The Simpson-Golabi-Behmel syndrome causative glypican-3, binds to and inhibits the dipeptidyl peptidase activity of CD26.

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition shown to be the result of deletions of the glypican-3 (GPC3) gene. GPC3 is a proteoglycan localized to the cell membrane via a glycosylphosphatidyl-inositol (GPI) anchor. To further elucidate the GPC3 function(s), we have screened various cell lines for proteins that interact with GPC3, resulting in the isolation of a 115 kDa protein, identified as CD26. The interaction occurred with both the glycosylated and unglycosylated forms of GPC3 and led to the inhibition of CD26 peptidase activity. Moreover, introduction of CD26 into Cos-1 cells was accompanied by the up-regulation of cell growth, while inclusion of recombinant GPC3 in the media reduced the growth of CD26 transfected Cos-1 cells, drastically. Furthermore, HepG2 C3A cells containing CD26 underwent apoptosis in the presence of recombinant GPC3 in both concentration and time-dependant manner. In light of the fact that inhibition of CD26 reduces the rate of cell proliferation, we propose that a number of physical findings observed in SGBS patients may be a consequence of a direct interaction of GPC3 with CD26. Furthermore, GPC3 without the GPI anchor is capable of inducing apoptosis indicating that neither the GPI anchor nor the membrane attachment is required for apoptosis induction.

Distribution of neuronal apoptosis inhibitory protein in human tissues.

The neuronal apoptosis inhibitory protein (NAIP) gene, also known as the baculovirus inhibitor of apoptosis repeat-containing protein 1 (BIRC1) gene, is a member of the inhibitors of apoptosis (IAP) family and was first characterized as a candidate gene for spinal muscular atrophy (SMA). The expression of NAIP has been thoroughly studied in the central nervous system and overlaps the pattern of neurodegeneration in SMA. Recent studies have pointed to a role for NAIP in non-neuronal cells. We report here the production of a specific anti-NAIP antibody and the profile of NAIP expression in human adult tissues by Western blot and immunohistochemical detection methods. NAIP was detected in a number of tissues by Western blot analysis, but immunohistochemistry revealed that NAIP's presence in certain tissues, such as liver, lung, and spleen, is most likely due to macrophage infiltration. In the small intestine, the expression of NAIP coincides with the expression of p21(WAF1). This observation, coupled with findings from other groups, suggests a role for NAIP in increasing the survival of cells undergoing terminal differentiation as well as the possibility that the protein serves as an intestinal pathogen recognition protein. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Histologic and transcriptional assessment of a mild SMA model.

Spinal muscular atrophy (SMA) is caused by survival of motor neuron (SMN) deficiency, leading to specific motor neuron attrition. The time course and molecular pathophysiologic etiology of motor neuron loss observed in SMA remains obscure. Mice heterozygous for Smn show up to 50% motor neuron attrition by 6 months of age and are used as a model for mild SMA in humans. To determine both the rate of cellular loss and the molecular events underlying motor neuron degeneration in SMA, motor neuron counts and mRNA quantification were performed in spinal cords of Smn(+/-) mice and wild-type littermates. Surprisingly, despite the chronic, subclinical nature of motor neuron loss, we find that the bulk of the loss occurs by 5 weeks of age. RNA isolated from the spinal cords of 5 week-old Smn(+/-) mice subjected to microarray analysis reveal alterations in genes involved in RNA metabolism, apoptosis and transcriptional regulation including a general perturbation of transcripts coding for calcium binding proteins. A subset of these changes in expression was further characterized by semi-quantitative RT-PCR and Western blot analysis at various time points. Taken together, these results indicate that spinal cord cells present the first signs of the apoptotic process consistent with a response to the stress of Smn depletion. A picture of comparatively rapid neuronal attrition in spite of the very mild nature of SMA is obtained. Furthermore, changes occur, which may be reactive to and not causative of the cellular loss, involving central cellular functions as well as calcium modulating proteins.

Hypoxia alters the expression of inhibitor of apoptosis proteins after brain trauma in the mouse.

Hypoxia worsens brain injury following trauma, but the mechanisms remain unclear. The purpose of this study was to determine the effect of traumatic brain injury (TBI) and secondary hypoxia (9% oxygen) on apoptosis-related protein expression, cell death, and behavior. Using a murine weight-drop model, TBI led to an early (6 h) increase followed by a later (24 h) decrease in neuronal apoptosis inhibitor protein (NAIP) expression in the olfactory and motor cortex; in contrast, TBI led to a sustained (6 h to 7 days) increase in NAIP in the striatum. The peak increase in the expression of NAIP (6-12 h) following TBI alone was delayed (1-7 days) when hypoxia was added to TBI. Hypoxia following TBI further depleted other apoptosis inhibitor proteins (IAPs) and activated caspases, as well as increased contusion size and worsened cell death. Hypoxia added to TBI also increased motor and feeding activity on days 2 and 4 compared to TBI alone. Hypoxia without TBI had no effect on the expression of IAPs or cell death. These findings show that IAPs have a potential role in the increased vulnerability of brain cells to hypoxia following TBI, and have implications for configuring future therapies for TBI.

Rapid detection of single nucleotide polymorphisms associated with spinal muscular atrophy by use of a reusable fibre-optic biosensor.

Rapid (<2 min) and quantitative genotyping for single nucleotide polymorphisms (SNPs) associated with spinal muscular atrophy was done using a reusable (approximately 80 cycles of application) fibre-optic biosensor over a clinically relevant range (0-4 gene copies). Sensors were functionalized with oligonucleotide probes immobilized at high density (approximately 7 pmol/cm2) to impart enhanced selectivity for SNP discrimination and used in a total internal reflection fluorescence detection motif to detect 202 bp PCR amplicons from patient samples. Real-time detection may be done over a range of ionic strength conditions (0.1-1.0 M) without stringency rinsing to remove non-selectively bound materials and without loss of selectivity, permitting a means for facile sample preparation. By using the time-derivative of fluorescence intensity as the analytical parameter, linearity of response may be maintained while allowing for significant reductions in analysis time (10-100-fold), permitting for the completion of measurements in under 1 min.

The neuronal apoptosis inhibitory protein is a direct inhibitor of caspases 3 and 7.

The neuronal apoptosis inhibitory protein (NAIP) was identified as a candidate gene for the inherited neurodegenerative disorder spinal muscular atrophy. NAIP is the founding member of a human protein family that is characterized by highly conserved N-terminal motifs called baculovirus inhibitor of apoptosis repeats (BIR). Five members of the human family of inhibitor of apoptosis proteins including NAIP have been shown to be antiapoptotic in various systems. To date, a mechanism for the antiapoptotic effect of NAIP has not been elucidated. To investigate NAIP function, we found cytoprotection of NAIP-expressing primary cortical neurons treated to undergo caspase-3-dependent apoptosis. The additional treatment of these neurons with the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone did not result in increased survival. Similar cytoprotective effects were obtained using HeLa cells transiently transfected with a NAIP N-terminal construct and treated to undergo a caspase-3-dependent cell death. To examine whether NAIP inhibits caspases directly, recombinant N-terminal NAIP protein containing BIR domains was overexpressed, purified, and tested for caspase inhibition potential. Our results demonstrate that inhibition of caspases is selective and restricted to the effector group of caspases, with K(i) values as low as approximately 14 nm for caspase-3 and approximately 45 nm for caspase-7. Additional investigations with NAIP fragments containing either one or two NAIP BIRs revealed that the second BIR and to a lesser extent the third BIR alone are sufficient to mediate full caspase inhibition.