Reg-1α, a New Substrate of Calpain-2 Depending on Its Glycosylation Status.

Reg-1α/lithostathine, a protein mainly associated with the digestive system, was previously shown to be overexpressed in the pre-clinical stages of Alzheimer's disease. In vitro, the glycosylated protein was reported to form fibrils at physiological pH following the proteolytic action of trypsin. However, the nature of the protease able to act in the central nervous system is unknown. In the present study, we showed that Reg-1α can be cleaved in vitro by calpain-2, the calcium activated neutral protease, overexpressed in neurodegenerative diseases. Using chemical crosslinking experiments, we found that the two proteins can interact with each other. Identification of the cleavage site using mass spectrometry, between Gln4 and Thr5, was found in agreement with the in silico prediction of the calpain cleavage site, in a position different from the one reported for trypsin, i.e., Arg11-Ile12 peptide bond. We showed that the cleavage was impeded by the presence of the neighboring glycosylation of Thr5. Moreover, in vitro studies using electron microscopy showed that calpain-cleaved protein does not form fibrils as observed after trypsin cleavage. Collectively, our results show that calpain-2 cleaves Reg-1α in vitro, and that this action is not associated with fibril formation.

Reg-1α Promotes Differentiation of Cortical Progenitors via Its N-Terminal Active Domain.

Reg-1α belongs to the Reg family of small, secreted proteins expressed in both pancreas and nervous system. Reg-1α is composed of two domains, an insoluble C-type lectin domain and a short soluble N-terminal peptide, which is released from the molecule upon proteolytic N-terminal processing, although the biological significance of this proteolysis remains unclear. We have previously shown that binding of Reg-1α to its receptor Extl3 stimulates axonal outgrowth. Reg-1α and Extl3 genes are expressed in the developing cortex but their expression decreases in adulthood, pointing to a possible function of this signaling system at the early developmental stages. Here, we demonstrate that recombinant Reg-1α increases migration and differentiation of cultured embryonic rat telencephalic progenitors via the activation of GSK-3ß activity. In vivo overexpression of Reg-1α by in utero electroporation, has a similar effect, favoring premature differentiation of cortical progenitors. Notably, the N-terminal soluble domain, but not the C-type lectin domain, is largely responsible for Reg-1α effects on cortical neuronal differentiation. We thus conclude that Reg-1α via its proteolytically generated N-terminal domain is required for basic development processes.

Regenerating islet-derived 1α (REG-1α) protein increases tau phosphorylation in cell and animal models of tauopathies.

REG-1α, a secreted protein containing a C-type lectin domain, is expressed in various organs and plays different roles in digestive system cells in physiological and pathological conditions. Like other members of the Reg family, REG-1α is expressed also in the brain where it has different functions. For instance, we previously reported that REG-1α regulates neurite outgrowth and is overexpressed during the very early stages of Alzheimer's disease (AD). However, REG-1α function in neural cells during neural degeneration remains unknown. First, REG-1α and phosphorylated tau expression were assessed in tissue sections from the hippocampus, representing neurofibrillary tangles (NFTs), from patients with AD, and from basal ganglia, representing subcortical NFTs, from patients with progressive supranuclear palsy (PSP). We found an association between REG-1α expression, tau hyperphosphorylation and NFTs in human brain samples from patients with these neurodegenerative diseases. Then, the effects of REG-1α overexpression on tau phosphorylation and axonal morphology were investigated i) in primary cultures of rat neurons that express human tau P301L and ii) in a transgenic zebrafish model of tauopathy that expresses human tau P301L. In the tau P301L cell model, REG-1α overexpression increased tau phosphorylation at the S202/T205 and S396 residues (early and late stages of abnormal phosphorylation, respectively) through the AKT/GSK3-ß pathway. This effect was associated with axonal defects both in tau P301L-expressing rat neurons and zebrafish embryos. Our findings suggest a functional role for REG-1α during tauopathy development and progression and, specifically, its involvement in the modification of tau phosphorylation temporal sequence.

High-resolution cryo-EM structure of the F-actin-fimbrin/plastin ABD2 complex.

Many actin binding proteins have a modular architecture, and calponin-homology (CH) domains are one such structurally conserved module found in numerous proteins that interact with F-actin. The manner in which CH-domains bind F-actin has been controversial. Using cryo-EM and a single-particle approach to helical reconstruction, we have generated 12-A-resolution maps of F-actin alone and F-actin decorated with a fragment of human fimbrin (L-plastin) containing tandem CH-domains. The high resolution allows an unambiguous fit of the crystal structure of fimbrin into the map. The interaction between fimbrin ABD2 (actin binding domain 2) and F-actin is different from any interaction previously observed or proposed for tandem CH-domain proteins, showing that the structural conservation of the CH-domains does not lead to a conserved mode of interaction with F-actin. Both the stapling of adjacent actin protomers and the additional closure of the nucleotide binding cleft in F-actin when the fimbrin fragment binds may explain how fimbrin can stabilize actin filaments. A mechanism is proposed where ABD1 of fimbrin becomes activated for binding a second actin filament after ABD2 is bound to a first filament, and this can explain how mutations of residues buried in the interface between ABD2 and ABD1 can rescue temperature-sensitive defects in actin.

