Lysosomal protease cathepsin D; a new driver of apoptosis during acute kidney injury.

Acute kidney injury (AKI) is an abrupt reduction in kidney function caused by different pathological processes. It is associated with a significant morbidity and mortality in the acute phase and an increased risk of developing End Stage Renal Disease. Despite the progress in the management of the disease, mortality rates in the last five decades remain unchanged at around 50%. Therefore there is an urgent need to find new therapeutic strategies to treat AKI. Lysosomal proteases, particularly Cathepsin D (CtsD), play multiple roles in apoptosis however, their role in AKI is still unknown. Here we describe a novel role for CtsD in AKI. CtsD expression was upregulated in damaged tubular cells in nephrotoxic and ischemia reperfusion (IRI) induced AKI. CtsD inhibition using Pepstatin A led to an improvement in kidney function, a reduction in apoptosis and a decrease in tubular cell damage in kidneys with nephrotoxic or IRI induced AKI. Pepstatin A treatment slowed interstitial fibrosis progression following IRI induced AKI. Renal transplant biopsies with acute tubular necrosis demonstrated high levels of CtsD in damaged tubular cells. These results support a role for CtsD in apoptosis during AKI opening new avenues for the treatment of AKI by targeting lysosomal proteases.

De novo designed library of linear helical peptides: an exploratory tool in the discovery of protein-protein interaction modulators.

Protein-protein interactions (PPIs) have emerged as important targets for pharmaceutical intervention because of their essential role in numerous physiological and pathological processes, but screening efforts using small-molecules have led to very low hit rates. Linear peptides could represent a quick and effective approach to discover initial PPI hits, particularly if they have inherent ability to adopt specific peptide secondary structures. Here, we address this hypothesis through a linear helical peptide library, composed of four sublibraries, which was designed by theoretical predictions of helicity (Agadir software). The 13-mer peptides of this collection fixes either a combination of three aromatic or two aromatic and one aliphatic residues on one face of the helix (Ac-SSEEX(5)ARNX(9)AAX(12)N-NH2), since these are structural features quite common at PPIs interfaces. The 81 designed peptides were conveniently synthesized by parallel solid-phase methodologies, and the tendency of some representative library components to adopt the intended secondary structure was corroborated through CD and NMR experiments. As proof of concept in the search for PPI modulators, the usefulness of this library was verified on the widely studied p53-MDM2 interaction and on the communication between VEGF and its receptor Flt-1, two PPIs for which a hydrophobic α-helix is essential for the interaction. We have demonstrated here that, in both cases, selected peptides from the library, containing the right hydrophobic sequence of the hot-spot in one of the protein partners, are able to interact with the complementary protein. Moreover, we have discover some new, quite potent inhibitors of the VEGF-Flt-1 interaction, just by replacing one of the aromatic residues of the initial F(5)Y(9)Y(12) peptide by W, in agreement with previous results on related antiangiogenic peptides. Finally, the HTS evaluation of the full collection on thermoTRPs has led to a few antagonists of TRPV1 and TRPA1 channels, which open new avenues on the way to innovative modulators of these channels.

Expression of orexin A and its receptor 1 in the choroid plexuses from buffalo brain.

The hypothalamic peptide orexin A, deriving from the proteolytic cleavage of the precursor molecule prepro-orexin, has a wide range of physiological effects including the regulation of feeding behaviour, neuroendocrine functions, sleep-wake cycle, and energy homeostasis. Lowered excretion of orexin A into the cerebrospinal fluid (CSF) plays a pathological role in animal and human narcolepsy. Altered levels of orexin A into the CSF have been also found in numerous disorders of the central nervous system, including Parkinson's and Huntington's disease, dementia, and depressive disorders. While the localization of orexin A and its receptor 1, OX(1), has been elicited in many regions of the mammalian brain and in peripheral organs, there are no information on the expression of the neuropeptide and its receptor 1 in the choroid plexuses (CPs) producing the CSF. In this study, we investigated the expression of orexin A and OX(1) in the CPs from the brain of an adult mammalian species, Bubalis bubalis, by immunogold-labelling in scanning electron microscopy. Both orexin A and OX(1) immuno-reactivity appeared to be widely distributed on the surface of choroid epithelium. Interestingly, a marked orexin A labelling was detected in the areas surrounding the CP blood capillaries. The expression of prepro-orexin and OX(1) mRNA transcripts of 200 and 300 bp, respectively, was assessed in the CPs by reverse-transcription polymerase chain reaction, while Western blotting analysis confirmed the presence of these two proteins in the tissue. Our findings provide the first evidence for orexin A and OX(1) expression in the CPs from mammalian brain, and suggest that the levels of orexin A into the CSF are probably regulated by CP activity.

