Conformational Studies of PACAP(1–27) and Its Fragments

Summary Conformational features of the neuropeptide pituitary adenylate cyclase activating polypeptide (1–27) (PACAP(1–27)) and its shorter fragments (1–5), (7–11) and (14–27) were studied by circular dichroism (CD) and fluorescence spectroscopy. The obtained CD spectra revealed that only PACAP(1–27) and the fragment (14–27) possess some content of an organized structure — the α-helix. This C-terminal, helical part of the peptides is important for receptor binding as it provides a stable structure that can reside in the ordered lipid region of the receptor site in the membrane, while the primary biological function of the hormone resides in the N-terminal, disordered part. Fluorescence studies have revealed that the tyrosine residue located in the helical region of PACAP has a higher quantum yield and a longer average lifetime than the tyrosine in the N-terminus, probably due to a ‘shielding’ effect of the hydrophobic cluster around Tyr²².     

Cytoskeletal elements are required for the formation and maturation of autophagic vacuoles.

We evaluated the role of cytoskeletal elements in the degradation of endogenous proteins via autophagy using biochemical and morphological techniques. In the absence of exogenous amino acids, degradation of endogenous proteins was enhanced in cultured normal rat kidney cells. This enhanced degradative state was accompanied by a 4-fold increase in the occurrence of autophagic vacuoles. In the presence of drugs that induce the depolymerization of microfilaments (cytochalasins B and D) or microtubules (nocodazole), protein degradation was not enhanced in nutrient-deprived cells. Although these drugs had similar inhibitory effects on the protein degradation, their effect on autophagy differed. Cytochalasins B and D interfered with the formation of the autophagosome. In cells treated with these drugs, the fractional volume represented by autophagic vacuoles was not substantially increased despite nutrient depletion. On the contrary, nocodazole appeared to have no effect on the formation of autophagosomes. Instead, this drug suppressed the delivery of hydrolytic enzymes, thereby resulting in an accumulation of acidic autophagic vacuoles containing undegraded cellular components.     

Copper(II) complexation by pituitary adenylate cyclase activating polypeptide fragments.

Results are reported from potentiometric and spectroscopic (UV-Vis, CD, and ESR) studies of the protonation constants and Cu2+ complex stability constants of pituitary adenylate cyclase activating polypeptide fragments (HSDGI-NH2, TDSYS-NH2, RKQMAVKKYLAAVL-NH2). With HSDGI-NH2, the formation of a dimeric complex Cu2H-2L2 was found in the pH range 5-8, in which the coordination of copper(II) is glycylglycine-like, while the fourth coordination site is occupied by the imidazole N3 nitrogen atom, forming a bridge between two copper(II) ions. The formation of dimeric species does not prevent the deprotonation and coordination of the amide nitrogen, and in pH above 8 the CuH-2L complex is formed. Aspartic acid in the third position of peptide sequence stabilizes the CuH-2L species and prevents the coordination of a fourth nitrogen donor. Aspartic acid residue in the second position of TDSYS-NH2 stabilizes the CuL (2N) complex but does not prevent deprotonation and binding of the second and third peptide nitrogens to give 3N and 4N complexes at higher pH. The tetradecapeptide amide forms with copper(II) ions unusually stable 3N and 4N complexes compared to pentaalanine amide.     

Ubiquitinated aldolase B accumulates during starvation-induced lysosomal proteolysis

We have previously shown that stress-induced protein degradation requires a functional ubiquitin-activating enzyme and the autophagic-lysosomal pathway. In this study, we examined the occurrence of ubiquitin-protein conjugates that form during nutrient starvation. Kidney and liver epithelial cells respond to nutrient stress by enhancing autophagy and protein degradation. We have shown that this degradative response was more dramatic in nondividing cultures. In addition, the onset of autophagy was suppressed by pactamycin, cycloheximide, and puromycin. We observed an accumulation of ubiquitinated proteins coincident with the degradative response to amino acid starvation. The stress-induced protein ubiquitination was not affected by cycloheximide, indicating that protein synthesis was not required. The ubiquitinated proteins were localized to the cytosol and subcellular fractions enriched with autophagosomes and lysosomes. The incorporation of the ubiquitinated proteins into autolysosomes was dramatically reduced by 3-methyladenine, an inhibitor of autophagy. The evidence suggests that ubiquitinated proteins are sequestered by autophagy for degradation. We next set out to identify those primary ubiquitinated proteins at 60 kDa and 68 kDa. Polyclonal antibodies were prepared against these proteins that had been immunopurified from rat liver lysosomes. The antibodies prepared against those 68 kDa proteins also recognized a 40 kDa protein in cytosolic fractions. Internal amino acid sequences obtained from two cyanogen bromide fragments of this 40 kDa protein were shown to be identical to sequences in liver fructose-1,6-bisphosphate aldolase B. Anti-Ub68 antibodies recognized purified aldolase A and aldolase B. Conversely, antibodies prepared against aldolase B recognized the 40 kDa aldolase as well as four to five high molecular weight forms, including a 68 kDa protein. Finally, we have shown that the degradation of aldolase B was enhanced during amino acid and serum starvation. This degradation was suppressed by chloroquine and 3-methyladenine, suggesting that aldolase B was being degraded within autolysosomes. We propose that aldolase B is ubiquitinated within the cytosol and then transported into autophagosomes and autolysosomes for degradation during nutrient stress. J Cell Physiol 178:17–27, 1999. © 1999 Wiley-Liss, Inc.