Calpain activation through galectin-3 inhibition sensitizes prostate cancer cells to cisplatin treatment

Prostate cancer will develop chemoresistance following a period of chemotherapy. This is due, in part, to the acquisition of antiapoptotic properties by the cancer cells and, therefore, development of novel strategies for treatment is of critical need. Here, we attempt to clarify the role of the antiapoptotic molecule galectin-3 in prostate cancer cells using siRNA and antagonist approaches. The data showed that Gal-3 inhibition by siRNA or its antagonist GCS-100/modified citrus pectin (MCP) increased cisplatin-induced apoptosis of PC3 cells. Recent studies have indicated that cisplatin-induced apoptosis may be mediated by calpain, a calcium-dependent protease, as its activation leads to cleavage of androgen receptor into an androgen-independent isoform in prostate cancer cells. Thus, we examined whether calpain activation is associated with the Gal-3 function of regulating apoptosis. Here, we report that Gal-3 inhibition by siRNA or GCS-100/MCP enhances calpain activation, whereas Gal-3 overexpression inhibits it. Inhibition of calpain using its inhibitor and/or siRNA attenuated the proapoptotic effect of Gal-3 inhibition, suggesting that calpain activation may be a novel mechanism for the proapoptotic effect of Gal-3 inhibition. Thus, a paradigm shift for treating prostate cancer is suggested whereby a combination of a non-toxic anti-Gal-3 drug together with a toxic chemotherapeutic agent could serve as a novel therapeutic modality for chemoresistant prostate cancers

Molecular Determinants of Survival Motor Neuron (SMN) Protein Cleavage by the Calcium-Activated Protease, Calpain

Spinal muscular atrophy (SMA) is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN) protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs). It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V), reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S), abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294) resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN

Ischemia-induced calpain activation causes eukaryotic (translation) initiation factor 4G1 (eIF4GI) degradation, protein synthesis inhibition, and neuronal death

Persistent protein synthesis inhibition (PSI) is a robust predictor of eventual neuronal death following cerebral ischemia. We thus tested the hypothesis that persistent PSI inhibition and neuronal death are causally linked. Neuronal viability strongly correlated with both protein synthesis and levels of eukaryotic (translation) initiation factor 4G1 (eIF4G1). We determined that in vitro ischemia activated calpain, which degraded eIF4G1. Overexpression of the calpain inhibitor calpastatin or eIF4G1 resulted in increased protein synthesis and increased neuronal viability compared with controls. The neuroprotective effect of eIF4G1 overexpression was due to restoration of cap-dependent protein synthesis, as well as protein synthesis-independent mechanisms, as inhibition of protein synthesis with cycloheximide did not completely prevent the protective effect of eIF4G1 overexpression. In contrast, shRNA-mediated silencing of eIF4G1 exacerbated ischemia-induced neuronal injury, suggesting eIF4G1 is necessary for maintenance of neuronal viability. Finally, calpain inhibition following global ischemia in vivo blocked decreases in eIF4G1, facilitated protein synthesis, and increased neuronal viability in ischemia-vulnerable hippocampal CA1 neurons. Collectively, these data demonstrate that calpain-mediated degradation of a translation initiation factor, eIF4G1, is a cause of both persistent PSI and neuronal death

Immunophenotyping of Rhesus CMV

A vaccine to ameliorate cytomegalovirus (CMV)‐related pathogenicity in transplantation patients is considered a top priority. A therapeutic vaccine must include components that elicit both neutralizing antibodies, and highly effective CD8 T‐cell responses. The most important translational model of vaccine development is the captive‐bred rhesus macaque ( Macaca mulatta ) of Indian origin. There is a dearth of information on rhesus cytomegalovirus (rhCMV)‐specific CD8 T cells due to the absence of well‐defined CD8 T‐cell epitopes presented by classical MHC‐I molecules. In the current study, we defined two CD8 T‐cell epitopes restricted by high‐frequency Mamu alleles: the Mamu‐A1*002:01 restricted VY9 (VTTLGMALY aa291‐299) epitope of protein IE‐1, and the Mamu‐A1*008:01 restricted NP8 (NPTDRPIP aa96‐103) epitope of protein phosphoprotein 65‐2. We developed tetramers and determined the level, phenotype, and functional capability of the two epitope‐specific T‐cell populations in circulation and various tissues. We demonstrated the value of these tetramers for in situ tetramer staining. Here, we first provided critical reagents and established a flow cytometric staining strategy to study rhCMV‐specific T‐cell responses in up to 40% of captive‐bred rhesus macaques. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry

Apurinic/apyrimidinic endonuclease APE1 is required for PACAP-induced neuroprotection against global cerebral ischemia

Inducible DNA repair via the base-excision repair pathway is an important prosurvival mechanism activated in response to oxidative DNA damage. Elevated levels of the essential base-excision repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1)/redox effector factor-1 correlate closely with neuronal survival against ischemic insults, depending on the CNS region, protective treatments, and degree of insult. However, the precise mechanisms by which this multifunctional protein affords protection and is activated by upstream signaling pathways in postischemic neurons are not well delineated. Here we show that intracerebral administration of pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenously occurring small neuropeptide, induces expression of APE1 in hippocampal neurons. Induction of APE1 expression requires PKA- and p38-dependent phosphorylation of cAMP response-element binding and activating transcription factor 2, which leads to transactivation of the APE1 promoter. We further show that PACAP markedly reduces oxidative DNA stress and hippocampal CA1 neuronal death following transient global ischemia. These effects occurred, at least in part, via enhanced APE1 expression. Furthermore, the DNA repair function of APE1 was required for PACAP-mediated neuroprotection. Thus, induction of DNA repair enzymes may be a unique strategy for neuroprotection against hippocampal injury