Despite increasing evidence of host phenotypic manipulation by parasites, the underlying mechanisms causing infected hosts to act in ways that benefit the parasite remain enigmatic in most cases. Here, we used proteomics tools to identify the biochemical alterations that occur in the head of the cricket Nemobius sylvestris when it is driven to water by the hairworm Paragordius tricuspidatus. We characterized host and parasite proteomes during the expression of the water-seeking behaviour. We found that the parasite produces molecules from the Wnt family that may act directly on the development of the central nervous system (CNS). In the head of manipulated cricket, we found differential expression of proteins specifically linked to neurogenesis, circadian rhythm and neurotransmitter activities. We also detected proteins for which the function(s) are still unknown. This proteomics study on the biochemical pathways altered by hairworms has also allowed us to tackle questions of physiological and molecular convergence in the mechanism(s) causing the alteration of orthoptera behaviour. The two hairworm species produce effective molecules acting directly on the CNS of their orthoptera hosts.
The positive transcription elongation factor (P-TEFb) is required for the transcription of most genes by RNA polymerase II. Hexim proteins associated with 7SK RNA bind to P-TEFb and reversibly inhibit its activity. P-TEFb comprises the Cdk9 cyclin-dependent kinase and a cyclin T. Hexim proteins have been shown to bind the cyclin T subunit of P-TEFb. How this binding leads to inhibition of the kinase activity of Cdk9 has remained elusive, however. Using a photoreactive amino acid incorporated into proteins, we show that in live cells, cell extracts, and in vitro reconstituted complexes, Hexim1 cross-links and thus contacts Cdk9. Notably, replacement of a phenylalanine, F208, belonging to an evolutionary conserved Hexim1 peptide ( 202 PYNTTQFLM 210 ) known as the "PYNT" sequence, cross-links a peptide within the activation segment that controls access to the Cdk9 catalytic cleft. Reciprocally, Hexim1 is cross-linked by a photoreactive amino acid replacing Cdk9 W193, a tryptophan within this activation segment. These findings provide evidence of a direct interaction between Cdk9 and its inhibitor, Hexim1. Based on similarities with Cdk2 3D structure, the Cdk9 peptide cross-linked by Hexim1 corresponds to the substrate bindingsite. Accordingly, the Hexim1 PYNT sequence is proposed to interfere with substrate binding to Cdk9 and thereby to inhibit its kinase activity.cyclin-dependent kinase inhibition | transcription factor regulation | regulatory noncoding RNA | benzoyl phenylalanine | protein-protein cross-linking
Calpains are intracellular proteases that play a key role in inflammation/immunity. Rare studies show that they are partially externalized. However, the mechanism of this secretion and the functions of exteriorized calpains remain poorly understood. In this study, we found that mouse and human lymphocytes secreted calpains through an ABCA1-driven process. In turn, extracellular calpains inhibited IL-17A expression. We were able to attribute this function to a cleavage of the TLR2 extracellular domain, which prevented TLR2-induced transcription of molecules essential for IL-17A induction. Calpain exteriorization and TLR2 cleavage were critical for the control of IL-17A expression by low doses of IL-2. By using newly developed transgenic mice in which extracellular calpains are specifically inactivated, we provide evidence for the relevance of calpain externalization in vivo in regulating IL-17A expression and function in experimental sterile peritonitis and autoimmune arthritis, respectively. Thus, this study identifies calpain exteriorization as a potential target for immune modulation.
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