Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani

The differentiation of Leishmania parasites from the insect stage, the promastigote, toward the pathogenic mammalian stage, the amastigote, is triggered primarily by the rise in ambient temperature encountered during the insect-to-mammal transmission. We show here that inactivation of heat shock protein (Hsp) 90, with the use of the drugs geldanamycin or radicicol, mimics transmission and induces the differentiation from the promastigote to the amastigote stage. Geldanamycin also induces a growth arrest of cultured promastigotes that can be forestalled by overexpression of the cytoplasmic Hsp90. Moreover, we demonstrate that Hsp90 serves as a feedback inhibitor of the cellular heat shock response in Leishmania. Our results are consistent with Hsp90 homeostasis serving as cellular thermometer for these primitive eukaryotes, controlling both the heat shock response and morphological differentiation.     

A novel role for 100 kD heat shock proteins in the parasite Leishmania donovani

Heat shock proteins of the 100 kD family have been known to confer general stress tolerance in yeast and plants. Several protozoan parasites possess genes for Hsp100 proteins. In Leishmania species the protein is expressed under heat stress and during the mammalian stage, the amastigote. We show here that replacement of the clpB gene which encodes Hsp100 does not affect thermotolerance or general viability in Leishmania donovani insect stages (promastigotes) nor in axenically cultured mammalian stages (amastigotes). However, its expression is required for normal development of the parasite inside mammalian host cells. Hsp100 appears to function as an antagonist of amastigote-to-promastigote differentiation and a promoter of full amastigote development.     

Expression of heat shock proteins in response to high and low temperature extremes in diapausing pharate larvae of the gypsy moth,Lymantria dispar

Diapausing pharate first instars of the gypsy moth, Lymantria dispar, respond to high temperature (37–41°C) by suppressing normal protein synthesis and synthesizing a set of seven heat shock proteins with M_rs of 90,000, 75,000, 73,000, 60,000, 42,000, 29,000, and 22,000 as determined by SDS-PAGE. During recovery at 25°C from heat shock, synthesis of the heat shock proteins gradually decreases over a period of 6 h, while normal protein synthesis is restored. A subset of these same heat shock proteins is also expressed during recovery at 4°C or 25°C from brief exposures to low temperature (-10 to 20°C), and its expression is more intense with increased severity of cold exposure. During recovery at 4°C after 24 h at ?20°C, both 90,000 and 75,000 M_r heat shock proteins are expressed for more than 96 h. While normal protein synthesis is suppressed during heat shock and recovery from heat shock, normal protein synthesis coincides with synthesis of the heat shock proteins during recovery from low temperatures, thus implying that expression of the heat shock proteins is not invariably linked to suppression of normal protein synthesis. Western transfer, using a monoclonal antibody that recognizes the inducible form of the human 70,000 M_r heat shock protein, demonstrates that immunologically related proteins in the gypsy moth are expressed at 4°C and during recovery from cold and heat shock.     

Heat-stress induced modulation of protein phosphorylation in virulent promastigotes of Leishmania donovani

In parasites such as Leishmania, the study of molecular events induced in response to heat stress is of immense interest since temperature increase is an integral part of the life cycle. Protein phosphorylation is known to control major steps of proliferation and differentiation in eukaryotic cells. Studies on intracellular signaling systems in protozoa are relatively recent. We have examined the effect of heat shock on the protein phosphorylation status in promastigotes of Leishmania donovani. The patterns of total protein phosphorylation and specific phosphorylation at tyrosine residues were examined using [32P]-orthophosphate labelling of the parasites and immunoblotting with a monoclonal anti-phosphotyrosine antibody. The major proteins of L. donovani that were phosphorylated at 24 degrees C had apparent molecular weights of 110, 105, 66-68, 55, 36-40 and 20 kDa. Heat shock (from 24 to 37 degrees C) led to a significant decrease in phosphorylation of the majority of phosphoproteins in the virulent promastigotes. On the other hand, the avirulent promastigotes did not show any decrease in protein phosphorylation on exposure to heat stress. Predominant phosphorylation at tyrosine residues was detectable in proteins of putative size 105-110 kDa in both virulent and avirulent parasites. Heat shock led to a reduction in the level of phosphotyrosine in both these proteins in the case of virulent parasites, while no such reduction was detectable in avirulent parasites. Significant modifications in the phosphorylation status of proteins in response to heat stress including that of tyrosine containing proteins, observed exclusively in virulent parasites, suggest that modulation of protein phosphorylation/dephosphorylation may play a role in signal transduction pathways in the parasite upon heat shock encountered on entering the mammalian host.     

