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Protein homeostasis in eukaryotes is controlled by proteasomal turnover of unstable proteins via the ubiquitin system and lysosomal turnover of the majority of stable proteins through macroautophagy (referred to hereafter [...], Autophagy, the process by which proteins and organelles are sequestered in autophagosomal vesicles and delivered to the lysosome/vacuole for degradation, provides a primary route for turnover of stable and defective cellular proteins. Defects in this system are linked with numerous human diseases. Although conserved protein kinase, lipid kinase and ubiquitin-like protein conjugation subnetworks controlling autophagosome formation and cargo recruitment have been defined, our understanding of the global organization of this system is limited. Here we report a proteomic analysis of the autophagy interaction network in human cells under conditions of ongoing (basal) autophagy, revealing a network of 751 interactions among 409 candidate interacting proteins with extensive connectivity among subnetworks. Many new autophagy interaction network components have roles in vesicle trafficking, protein or lipid phosphorylation and protein ubiquitination, and affect autophagosome number or flux when depleted by RNA interference. The six ATG8 orthologues in humans (MAP1LC3/GABARAP proteins) interact with a cohort of 67 proteins, with extensive binding partner overlap between family members, and frequent involvement of a conserved surface on ATG8 proteins known to interact with LC3-interacting regions in partner proteins. These studies provide a global view of the mammalian autophagy interaction landscape and a resource for mechanistic analysis of this critical protein homeostasis pathway.

The cupredoxin fold is an important motif in numerous proteins that are central to several critical cellular processes ranging from aerobic and anaerobic respiration to catalysis and iron homeostasis. Three types of copper sites have been found to date within cupredoxin folds: blue type 1 (T1) copper, red type 2 (T2) copper, and purple CUA. Although as much as 90% sequence difference has been observed among some members of this superfamily of proteins that span several kingdoms, sequence alignment and phylogenic trees strongly suggest an evolutionary link and common ancestry. However, experimental evidence for such a link has been lacking. We report herein the observation of pH-dependent transformation between blue T1 copper, red T2 copper, and the native purple CUA centers of nitrous oxide reductase (N2OR) from Paracoccus denitrificans. The blue and red copper centers form initially before they are transformed into purple CUA center. This transformation process is pH-dependent, with lower pH resulting in fewer trapped T1 and T2 coppers and faster transition to purple CUA. These observations suggest that the purple CUA site contains the essential elements of T1 and T2 copper centers and that the CUA center is preferentially formed at low pH. Therefore, this work provides an underlying link between the various cupredoxin copper sites and possible experimental evidence in vitro for the evolutionary relationship between the cupredoxin proteins. The findings also lend physiological relevance to cupredoxin site biosynthesis. blue type | copper | purple copper [Cu.sub.A] | red type 2 copper | folding | kinetics

The sirtuins are a group of proteins linked to aging, metabolism and stress tolerance in several organisms. Among the many genes that have been shown to affect aging in model organisms, sirtuin genes are unique in that their activity level is positively correlated with lifespan (i.e. they are anti-aging genes). Sirtuins are a druggable class of enzymes (i.e. amenable to intervention by small molecules) that could have beneficial effects on a variety of human diseases. In view of the many functions of Sirtuin 1 (SIRT1) in cells, this review focuses on its role in regulating important aspects of mitochondrial biology. Mitochondria have been linked to aging, and also to diseases of aging. Thus, sirtuins might provide a key link between mitochondrial dysfunction, aging and metabolic disease.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.10.025 Byline: R. Luke Wiseman (1)(2), Evan T. Powers (1)(2), Joel N. Buxbaum (3), Jeffery W. Kelly (1)(2), William E. Balch (4)(5) Keywords: CELLBIO; PROTEINS; HUMDISEASE Abstract: To provide an integrated view of endoplasmic reticulum (ER) function in protein export, we have described the interdependence of protein folding energetics and the adaptable biology of cellular protein folding and transport through the exocytic pathway. A simplified treatment of the protein homeostasis network and a formalism for how this network of competing pathways interprets protein folding kinetics and thermodynamics provides a framework for understanding cellular protein trafficking. We illustrate how folding and misfolding energetics, in concert with the adjustable biological capacities of the folding, degradation, and export pathways, collectively dictate an adaptable standard for protein export from the ER. A model of folding for export (FoldEx) establishes that no single feature dictates folding and transport efficiency. Instead, a network view provides insight into the basis for cellular diversity, disease origins, and protein homeostasis, and predicts strategies for restoring protein homeostasis in protein-misfolding diseases. Author Affiliation: (1) Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (2) The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (3) Department of Molecular and Experimental Medicine, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (4) Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (5) The Institute for Childhood and Neglected Diseases, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Article History: Received 9 March 2007; Revised 31 July 2007; Accepted 10 October 2007 Article Note: (miscellaneous) Published: November 15, 2007

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heteroeysts in a semiregnlar pattern when it is grown on [N.sub.2] as the sole nitrogen source. The transition from vegetative cells to heteroeysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane [beta]-barrel protein belonging to the TolC family, Air2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Air2887 did not form the specific glycolipid layer of the heterocyst of cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glyeolipid contents wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Air2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glyeolipid layer. We designate the air2887 gene hgdD (heterocyst glycolipid deposition protein).

In the present study, we have investigated gender differences in rat liver mitochondrial oxidative metabolism. Total mitochondrial population (M) as well as the heavy (M1), medium (M3), and light (M8) mitochondrial fractions obtained by means of differential centrifugation steps at 1,000, 3,000, and 8,000 g, respectively, were isolated. Electron microscopic analysis was performed and mitochondrial protein content and cardiolipin levels, mitochondrial [O.sub.2] flux, ATP synthase activity, mitochondrial membrane potential, and mitochondrial transcription factor A (TFAM) protein levels were measured in each sample. Our results indicate that mitochondria from females have higher protein content and higher cardiolipin levels, greater respiratory and phosphorylative capacities, and more-energized mitochondria in respiratory state 3. Moreover, protein levels of TFAM were four times greater in females than in males. Gender differences in the aforementioned parameters were more patent in the isolated heavy M1 and M3 mitochondrial fractions. The present study demonstrates that gender-related differences in liver mitochondrial function are due mainly to a higher capacity and efficiency of substrate oxidation, likely related to greater mitochondrial machinery in females than in males, which is in accord with greater mitochondrial differentiation in females. energy metabolism; mitochondrial biogenesis; high-resolution respirometry; mitochondrial membrane potential; mitochondrial transcription factor A

Excessive accumulation of amyloid [beta]-peptide (A[beta]) plays an early and critical role in synapse and neuronal loss in Alzheimer's Disease (AD). Increased oxidative stress is one of the mechanisms whereby A[beta] induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in A[beta] toxicity. Treatment of cells and primary neuronal cultures with A[beta] caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced A[beta]-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. A[beta] induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against A[beta]-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies A[beta] toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD.

Byline: Ganiraju Manyam, Ancha Baranova, Mikhail Skoblov, Rakesh K. Mishra Genomic sequences for many animal species are now available in the public domain. Protein similarity search in evolutionarily distant organisms by sequence comparison often turns out to be difficult. Here, we present the Structure and Sequence Alignment (SnS-Align) tool that graphically presents pairwise local alignment of sandwiched protein sequences, a hybrid of the primary protein sequence and its secondary structure. The utility of the tool is demonstrated by sample analysis of the gap junction protein superfamily of innexins/pannexins and the classic myoglobin family. SnS-Align can also be used for demarcation of the structurally conserved domains within superfamilies of paralogous genes.