Calcium-activated Calpain-2 Is a Mediator of Beta Cell Dysfunction and Apoptosis in Type 2 Diabetes

The islet in type 2 diabetes (T2DM) and the brain in neurodegenerative diseases share progressive cell dysfunction, increased apoptosis, and accumulation of locally expressed amyloidogenic proteins (islet amyloid polypeptide (IAPP) in T2DM). Excessive activation of the Ca²⁺-sensitive protease calpain-2 has been implicated as a mediator of oligomer-induced cell death and dysfunction in neurodegenerative diseases. To establish if human IAPP toxicity is mediated by a comparable mechanism, we overexpressed human IAPP in rat insulinoma cells and freshly isolated human islets. Pancreas was also obtained at autopsy from humans with T2DM and nondiabetic controls. We report that overexpression of human IAPP leads to the formation of toxic oligomers and increases beta cell apoptosis mediated by increased cytosolic Ca²⁺ and hyperactivation of calpain-2. Cleavage of α-spectrin, a marker of calpain hyperactivation, is increased in beta cells in T2DM. We conclude that overactivation of Ca²⁺-calpain pathways contributes to beta cell dysfunction and apoptosis in T2DM.     

DNA Damage-Induced Phosphorylation of TRF2 Is Required for the Fast Pathway of DNA Double-Strand Break Repair

Protein kinases of the phosphatidylinositol 3-kinase-like kinase family, originally known to act in maintaining genomic integrity via DNA repair pathways, have been shown to also function in telomere maintenance. Here we focus on the functional role of DNA damage-induced phosphorylation of the essential mammalian telomeric DNA binding protein TRF2, which coordinates the assembly of the proteinaceous cap to disguise the chromosome end from being recognized as a double-stand break (DSB). Previous results suggested a link between the transient induction of human TRF2 phosphorylation at threonine 188 (T188) by the ataxia telangiectasia mutated protein kinase (ATM) and the DNA damage response. Here, we report evidence that X-ray-induced phosphorylation of TRF2 at T188 plays a role in the fast pathway of DNA DSB repair. These results connect the highly transient induction of human TRF2 phosphorylation to the DNA damage response machinery. Thus, we find that a protein known to function in telomere maintenance, TRF2, also plays a functional role in DNA DSB repair.Telomeres act as protective caps to disguise the chromosome end from being recognized as a DNA double-strand break (DSB) and play other important roles in maintaining genomic integrity (2, 21, 26). Telomere capping dysfunction resulting in genomic instability is likely a major pathway leading to human cancers and other age-related diseases (8, 27).An increasing number of proteins known to play important roles in DNA repair have also been found to be critical for telomere maintenance (6). Specifically, phosphatidylinositol (PI) 3-kinase-like kinase family members, such as ataxia telangiectasia mutated protein kinase (ATM) and the DNA-dependent protein kinase catalytic subunit in mammals, originally known to act in maintaining genomic stability via DNA repair pathways, have been shown to be important in telomere maintenance (1, 4, 7, 9, 10, 16, 25). Previous reports indicate that ATM is required for the DNA damage-induced phosphorylation of two major telomere-associated proteins in mammals, human TRF1 and TRF2 (16, 28). The specific molecular roles played by the DNA damage-induced phosphorylation of TRF1 and TRF2 in telomere maintenance and/or DNA repair are unclear and under active investigation. We previously reported that upon DNA damage, human TRF2 was rapidly and transiently phosphorylated at threonine 188 (T188) (28). Here, we report that X-ray-induced phosphorylation of human TRF2 at T188 plays a functional role in the fast pathway of DNA DSB repair.     

Investigation of DNA complexes with histones F3 and F3+F2a2]

Characteristic viscosity, sedimentation constant and optical anisotropy were studied of the complexes formed between DNA and histone fractions F3 and F3+F2a2. The parameters mentioned continuously change with the increase of protein content within the complex. Analysis of experimental data shows that binding of a histone bads to a decrease of size and thermodynamic rigidity of the DNA molecule. On the basis of results obtained a model of F3 histone binding with DNA is suggested, amino acid sequence of this protein being taken into account. Comparison of behaviour of nucleohistones DNA+F3 and DNA+F1 studied previously testifies different way of binding of these histones to DNA.     

Specificity of E2F1, E2F2, and E2F3 in mediating phenotypes induced by loss of Rb.

The Rb/E2F pathway plays a critical role in the control ofcellular proliferation. Here, we report that E2F1, E2F2, and E2F3 make major individual contributions toward the in vivo phenotypic consequences of Rb deficiency. In the developing lens of Rb(-/-) embryos, loss of E2F1, E2F2, or E2F3 reduces the unscheduled proliferation of fiber cells, with the loss of E2F3 having the most pronounced effect. In Rb-deficient retinas, all three E2Fs contribute equally to the ectopic proliferation of postmitotic neuronal cells. In contrast, E2F1 is unique in mediating apoptosis in both Rb(-/-) lenses and retinas. In the central nervous system, loss of E2F1 or E2F3 can almost completely eliminate the ectopic DNA replication and apoptosis observed in Rb(-/-) embryos, and loss of E2F2 partially reduces the unscheduled DNA replication and has no effect on apoptosis. These results provide clear evidence for functional specificity among E2Fs in the control of Rb-dependent proliferation and apoptosis in a tissue-specific manner.     

Ribosomal Protein S3, a New Substrate of Akt,Serves as a Signal Mediator between Neuronal Apoptosis and DNA Repair

RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation.     

14-3-3 proteins integrate E2F activity with the DNA damage response

The E2F family is composed of at least eight E2F and two DP subunits, which in cells exist as E2F/DP heterodimers that bind to and regulate E2F target genes. While DP-1 is an essential and widespread component of E2F, much less is known about the DP-3 subunit, which exists as a number of distinct protein isoforms that differ in several respects including the presence of a nuclear localisation signal (NLS). We show here that the NLS region of DP-3 harbours a binding site for 14-3-3epsilon, and that binding of 14-3-3epsilon alters the cell cycle and apoptotic properties of E2F. DP-3 responds to DNA damage, and the interaction between DP-3 and 14-3-3epsilon is under DNA damage-responsive control. Further, 14-3-3epsilon is present in the promoter region of certain E2F target genes, and reducing 14-3-3epsilon levels induces apoptosis. These results identify a new level of control on E2F activity and, at a more general level, suggest that 14-3-3 proteins integrate E2F activity with the DNA damage response.     

Smad about E2F. TGFbeta repressionof c-Myc via a Smad3/E2F/p107 complex

Signaling by TGFbeta regulates the expression of hundreds of genes. The rapid repression of c-Myc stands out because of its roles in growth control and cancer. Recent work shows that c-Myc repression by TGFbeta is mediated by the nuclear translocation of a novel, preformed complex composed of Smad3, E2F4 or E2F5, and the Rb-related factor p107.