0.61 M, and 0.27 M, respectively. These compounds were even more effective against parasites growing in acidic medium, in which their LD 50 values were severalfold lower. Intracellular acidosis leading to apoptotic and necrotic death of L. major promastigotes was found to be the basis of their leishmanicidal activity. Maneb, zineb, and propineb also efficiently reduced the intracellular parasite burden, suggesting that amastigote forms of the parasite are also susceptible to these metal dithiocarbamates. Interestingly, mammalian cells were unaffected by these compounds even at concentrations which are severalfold higher than their antileishmanial LD 50 s). Our data thus establish maneb, zineb, and propineb as a new class of antileishmanial compounds having broad therapeutic indices. Leishmaniasis is a vector-borne disease caused by the protozoan parasite of the genus Leishmania. The disease is manifested in various clinical forms, ranging from self-healing skin ulcers to fatal infection of the visceral organs. With an estimated 1.3 million new cases and more than 20,000 deaths every year, leishmaniasis continues to be a threat to a huge population living in tropical and subtropical countries (1).To date, there is no antileishmanial vaccine for clinical use (2). Treatment options are also few. After several decades of successful use against visceral leishmaniasis, the pentavalent antimonials have become almost obsolete because of resistance developed against these drugs (3). This has led to the emergence of a second line of defense, including amphotericin B, paromomycin, and miltefosine (4). However, severe side effects, cases of disease relapse after an initial cure, and increasing signs of resistance have limited their efficacy (5-7). The liposomal formulation of amphotericin B (AmBisome) is by far the most effective treatment for leishmaniasis, having minimal side effects (8). Despite the effectiveness of AmBisome, its cost is a major point of concern, especially since this disease is prevalent in poorer sections of communities. Novel therapies against all forms of leishmaniasis are therefore urgently needed.Carbonic anhydrases (CAs) are a family of metalloenzymes that catalyze reversible hydration of CO 2 . By catalyzing this simple reaction, they play vital roles in a ...

Missense mutations in the carbonic anhydrase IV (CA IV) gene have been identified in patients with an autosomal dominant form of retinitis pigmentosa (RP17). We used two transient expression systems to investigate the molecular mechanism by which the newly identified CA IV mutations, R69H and R219S, contribute to retinal pathogenesis. Although the R219S mutation drastically reduced the activity of the enzyme, the R69H mutation had a minimal effect, suggesting that loss of CA activity is not the molecular basis for their pathogenesis. Defective processing was apparent for both mutant proteins. Cell surface-labeling techniques showed that the R69H and R219S mutations both impaired the trafficking of CA IV to the cell surface, resulting in their abnormal intracellular retention. Expression of both CA IV mutants induced elevated levels of the endoplasmic reticulum (ER) stress markers, BiP and CHOP, and led to cell death by apoptosis. They also had a dominant-negative effect on the secretory function of the ER. These properties are similar to those of R14W CA IV, the signal sequence variant found in the original patients with RP17. These findings suggest that toxic gain of function involving ER stress-induced apoptosis is the common mechanism for pathogenesis of this autosomal-dominant disease. Apoptosis induced by the CA IV mutants could be prevented, at least partially, by treating the cells with dorzolamide, a CA inhibitor. Thus, the use of a CA inhibitor as a chemical chaperone to reduce ER stress may delay or prevent the onset of blindness in RP17.autosomal dominant ͉ chemical chaperone ͉ ER stress ͉ missense mutation ͉ Naϩ/bicarbonate co-transporter 1

Leishmania parasites have evolved to endure the acidic phagolysosomal environment within host macrophages. How Leishmania cells maintain near-neutral intracellular pH and proliferate in such a proton-rich mileu remains poorly understood. We report here that, in order to thrive in acidic conditions, Leishmania major relies on a cytosolic and a cell surface carbonic anhydrase, LmCA1 and LmCA2, respectively. Upon exposure to acidic medium, the intracellular pH of the LmCA1 +/− , LmCA2 +/− and LmCA1 +/− :LmCA2 +/− mutant strains dropped by varying extents that led to cell cycle delay, growth retardation and morphological abnormalities. Intracellular acidosis and growth defects of the mutant strains could be reverted by genetic complementation or supplementation with bicarbonate. When J774A.1 macrophages were infected with the mutant strains, they exhibited much lower intracellular parasite burdens than their wild-type counterparts. However, these differences in intracellular parasite burden between the wild-type and mutant strains were abrogated if, before infection, the macrophages were treated with chloroquine to alkalize their phagolysosomes. Taken together, our results demonstrate that haploinsufficiency of LmCA1 and/or LmCA2 renders the parasite acid-susceptible, thereby unravelling a carbonic anhydrase-mediated pH homeostatic circuit in Leishmania cells.

Autosomal dominant familial isolated hypoparathyroidism (AD-FIH) is caused by a Cys 3 Arg mutation (C18R) in the hydrophobic core of the signal peptide of human preproparathyroid hormone (PPTH). Although this mutation impairs secretion of the hormone, the mechanism by which one mutant allele produces the autosomal-dominant disease is unexplained. Using transfected HEK293 cells, we demonstrate that the expressed mutant hormone is trapped intracellularly, predominantly in the endoplasmic reticulum (ER). This ER retention was found to be toxic for the cells, which underwent apoptosis, as evident from the marked increase in the number of cells staining positive for Annexin V binding and for the TUNEL reaction. The cells producing mutant hormone also had marked up-regulation of the ER stress-responsive proteins, BiP and PERK, as well as the proapoptotic transcription factor, CHOP. Up-regulation of these markers of the unfolded protein response supported a causal link between the ER stress and the cell death cascade. When the C18R PPTH was expressed in the presence of 4-phenylbutyric acid, which is a pharmacological chaperone, intracellular accumulation was reduced and normal secretion was restored. This treatment also produced remarkable reduction of ER stress signals and protection against cell death. These data implicate ER stress-induced cell death as the underlying mechanism for AD-FIH and suggest that the pharmacological manipulation of this pathway by using chemical chaperones offers a therapeutic option for treating this disease.endoplasmic reticulum stress ͉ parathyroid hormone ͉ unfolded protein response ͉ 4-phenylbutyric acid F amilial isolated hypoparathyroidism (FIH) represents a heterogeneous group of genetic disorders characterized by low calcium and high phosphorus levels in the blood serum, where the amount of immunoreactive parathyroid hormone (PTH) is low or undetectable. Clinical symptoms include neuromuscular irritability, tingling of the fingers and toes, and spontaneous or latent tetany (1, 2). These diseases vary in mode of inheritance. Mutations in the preproparathyroid hormone (PPTH) gene are found to be associated with the disease in some pedigrees (3-5).PPTH is synthesized as a 115-aa precursor in the parathyroid gland and is converted to the pro-PTH upon removal of the signal peptide (by signal peptidase) within the endoplasmic reticulum (ER). After trafficking to the Golgi apparatus, the pro-PTH is cleaved to the 84-aa mature PTH (Fig. 1). Then the mature PTH is packaged into secretory granules and, depending on needs, released into the blood, where it functions as a major regulator of the calcium level. The release of the PTH by the parathyroid gland is strictly calcium-dependent (6).In 1990, Arnold et al. (3) identified a T to C point mutation in the signal peptide-encoding region of the PPTH gene in a family with autosomal dominant FIH (AD-FIH) (7). This mutation disrupts the hydrophobic core of the signal sequence by changing the codon at position 18 (Ϫ8 position with respect to the s...