Protozoan Kinetoplastida, including the pathogenic trypanosomatids of the genera Trypanosoma and Leishmania, compartmentalize several important metabolic systems in their peroxisomes which are designated glycosomes. The enzymatic content of these organelles may vary considerably during the life-cycle of most trypanosomatid parasites which often are transmitted between their mammalian hosts by insects. The glycosomes of the Trypanosoma brucei form living in the mammalian bloodstream display the highest level of specialization; 90% of their protein content is made up of glycolytic enzymes. The compartmentation of glycolysis in these organelles appears essential for the regulation of this process and enables the cells to overcome short periods of anaerobiosis. Glycosomes of all other trypanosomatid forms studied contain an extended glycolytic pathway catalyzing the aerobic fermentation of glucose to succinate. In addition, these organelles contain enzymes for several other processes such as the pentose-phosphate pathway, beta-oxidation of fatty acids, purine salvage, and biosynthetic pathways for pyrimidines, ether-lipids and squalenes. The enzymatic content of glycosomes is rapidly changed during differentiation of mammalian bloodstream-form trypanosomes to the forms living in the insect midgut. Autophagy appears to play an important role in trypanosomatid differentiation, and several lines of evidence indicate that it is then also involved in the degradation of old glycosomes, while a population of new organelles containing different enzymes is synthesized. The compartmentation of environment-sensitive parts of the metabolic network within glycosomes would, through this way of organelle renewal, enable the parasites to adapt rapidly and efficiently to the new conditions.

Protozoan Kinetoplastida, a group that comprises the pathogenic Trypanosoma brucei, compartmentalize several metabolic systems such as the major part of the glycolytic pathway, in multiple peroxisome-like organelles, designated glycosomes. Trypanosomes have a complicated life cycle, involving two major, distinct stages living in the mammalian bloodstream and several stages inhabiting different body parts of the tsetse fly. Previous studies on non-differentiating trypanosomes have shown that the metabolism and enzymatic contents of glycosomes in bloodstream-form and cultured procyclic cells, representative of the stage living in the insect's midgut, differ considerably. In this study, the morphology of glycosomes and their position relative to the lysosome were followed, as were the levels of some glycosomal enzymes and markers for other subcellular compartments, during the differentiation from bloodstream-form to procyclic trypanosomes. Our studies revealed a small tendency of glycosomes to associate with the lysosome when a population of long-slender bloodstream forms differentiated into short-stumpy forms which are pre-adapted to live in the fly. The same phenomenon was observed during the short-stumpy to procyclic transformation, but then the process was fast and many more glycosomes were associated with the dramatically enlarged degradation organelle. The observations suggested an efficient glycosome turnover involving autophagy. Changes observed in the levels of marker enzymes are consistent with the notion that, during differentiation, glycosomes with enzymatic contents specific for the old life-cycle stage are degraded and new glycosomes with different contents are synthesized, causing that the metabolic repertoire of trypanosomes is, at each stage, optimally adapted to the environmental conditions encountered.

The JAK2 V617F mutation present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients renders the kinase constitutively active. In the absence of a three-dimensional structure for the full-length protein, the mechanism of activation of JAK2 V617F has remained elusive. In this study, we used functional mutagenesis to investigate the involvement of the JH2 αC helix in the constitutive activation of JAK2 V617F. We show that residue F595, located in the middle of the αC helix of JH2, is indispensable for the constitutive activity of JAK2 V617F. Mutation of F595 to Ala, Lys, Val or Ile significantly decreases the constitutive activity of JAK2 V617F, but F595W and F595Y are able to restore it, implying an aromaticity requirement at position 595. Substitution of F595 to Ala was also able to decrease the constitutive activity of two other JAK2 mutants, T875N and R683G, as well as JAK2 K539L, albeit to a lower extent. In contrast, the F595 mutants are activated by erythropoietin-bound EpoR. We also explored the relationship between the dimeric conformation of EpoR and several JAK2 mutants. Since residue F595 is crucial to the constitutive activation of JAK2 V617F but not to initiation of JAK2 activation by cytokines, we suggest that small molecules that target the region around this residue might specifically block oncogenic JAK2 and spare JAK2 wild-type.

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