Importance of serotonin (5-HT) and its precursor l-tryptophan for homeostasis and function of skeletal muscle in rats. A morphological and endocrinological study.

Serotonin (5-HT) is a neurotransmitter, synthesized in serotonergic neurons of the central nervous system and in enterochromaffin cells of the gastrointestinal tract, which is involved in the regulation of several body functions, including muscle tissue development and growth and its contractile response. l-Tryptophan (l-Trp) is an essential amino acid and precursor of 5-HT. The aim of the present study was to better understand the mechanisms that govern neuroendocrine homeostasis of muscle tissue and emphasize the importance of a diet, complete in all its elements, referring specifically to the essential amino acids such as l-Trp, crucial in several neuroendocrine functions.We analyzed the possible consequences of l-Trp-free diet on 5-HT production and on skeletal muscle morphology and function in young female rats. We also evaluated the eventual alterations of hormone production such as growth hormone (GH), thyroid stimulating hormone (TSH) and thyroid hormones (T3 and T4) that control and regulate growth, metabolism and efficiency of the skeletal muscle. Our results showed a strong decrease of 5-HT, GH, TSH, T3 and T4 levels associated to a clear difference in body weight between experimental and control rats. Moreover, the muscle samples of experimental rats showed histological and ultrastructural alterations. These findings thus supported a strong link between l-Trp, serotonergic system, hormone secretion and morphology of skeletal muscle tissue and thus, the importance of a balanced daily diet.

IRF5 is a target of BCR-ABL kinase activity and reduces CML cell proliferation.

Interferon regulatory factor 5 (IRF5) modulates the expression of genes controlling cell growth and apoptosis. Previous findings have suggested a lack of IRF5 transcripts in both acute and chronic leukemias. However, to date, IRF5 expression and function have not been investigated in chronic myeloid leukemia (CML). We report that IRF5 is expressed in CML cells, where it interacts with the BCR-ABL kinase that modulates its expression and induces its tyrosine phosphorylation. Tyrosine-phosphorylated IRF5 displayed reduced transcriptional activity that was partially restored by imatinib mesylate (IM). Interestingly, a mutant devoid of a BCR-ABL consensus site (IRF5(Y104F)) still presented significant tyrosine phosphorylation. This finding suggests that the oncoprotein phosphorylates additional tyrosine residues or induces downstream signaling pathways leading to further IRF5 phosphorylation. We also found that ectopic expression of IRF5 decreases the proliferation of CML cell lines by slowing their S-G2 transition, increasing the inhibition of BCR-ABL signaling and enhancing the lethality effect observed after treatment with IM, α-2-interferon and a DNA-damaging agent. Furthermore, IRF5 overexpression successfully reduced the clonogenic ability of CML CD34-positive progenitors before and after exposure to the above-indicated cytotoxic stimuli. Our data identify IRF5 as a downstream target of the BCR-ABL kinase, suggesting that its biological inactivation contributes to leukemic transformation.

BCR-ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein.

Patients with chronic myeloid leukemia in whom tyrosine kinase inhibitors (TKIs) fail often present mutations in the BCR-ABL catalytic domain. We noticed a lack of substitutions involving 4 amino acids (E286, M318, I360, and D381) that form hydrogen bonds with ponatinib. We therefore introduced mutations in each of these residues, either preserving or altering their physicochemical properties. We found that E286, M318, I360, and D381 are dispensable for ABL and BCR-ABL protein stability but are critical for preserving catalytic activity. Indeed, only a "conservative" I360T substitution retained kinase proficiency and transforming potential. Molecular dynamics simulations of BCR-ABL(I360T) revealed differences in both helix αC dynamics and protein-correlated motions, consistent with a modified ATP-binding pocket. Nevertheless, this mutant remained sensitive to ponatinib, imatinib, and dasatinib. These results suggest that changes in the 4 BCR-ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Notably, amino acids equivalent to those identified in BCR-ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.

Genomics, evolution, and expression of TBPL2, a member of the TBP family.

TBPL2 is the most recently discovered and less characterized member of the TATA box binding protein (TBP) family that also comprises TBP, TATA box binding protein-like 1 (TBPL1), and Drosophila melanogaster TBP related factor (TRF). In this paper we report our in silico and in vitro data on (i) the genomics of the TBPL2 gene in Homo sapiens, Pan troglodytes, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus tropicalis, and Takifugu rubripes; (ii) its evolution and phylogenetic relationship with TBP, TBPL1, and TRF; (iii) the structure of the TBPL2 proteins that belong to the recently identified group of the intrinsically unstructured proteins (IUPs); and (iv) TBPL2 expression in different organs and cell types of Homo sapiens and Rattus norvegicus. Similar to TBP, both the TBPL2 gene and protein are bimodular. The 3' region of the gene encoding the DNA binding domain (DBD) was well conserved during evolution. Its high homology to vertebrate TBP suggests that TBPL2 also should bind to the TATA box and interact with the proteins binding to TBP carboxy-terminal domain, such as the TBP associated factors (TAFs). As already demonstrated for TBP, TBPL2 amino-terminal segment is intrinsically unstructured and, even though variable among vertebrates, comprises a highly conserved motif not found in any other known protein. Absence of TBPL2 from the genome of invertebrates and plants demonstrates its specific origin within the subphylum of vertebrates. Our RT-PCR analysis of human and rat RNA shows that, similar to TBP, TBPL2 is ubiquitously synthesized even though at variable levels that are at least two orders of magnitude lower. Higher expression of TBPL2 in the gonads than in other organs suggests that it could perform important functions in gametogenesis. Our genomic and expression data should contribute to clarify why TBP has a general master role within the transcription apparatus (TA), whereas both TBPL1 and TBPL2 perform tissue-specific functions.

Modelling of the ABL and ARG proteins predicts two functionally critical regions that are natively unfolded.

The ABL and ARG tyrosine kinases regulate many pivotal cellular processes and are implicated in the pathogenesis of several forms of leukemia. We have modelled the previously uncharacterized core domain (SH3-SH2-tyrosine kinase) and C-terminal actin-binding domain of ARG. We have also investigated the structural arrangement of the ABL and ARG Cap region and of the long multifunctional region located downstream of the tyrosine kinase domain. We report that the ARG core domain is homologous to the corresponding ABL region, therefore suggesting that ARG catalytic activity is likely regulated by the same SH3-SH2 clamp described for ABL. We also report that the Cap of both ABL and ARG is natively unfolded. Hence, biological events determining the folding of the Cap are critical to allow its interaction with the tyrosine kinase C-lobe. Furthermore, our results show that, with the exception of the C-terminal actin-binding domain, the entire region encoded by the ABL and ARG last exon is natively unfolded. Phosphorylation events or protein-protein interactions regulating the folding of this region will therefore modulate the activity of its numerous functional domains. Finally, our analyses show that the C-terminal actin-binding domain of ARG displays a four-helix bundle structure similar to the one reported for the corresponding ABL region. Our findings imply that many biological activities attributed to ABL, ARG, and their oncogenic counterparts are regulated by natively unfolded regions.