TMV protein synthesis is not translationally regulated by heat shock

Summary Tobacco mosaic virus (TMV) protein synthesis in tobacco leaf tissue was not translationally regulated under conditions of heat shock as were most of the other proteins that were produced at 25°C. Upon shift from 25°C to 37–40°C, most host protein synthesis was inhibited followed by initiation of synthesis of heat shock proteins. In contrast, TMV protein synthesis continued after the temperature shift. This phenomenon allowed the enhancement of detection of TMV protein synthesis in tobacco leaves. The most prominent proteins labeled were viral when tissue was labeled during the first hr following the shift to 40°C, a period after heat shock repression of host protein synthesis, but before the onset of most heat shock protein synthesis. Another method to predominately label viral proteins was to incubate infected leaves for periods at 35°C which induced repression of preexisting host protein synthesis without inducing synthesis of heat shock proteins.     

Involvement of the Leishmania donovani virulence factor A2 in protection against heat and oxidative stress

Leishmania is an obligate intracellular protozoan parasite that infects cells of the reticulo-endothelial system. Host defences against Leishmania include fever and oxidant production, and the parasite has developed a number of defence mechanisms to neutralize the host response. The Leishmania donovani A2 family of proteins has been shown to be essential for survival in mammalian visceral organs. Here we provide evidence that A2 proteins protect the parasite against host defences, namely heat stress (fever) and oxidative stress. A2 is however unable to protect the cells from endoplasmic reticulum stress induced by dithiothreitol. To downregulate A2 protein expression, L. donovani was transfected with an A2 antisense RNA expressing-vector, resulting in significant reduction of A2 levels. The resulting A2-deficient cells were more sensitive to heat shock and this was associated with increased production of internal oxidants during heat shock. Moreover, axenic amastigotes with downregulated A2 expression had increased internal oxidants and decreased viability following treatment with hydrogen peroxide or a nitric oxide donor when compared to control cells. Overall, these results suggest that A2 protects L. donovani from a variety of stresses, thereby allowing it to survive in the internal organs of the mammalian host and to cause visceral disease.     

Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani

In order to proceed through their life cycle, protozoan parasites of the genus Leishmania cycle between sandflies and mammals. This change of environment correlates with the differentiation from the promastigote stage (insect form) to the amastigote stage (intracellular mammalian form). The molecular basis underlying this major transformation is poorly understood so far; however, heat shock protein 90 (HSP90) appears to play a pivotal role. To further elucidate this process we identified proteins expressed preferentially in either of the two life cycle stages. By using two-dimensional (2-D) gel electrophoresis we observed defined changes in the protein pattern. A total of approximately 2000 protein spots were visualized. Of these, 31 proteins were present only in promastigotes. The abundance of 65 proteins increased during heat-induced in vitro amastigote differentiation, while a decreased abundance is observed for four proteins late in amastigote differentiation. Further analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting 67 protein spots were identified representing 41 different proteins known from databases and eight hypothetical proteins. Further studies showed that most of the stage-specific proteins fall into five groups of functionally related proteins. These functional categories are: (i) stress response (e.g. heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) energy metabolism and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism. Very similar changes in the 2-D protein pattern were obtained when in vitro amastigote differentiation was induced either by pharmacological inhibition of HSP90 or by a combination of heat stress and acidic pH supporting the critical role for HSP90 in life cycle control.     

Heat shock response of Dictyostelium

In response to a shift from 22 to 30°C the relative rate of synthesis of a small number of proteins is dramatically increased in Dictyostelium discoideum. The cells neither grow nor develop at this temperature but die slowly with a half-life of 18 hr. The major protein synthesized in response to a heat shock to 30°C in either growing cells or developing cells has an apparent molecular weight of 70,000 (70K). An increase in the relative rate of synthesis of 70K can be seen as early as 20 min following heat shock. Synthesis of 70K remains high for 4 hr at 30°C and then decreases. Similar kinetics of 70K synthesis occur during recovery at 22°C following a 1-hr heat shock. RNA synthesis during the first half-hour of heat shock is essential for the high rate of 70K measured 2 hr later. By isoelectric focusing the 70K protein can be separated into two spots, one of which overlaps one of the major heat shock proteins of Drosophila melanogaster. The relative rate of synthesis of several other proteins (82K, 60K, 43K) increases less dramatically in Dictyostelium during heat shock at 30°C. A heat shock to 34°C results in rapid synthesis of these proteins but not of 70K. The relative rates of synthesis of most other proteins made at 22°C decreases, most notably that of actin. Synthesis of heat shock proteins at 30°C does not significantly affect viability at 30°C but dramatically prolongs the period of time the cells can survive at 34°C. Thus, 30°C appears to be a stasis condition for Dictyostelium which elicits a response essential for protection from lethal temperatures. The similarity of the heat shock response in Dictyostelium to that in Drosophila and vertebrate cells suggests that certain aspects of the response may be universal in eukaryotes.     