Calpain involvement in the remodeling of cytoskeletal anchorage complexes.

Cells offer different types of cytoskeletal anchorages: transitory structures such as focal contacts and perennial ones such as the sarcomeric cytoskeleton of muscle cells. The turnover of these structures is controlled with different timing by a family of cysteine proteases activated by calcium, the calpains. The large number of potential substrates present in each of these structures imposes fine tuning of the activity of the proteases to avoid excessive action. This phenomenon is thus guaranteed by various types of regulation, ranging from a relatively high calcium concentration necessary for activation, phosphorylation of substrates or the proteases themselves with either a favorable or inhibitory effect, possible intervention of phospholipids, and the presence of a specific inhibitor and its possible degradation before activation. Finally, formation of multiprotein complexes containing calpains offers a new method of regulation.

Evidence in oyster of a plasma extracellular superoxide dismutase which binds LPS.

We have characterized in the oyster Crassostrea gigas an extracellular superoxide dismutase (Cg-EcSOD) which appears to bind lipopolysaccharides (LPS). The protein has been purified from the oyster plasma and identified as a Cu/ZnSOD according to its N-terminal sequencing and biological activity. Cg-EcSOD expression and synthesis are restricted to hemocytes as revealed by in situ hybridization and immunocytochemistry. Cg-EcSOD-expressing hemocytes were seen in blood circulation, in connective tissues, and closely associated to endothelium blood vessels. Cg-EcSOD presents in its amino acid sequence a LPS-binding motif found in the endotoxin receptor CD14 and we show that the protein displays an affinity to Escherichia coli bacteria and with LPS and Lipid A. Additionally, an RGD motif known to be implicated in the association to membrane integrin receptor is present in the amino acid sequence. The purified Cg-EcSOD was shown to bind to oyster hemocytes and to be immunocolocalized with a beta-integrin-like receptor.

The calpain 1-alpha-actinin interaction. Resting complex between the calcium-dependent protease and its target in cytoskeleton.

Calpain 1 behaviour toward cytoskeletal targets was investigated using two alpha-actinin isoforms from smooth and skeletal muscles. These two isoforms which are, respectively, sensitive and resistant to calpain cleavage, interact with the protease when using in vitro binding assays. The stability of the complexes in EGTA [Kd(-Ca2+) = 0.5 +/- 0.1 microM] was improved in the presence of 1 mm calcium ions [Kd(+Ca2+) = 0.05 +/- 0.01 microM]. Location of the binding structures shows that the C-terminal domain of alpha-actinin and each calpain subunit, 28 and 80 kDa, participates in the interaction. In particular, the autolysed calpain form (76/18) affords a similar binding compared to the 80/28 intact enzyme, with an identified binding site in the catalytic subunit, located in the C-terminal region of the chain (domain III-IV). The in vivo colocalization of calpain 1 and alpha-actinin was shown to be likely in the presence of calcium, when permeabilized muscle fibres were supplemented by exogenous calpain 1 and the presence of calpain 1 in Z-line cores was shown by gold-labelled antibodies. The demonstration of such a colocalization was brought by coimmunoprecipitation experiments of calpain 1 and alpha-actinin from C2.7 myogenic cells. We propose that calpain 1 interacts in a resting state with cytoskeletal targets, and that this binding is strengthened in pathological conditions, such as ischaemia and dystrophies, associated with high calcium concentrations.

Transient expression of the t-isoform of plastins/fimbrin in the stereocilia of developing auditory hair cells.

The transduction of auditory signals by cochlear hair cells depends upon the integrity of hair cell stereociliary bundles. Stereocilia contain a central core of actin filaments, cross-linked by actin bundling proteins. In the cochlea, the two proteins described to date as responsible for the spatial arrangement of actin filaments in sterocilia are fimbrin and the recently discovered espin. Fimbrin (the chick homolog of human I-plastin) belongs to the plastins/fimbrin family that includes two additional isoforms of plastins, T- and L-plastin. In the present study, we used isoform specific antibodies to investigate the presence of the T- and L-isoforms of plastin/fimbrin in the adult and developing rat cochlea. We found that T-plastin, but not L-plastin, is expressed in the rat cochlea. During postnatal development of the rat organ of Corti, T-plastin can be detected in the core of stereocilia from early stages of hair cell differentiation, and its expression gradually increases in stereocilia as hair cells mature. However, as opposed to other actin-binding proteins expressed in stereocilia, T-plastin is absent from the stereocilia of mature hair cells. Such temporally restricted expression strengthens the idea of functional differences between plastins isoforms, and suggests that T-plastin could have a specific role in stereocilia formation.