Emerging functional roles of cathepsin E

Cathepsin E is an intracellular aspartic protease of the endolysosomal pathway. It has been implicated in several physiological and pathological processes however, its exact functional role is yet to be elucidated. The present review gives an account of the major physiological functions that are associated to cathepsin E by various research groups and highlights the conditions developed in cathepsin E deficiency or the conditions where overexpression of cathepsin E is observed.     

Cathepsin E regulates the presentation of tetanus toxin C-fragment in PMA activated primary human B cells

Processing of antigens by proteases in the endocytic compartments of antigen presenting cells (APC) is essential to make them suitable for presentation as antigenic peptides to T lymphocytes. Several proteases of the cysteine, aspartyl and serine classes are involved in this process. It has been speculated, that the aspartyl protease cathepsin E (CatE) is involved in antigen processing in B cell line, monocyte-derived dendritic cells (DC) and murine DC. Here we show the expression of CatE in primary human B cells and DC, which was only elevated in B cells after induction with phorbol 12-myristate 13-acetate (PMA), resulted in enhanced presentation of tetanus toxin C-fragment (TTC) to the respective T cells. Inhibition of aspartyl proteases using pepstatin-A-penetratin (PepA-P), a highly efficient, cell-permeable aspartyl protease inhibitor, reduced significantly T cell activation in PMA activated B cells but not in PMA activated myeloid DC (mDC). Thus we suggest that CatE is important in the processing of TTC in primary human B cells.     

Development and precise characterization of phospho-site-specific antibody of Ser(357) of IRS-1: elimination of cross reactivity with adjacent Ser(358)

Antibodies that recognize specifically phosphorylated sites on proteins are widely utilized for studying the regulation and biological function of phosphoproteins. The proposed strategy is a powerful, analytical tool allowing the generation of phospho-site specific antibodies albeit adjacent phosphorylation sites are present. Here, we demonstrate the assessment and elimination of cross reactivity of phospho-site-specific-Ser³⁵⁷ IRS-1 antibody. While determining the specificity of p-Ser³⁵⁷ antiserum we came across the cross reactivity of the antiserum with adjacent Ser³⁵⁸ which was successfully abolished by an improved immuno-purification method. The specificity of the purified antiserum was then verified by indirect ELISA, results of ELISA were also mirrored in the experiments carried out in BHK-IR cells using different mutants of IRS-1 carrying mutations at either Ser³⁵⁷/Ser³⁵⁸/Ser^357/358. Immuno-purified-p-Ser³⁵⁷ did not react with IRS-1 Ala³⁵⁷ and IRS-1 Ala^357/358. In conclusion, the present study describes generation and characterization of p-Ser³⁵⁷ IRS-1 antibody, which reacts with IRS-1 in site specific and phosphorylation state-dependent manner without showing cross reactivity to adjacent Ser³⁵⁸. This antibody can be effectively used to further clarify the inhibitory role of Ser³⁵⁷ in insulin signal transduction.     

'Calcium-activated' intracellular calcium elevation: a novel mechanism of osteoclast regulation

The osteoclast is unique in its capacity to resorb bone. An unbalanced increase in this activity causes osteoporosis, a crippling bone disease that poses a major public health problem. Despite this, our understanding of osteoclast regulation is very limited. Calcitonin is the only known physiological inhibitor of osteoclast function. We demonstrate here for the first time that the concentration of calcium ions at the resorptive site directly regulates osteoclast function by modulating the intracellular free calcium concentration. This represents an important feedback mechanism of osteoclast control.     

Selective antagonism of calcitonin-induced osteoclastic quiescence (Q effect) by human calcitonin gene-related peptide-(Val8Phe37)

Exposure of isolated rat osteoclasts to calcitonin (CT) leads to an abrupt cessation of cell motility (Q effect) followed by cell retraction (R effect). We have previously shown that these effects are mediated by two G proteins that appear to activate separate post-receptor pathways. The present study demonstrates that the Q but not the R effect of CT (0.006 microM) is abolished in the presence of human calcitonin gene-related peptide (CGRP)-(Val8Phe37) (0.5 microM), a fragment analogue of human CGRP. This selective antagonism suggests that the Q effect could result from an action of CT upon a site that is distinct from that producing the R effect. The former site ('amylin site') also appears to interact with related peptides, amylin and CGRP, whilst the latter site ('CT site') specifically interacts with CT.     

Response of instream animal communities to a short-term extreme event and to longer-term cumulative impacts in a strategic water resource area,South Africa

Disturbance plays an integral part in generating heterogeneity required for ecosystem persistence, but the increased amplitude and duration of disturbances linked to drivers of global change could result in ecosystem shifts or collapse. Biomonitoring over time provides insights into trajectories of ecosystem change. The responses of two instream animal taxa to two contrasting disturbance events, a major flood event and the long-term cumulative effects of land-use changes, were assessed in 1999–2012 by quantifying variation and change in abundance of functional groups based on flow rate sensitivity, water quality and metrics of ecological condition. All metrics recovered to pre-flood conditions within seven months after the flood event. Similarly, cumulative impacts of land use effected significant decreases in some but not all metrics. Indices that did not change, including SASS total score and ASPT, were the result of insufficient consideration of the decrease in the abundance of sensitive taxa specifically, and the abundance of all taxa in general. The decrease in abundance of sensitive taxa could signal imminent collapse in certain metrics. Evidence is also provided for a shift in the structure of fish assemblages linked to the decrease and loss of taxa sensitive to ecosystem degradation caused by the longer-term impacts of land-use change.