The expression of Candida albicans enolase is not heat shock inducible

Abstract An isoprotein of enolase from the yeast Saccharomyces cerevisiae was reported to be a heat shock protein. The possible role of the C. albicans enolase as a heat shock protein was therefore investigated. The de novo synthesis of C. albicans enolase protein and mRNA did not increase during heat stress, but remained constitutively expressed. Amino acid similarity to the heat shock proteins suggests that although the C. albicans enolase is not a classical heat shock protein, it may be a memberof a group of constitutively expressed, structurally related proteins, the heat shock cognate proteins.     

Response of Higher Plants to Heat Shock

When sorghum seedlings were rapidly shifted from the cultural temperature of 30℃ to 40℃ and 45℃, a set of abnormal proteins, generally referred to as heat shock proteins were induced. They are a group of high molecular weight proteins (about 66–117 kD), a few intermediate molecular weight proteins (33–66kD) and a low molecular weight protein of 18 kD. At the same time, the synthesis of normal proteins was relatively depressed. The res ponse of the shoot tissues of sorghum seedings to heat shock is similar to that of the root tissues, but there are some differences in more detail between the two tissues. The synthesis of heat shock proteins in sorghum seedlings was rapid. After one-hour exposure at 45℃ their synthesis in the roots was detectable. Maximum induction took place in the second hour of exposure, thereafter their synthesis began to decline markedly. Finally, there appear to be some proteins whose synthesis was not supressed during heat shock, It is not yet known why the synthesis of these proteins is so stable.     

Absence of heat shock protein synthesis in isolated mitochondria and plastids from maize

Examination of the proteins synthesized by isolated mitochondria, chloroplasts, or proplastids from maize tissues showed that a heat treatment at 40 degrees C does not induce or enhance the synthesis of any protein when compared to preparations treated at the control temperature of 28 degrees C. These observations are consistent with the results obtained by labeling proteins in vivo under sterile conditions. In vivo labeling in the presence of cycloheximide during heat shock showed no heat shock protein synthesis. Labeling in the presence of chloramphenicol during heat shock showed a similar heat shock protein pattern as in the absence of the inhibitor. It is concluded that maize organelles do not synthesize heat shock proteins and that, if present, they may be due to bacterial contamination.     

Expression of the Heat Shock Response in a Tomato Interspecific Hybrid Is Not Intermediate between the Two Parental Responses

While it is apparent that the heat shock response is ubiquitous, variabilities in the nature of the heat shock response between closely related species have not been well characterized. The heat shock response of three genotypes of tomato, Lycopersicon esculentum, Lycopersicon pennellii, and the interspecific sexual hybrid was characterized. The two parental genotypes differed in the nature of the heat shock proteins synthesized; the speciesspecific heat shock proteins were identified following in vivo labeling of leaf tissue with [³⁵S]methionine and cysteine. The duration of, and recovery from, heat shock varied between the two species: L. esculentum tissue recovered more rapidly and protein synthesis persisted longer during a heat shock than in the wild species, L. pennellii. Both species induced heat shock protein synthesis at 35°C and synthesis was maximal at 37°C. The response of the F1 to heat shock was intermediate to the parental responses for duration of, and recovery from, heat shock. In other aspects, the response of the F1 to heat shock was not intermediate to the parental responses: the F1 induced only half of the L. esculentum specific heat shock proteins, and all of the L. pennellii specific heat shock proteins. A discussion of the inheritance of the regulation of the heat shock response is presented.     

Heat shock mRNA accumulation during recovery from cold shock in Drosophila melanogaster

Heat shock protein synthesis is induced in response to a variety of chemical and physical stresses. Among these are heating above normal growing temperatures, treatment with heavy metals, amino acid analogues, steroid hormones and a variety of other chemicals (CRC Crit. Rev. Biochem. 18, 239–280). We have shown previously that heat shock proteins are also synthesized during recovery from prolonged 0°C treatment in Drosophila larval salivary glands. In this paper we describe the cold treatments which induce heat shock protein synthesis in more detail, and show that heat shock mRNA does not accumulate during the cold treatment, but rather during the recovery period when the larvae are returned to 25°C. The implications of these results for the regulation of heat shock mRNA levels, and for the role of heat shock proteins in recovery from cold shock are discussed.