Molecular chaperones guide peptide fold conformation throughout the lifetime of the peptide. One network of chaperone proteins involved in this activity, Heat shock protein 70s (Hsp70s), are well characterized at restoring peptide fold, utilizing J-domain containing protein chaperone cofactors to activate Hsp70 activity. DnaJ (Hsp40) homolog, subfamily C, member 25 (DNAJC25) is a class III transmembrane J-domain containing protein that to date is underrepresented in the literature. Recently, Hejtmancik et al. 2012. (unpublished data) have revealed that missense mutation to DNACJ25 at Pro90Leu (P90L) is strongly correlated with inherited Closed-Angle Glaucoma. Inherited mutations are well characterized for Open-Angle Glaucoma, however, prior to this finding, were unknown for Closed-Angle Glaucoma. In this report, analysis of the in vitro chaperone activity of DNAJC25 w+ and P90L is assessed utilizing an Hsp70 mediated Glucose-6-Phosphate Dehydrogenase refolding system, SWISS-MODEL predictions are performed for the J-domain structure of DNAJC25 w+ and P90L with consequent analysis of DNAJC25 Pro90 conservation relative to other type I, II, and III J-domain containing proteins. DNAJC25 P90L demonstrated decreased chaperone activity in vitro compared to w+ DNAJC25. / by Daniel C. Chauss. / Vita. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Indiana University-Purdue University Indianapolis (IUPUI) / Autophagy is a conserved cellular process that involves sequestration and degradation of cytosolic contents. The cell can engulf autophagic cargo (lipids, long-lived proteins, protein aggregates, and pathogens) through a double bound membrane called an autophagosome that fuses with a lysosome where hydrolases then degrade these contents. This process is one of the main defenses against starvation and is imperative for newborns at birth. Research on this process has increased exponentially in the last decade since its discovery almost a half a century ago. It has been found that autophagy is an important process in many diseases, continues to be at the forefront of research, and is clearly not fully understood. Our preliminary cell culture data in endothelial and epithelial cells show that a blockade of the de novo ceramide synthesis pathway, during treatment with an autophagy stimulus (cigarette smoke extract exposure), does not result in any reduction in autophagy or autophagic flux. Conversely, when acid sphingomyelinase (ASM) is pharmacologically inhibited, which prevents the generation of ceramide from sphingomyelin in an acidic environment, a profound increase in autophagy is observed. In this work, we hypothesize that (ASM) is an endogenous inhibitor of autophagy. ASM has two forms, a secreted form and a lysosomal form. N-terminal processing in the Golgi determines its cellular fate. In the lysosomal form, the phosphodiesterase is bound in the lysosomal membrane. The pharmacological inhibition mechanism is to release ASM from the membrane and allow other hydrolases to actively degrade the enzyme which, in turn, decreases the activity of ASM. This suggests that either the activity of ASM is a regulator of autophagy or that the presence of ASM, activity aside, is required for the lysosomal nutrient sensing machinery (LYNUS) to function properly. Here, we show that ASM is, in fact, an endogenous inhibitor of autophagy in vitro. The phosphorylation status of P70 S6k, a downstream effector of mammalian target of rapamycin (mTOR), which is part of the LYNUS, shows that dissociation of ASM from the membrane regulates mTOR and disturbs the LYNUS in such a manner as to signal autophagy.