Effects of ageing and senescence on pancreatic ß-cell function.

Ageing is generally associated with deterioration of organ function and regenerative potential. In the case of pancreatic ß-cells, an age-related decline in proliferative potential is well documented, and was proposed to contribute to the increased prevalence of type 2 diabetes in the elderly. The effects of ageing on ß-cell function, namely glucose-stimulated insulin secretion (GSIS), have not been studied as extensively. Recent work revealed that, surprisingly, ß-cells of mature mice and humans secrete more insulin than young ß-cells in response to high glucose concentrations, potentially serving to counteract age-related peripheral insulin resistance. This functional change appears to be orchestrated by p16(Ink4A) -driven cellular senescence and downstream remodelling of chromatin structure and DNA methylation, enhancing the expression of genes controlling ß-cell function. We propose that activation of the cellular senescence program drives life-long functional maturation of ß-cells, due to ß-cell hypertrophy, enhanced glucose uptake and more efficient mitochondrial metabolism, in parallel to locking these cells in a non-replicative state. We speculate that the beneficial aspects of this process can be harnessed to enhance GSIS. Other age-related mechanisms, which are currently poorly understood, act to increase basal insulin secretion levels also in low glucose conditions. This leads to an overall reduction in the amplitude of insulin secretion between low and high glucose at old age, which may contribute to a deterioration in metabolic control.

Effect of multiple aliphatic amino acids substitutions on the structure, function, and mode of action of diastereomeric membrane active peptides.

The initial stages leading to the binding and functioning of membrane-active polypeptides including hormones, signal sequences, and lytic peptides are mainly governed by electrostatic attraction and hydrophobic partitioning between water and lipid bilayers. Antimicrobial peptides serve as an important model for studying the details of these initial steps. However, a systematic analysis of the contribution of multiple hydrophobic amino acids to these steps have been hindered by the propensity of many peptides to aggregate and become inactivated in solution. To this end, we synthesized a series of model amphipathic all L-amino acid peptides and their diastereomers with the sequence KX(3)KWX(2)KX(2)K, where X = Gly, Ala, Val, Ile, or Leu. The effect of the aliphatic amino acids on the biological activity, binding, structure, membrane localization, and mode of action of these peptides was investigated. Most of the L-amino acid peptides oligomerized and adopted distinct structures in solution and in a membrane mimetic environment. Among this group only the Leu containing peptide was hemolytic and highly active on most bacteria tested. The Val- and Leu-containing peptides were hemolytic but inactive toward most bacteria tested. In contrast, the diastereomeric peptides were monomeric and unstructured in solution, but they adopted distinct structures upon membrane binding. While hemolytic activity was drastically reduced, the spectrum of antibacterial activity was preserved or increased. Importantly, we found a direct correlation with the diastereomers between hydrophobicity and propensity to form a helical/distorted-helix and activity (induced membrane leakage and antibacterial activity), despite the fact that they contained 30% D-amino acids. Furthermore, efficient increase in membrane permeability can proceed through different mechanisms. Specifically, the Leu-containing diastereomeric peptide micellized vesicles and possibly bacterial membranes while the Ile-containing diastereomeric peptide fused model membranes and irregularly disrupted bacterial membranes.

A single-stranded DNA binding protein binds the origin of replication of the duplex kinetoplast DNA.

Replication of the kinetoplast DNA (kDNA) minicircle of trypanosomatids initiates at a conserved 12-nt sequence, 5'-GGGGTTGGTGTA-3', termed the universal minicircle sequence (UMS). A sequence-specific single-stranded DNA-binding protein from Crithidia fasciculata binds the heavy strand of the 12-mer UMS. Whereas this UMS-binding protein (UMSBP) does not bind a duplex UMS dodecamer, it binds the double-stranded kDNA minicircle as well as a duplex minicircle fragment containing the origin-associated UMS. Binding of the minicircle origin region by the single-stranded DNA binding protein suggested the local unwinding of the DNA double helix at this site. Modification of thymine residues at this site by KMnO4 revealed that the UMS resides within an unwound or otherwise sharply distorted DNA at the minicircle origin region. Computer analysis predicts the sequence-directed curving of the minicircle origin region. Electrophoresis of a minicircle fragment containing the origin region in polyacrylamide gels revealed a significantly lower electrophoretic mobility than expected from its length. The fragment anomalous electrophoretic mobility is displayed only in its native conformation and is dependent on temperature and gel porosity, indicating the local curving of the DNA double helix. We suggest that binding of UMSBP at the minicircle origin of replication is possible through local unwinding of the DNA double helix at the UMS site. It is hypothesized here that this local melting is initiated through the untwisting of unstacked dinucleotide sequences at the bent origin site.

Universal minicircle sequence binding protein, a CCHC-type zinc finger protein that binds the universal minicircle sequence of trypanosomatids. Purification and characterization.

Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5'-GGGGTTGGTGTA-3'). A single-stranded nucleic acid binding protein that binds specifically to this origin-associated sequence was purified to apparent homogeneity from Crithidia fasciculata cell extracts. This UMS-binding protein (UMSBP) is a dimer of 27.4 kDa with a 13.7-kDa protomer. UMSBP binds single-stranded DNA as well as single-stranded RNA but not double-stranded or four-stranded DNA structures. Stoichiometry analysis indicates the binding of UMSBP as a protein dimer to the UMS site. The five CCHC-type zinc finger motifs of UMSBP, predicted from its cDNA sequence, are similar to the CCHC motifs found in retroviral Gag polyproteins. The remarkable conservation of this motif in a family of proteins found in eukaryotic organisms from yeast and protozoa to mammals is discussed.

Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform.

After the cellular prion protein (PrPC) transits to the cell surface where it is bound by a glycophosphatidyl inositol (GPI) anchor, PrPC is either metabolized or converted into the scrapie isoform (PrPSc). Because most GPI-anchored proteins are associated with cholesterol-rich membranous microdomains, we asked whether such structures participate in the metabolism of PrPC or the formation of PrPSc. The initial degradation of PrPC involves removal of the NH2 terminus of PrPC to produce a 17-kD polypeptide which was found in a Triton X-100 insoluble fraction. Both the formation of PrPSc and the initial degradation of PrPC were diminished by lovastatin-mediated depletion of cellular cholesterol but were insensitive to NH4Cl. Further degradation of the 17-kD polypeptide did occur within an NH4Cl-sensitive, acidic compartment. Replacing the GPI addition signal with the transmembrane and cytoplasmic domains of mouse CD4 rendered chimeric CD4PrPC soluble in cold Triton X-100. Both CD4PrPC and truncated PrPC without the GPI addition signal (Rogers, M., F. Yehieley, M. Scott, and S. B. Prusiner. 1993. Proc. Natl. Acad. Sci. USA. 90:3182-3186) were poor substrates for PrPSc formation. Thus, it seems likely that both the initial degradation of PrPC to the 17-kD polypeptide and the formation of PrPSc occur within a non-acidic compartment bound by cholesterol-rich membranes, possibly glycolipid-rich microdomains, where the metabolic fate of PrPC is determined. The pathway remains to be identified by which the 17-kD polypeptide and PrPSc are transported to an acidic compartment, presumably endosomes, where the 17-kD polypeptide is hydrolyzed and limited proteolysis of PrPSc produces PrP 27-30.

Biosynthesis of the prion proteins in scrapie-infected cells in culture.

Prions are small proteinaceous particles that transmit scrapie and other fatal encephalopathies of humans and animals, and that appear to be devoid of nucleic acids. The only known--and perhaps the sole--component of the scrapie prion is an abnormal host-encoded protein, the scrapie prion protein PrPSc. The biosynthesis of this pathological protein in the host cell, which is thus of paramount importance to prion replication, is still poorly understood. We are studying the biosynthesis and degradation of the scrapie prion protein PrPSc and of its normal isoform PrPC in scrapie-infected rodent cells in culture. PrPC is anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI) moiety. In scrapie-infected mouse neuroblastoma N2a cells, PrPSc is formed post-translationally, probably from plasma membrane PrPC, in an unknown subcellular compartment that is readily accessible from the plasma membrane. Transport along the secretory pathway is necessary for PrPSc synthesis. In contrast to PrPC, PrPSc accumulates intracellularly, primarily in secondary lysosomes. The subcellular compartment(s) in which PrPSc is formed remain to be determined.

Biosynthesis of the prion proteins in scrapie-infected cells in culture

Prions are small proteinaceous particles that transmit scrapie and other fatal encephalopathies of humans and animals, and that appear to be devoided of nucleic acids. The only known-and perhaps the sole-component of the scrapie prion is an abnormal host-encoded protein, the scrapie prion protein PrPSc. The biosynthesis of this pathological protein in the host cell, which is thus of paramount importance to prion replication, is still poorly understood. We are studying the biosynthesis and degradation of the scrapie prion protein PrPSc and of its normal isoform PrPC in scrapie-infected rodent cells in culture. PrPC is anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI) moiety. In scrapie-infected mouse neuroblastoma N2a cells, PrPSc is formed post-translationally, probably from plasma membrane PrPC, in an unknown subcellular compartment that is readily accessible from the plasma membrane. Transport along the secretory pathway is necessary for PrPSc synthesis. In contrast to PrPC, PrPSc accumulates intracellularly, primarily in secondary lysosomes. The subcellular compartment(s) in which PrPSc is formed remain to be determined

Mutation of the prion protein in Libyan Jews with Creutzfeldt-Jakob disease.

BACKGROUND: Creutzfeldt-Jakob disease is a transmissible neurodegenerative disorder that occurs more than 100 times more frequently among Libyan Jews than in the worldwide population. We examined 11 patients with the disease--10 Libyan Jews from Israel and 1 Libyan Jew from Italy--to determine whether abnormalities of the prion protein could be detected in them. Abnormal forms of this host-encoded protein are the predominant if not sole components of the transmissible agent that causes the disease. METHODS: The prion-protein open-reading frame in peripheral-leukocyte DNA from the Italian patient was amplified with the polymerase chain reaction and sequenced. Allele-specific oligonucleotide hybridization was used to assess a prion-protein codon 200 lysine mutation in the 10 Israeli patients and 37 control subjects. RESULTS: The prion-protein sequence in DNA from the Italian patient revealed a single nucleotide change (G----A) at the first position of codon 200 that resulted in a substitution of lysine for glutamate. This substitution was detected in all 10 Israeli patients, 8 of whom had a positive family history of Creutzfeldt-Jakob disease. One patient was homozygous for the lysine mutation, and her clinical course did not differ from that of the patients heterozygous for the mutation. The lysine mutation was not found in one Moroccan Jew from Israel with Creutzfeldt-Jakob disease. CONCLUSIONS: The codon 200 lysine mutation of the prion-protein gene is consistently present among Libyan Jews with Creutzfeldt-Jakob disease, strongly supporting a genetic pathogenesis of their illness. The similarity of the clinical courses of the patient homozygous for this mutation and the patients heterozygous for it argues that familial Creutzfeldt-Jakob disease is a true dominant disorder.