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 fructose1,6-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.

Evaluation of cyropreserved internal thoracic artery as an alternative coronary graft: evidence for preserved functional, metabolic and structural integrity.

The internal thoracic artery (ITA) is the conduit of choice for coronary artery bypass grafting (CABG). This study, utilizing a canine model, evaluates cryopreserved ITA. Sixteen ITAs were harvested and cryopreserved according to United CryoInstitute protocol. Test conduits, 5 cm long and 4 mm mean diameter, were anastomosed to the ligated carotid artery of an unmatched mongrel recipient, above and below the site of native artery ligation. Graft patency was assessed by angiography at 14 days (early) and 980 days (late) postoperatively. Catheterization of the 16 vessels identified three (18%) early and one (6%) late graft occlusion. Ninety days postoperatively, each dog was killed and the graft harvested for histopathological and functional evaluation. Morphologic evaluation, using conventional staining, showed preserved cellular structure, decrease in smooth muscle cells and distorted endothelial layer. Immunocytochemistry, using an antibody against prostacyclin (PGI2), detected PGI2 immunoactivity in the ITA smooth muscle cells. An in vitro assay performed on the arterial rings confirmed preserved functional integrity of the vascular endothelium and smooth muscle. These findings suggest that cryopreserved ITA may have potential as a substitute graft, in devising conduit strategies for primary or reoperative coronary bypass surgery.

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.