Expression of the serotonin transporter SERT in the genital tract of cattle.

Both prostate and vestibular glands of mammals contain neuroendocrine cells which synthesize, store and release growth factors including neuropeptides and biogenic amines such as serotonin. An increase of the secretory products by these cells has been correlated to tumour progression and poor prognosis. Serotonin mediates a wide range of physiological functions by binding to multiple receptors on cell surface. However, the entire serotonergic system is mainly regulated by the serotonin transporter SERT which modulates serotonin concentration in extracellular fluid. Primarily located in serotonergic neurons, SERT is also expressed in various cell types in the periphery. In this study, we found a wide distribution of SERT in the parenchymal cells of both the prostate and the vestibular glands of cattle using immunohistochemistry. The expression of SERT mRNA transcripts was assessed by reverse-transcription polymerase chain reaction, thus suggesting that SERT is locally synthesized. Furthermore, Western blotting analysis showed the presence of two isoforms of the protein (70 and 140 kDa), probably corresponding to the high mannose-type SERT and its dimeric form. Our results provide the first evidence for SERT expression in the mammalian genital tract, thus highlighting a new potential target for the therapy of the genital tract cancers.

Expression of orexin A and its receptor 1 in the rat epididymis.

The hypothalamic peptide orexin A (oxA) derives from the proteolytic cleavage of the precursor molecule prepro-orexin. It binds with the high affinity G-protein-coupled orexin receptor 1 (OX1R). Here, we report the detection of oxA and OX1R in the principal cells of the rat caudal epididymis by immunohistochemistry. Both oxA and OX1R immunolabelling showed cytoplasmic supranuclear localization, filling the apical portion of the cells. The expression of prepro-orexin and OX1R mRNA transcripts in the rat epididymis was assessed by reverse-transcriptase polymerase chain reaction, while the presence of both these proteins in the tissue was confirmed by Western blotting analysis. Our findings provide the evidence for the presence of oxA and OX1R in the rat epididymis, and demonstrate that both proteins are locally synthesised, thus suggesting a role for oxA in governing the fertilizing capability of the immature male gamete.

Expression of the serotonin transporter (SERT) in the choroid plexuses from buffalo brain.

Choroid plexuses (CPs) play pivotal roles in a wide range of processes that establish, survey, and maintain the biochemical and cellular status of the central nervous system. Mammalian CPs contain a very high density of serotonin receptors, and serotonin has been shown to affect CP functions. The serotonin transporter (SERT) regulates the entire serotonergic system, including serotonin receptors by means of modulation of serotonin concentration in the extracellular fluid. In this study, the expression of SERT in the CPs from the brain of a mammalian species, Bubalis bubalis, was established. By immunogold labeling in scanning electron microscopy, SERT immunoreactivity was found to be localized on the apical surface of the choroid epithelium. In particular, SERT positivity was detected on the apical portion of villi, and both on the membrane and in the cytoplasm of grouped cells on the surface of the choroid epithelium. Significantly, no SERT was detected in blood vessels irrigating the CPs. The expression of SERT mRNA transcripts of 440 bp in the CPs was detected by reverse-transcription polymerase chain reaction, and Western blotting analysis revealed the presence of three isoforms of the protein with molecular masses of approximately 70, 80, and 140 kDa, respectively, probably corresponding to differently glycosylated SERT. Our findings provide the first report of SERT detection in the CPs of buffalo brain and indicate that this protein is locally synthesized from the choroid epithelial cells. We suggest that SERT might have an important role in mammalian CPs, possibly regulating the serotonin flow between brain and rest of the body.