In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

H2S belongs to the class of molecules known as gasotransmitters, which also includes nitric oxide (NO) and carbon monoxide (CO). Three enzymes are recognized as endogenous sources of H2S in various cells and tissues: cystathionine g-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). The current article reviews the regulation of these enzymes as well as the pathways of their enzymatic and non-enzymatic degradation and elimination. The multiple interactions of H2S with other labile endogenous molecules (e.g. NO) and reactive oxygen species are also outlined. The various biological targets and signaling pathways are discussed, with special reference to H2S and oxidative posttranscriptional modification of proteins, the effect of H2S on channels and intracellular second messenger pathways, the regulation of gene transcription and translation and the regulation of cellular bioenergetics and metabolism. The pharmacological and molecular tools currently available to study H2S physiology are also reviewed, including their utility and limitations. In subsequent sections, the role of H2S in the regulation of various physiological and cellular functions is reviewed. The physiological role of H2S in various cell types and organ systems are overviewed. Finally, the role of H2S in the regulation of various organ functions is discussed as well as the characteristic bell-shaped biphasic effects of H2S. In addition, key pathophysiological aspects, debated areas, and future research and translational areas are identified A wide array of significant roles of H2S in the physiological regulation of all organ functions emerges from this review.

Physiological roles of hydrogen sulfide in mammalian cells, tissues and organs.

Background The global rise of metabolic disorders, such as obesity, type 2 diabetes, and cardiovascular disease, demands a thorough molecular understanding of the cellular mechanisms that govern health or disease. The endoplasmic reticulum (ER) is a key organelle for cellular function and metabolic adaptation and, therefore disturbed ER function, known as “ER stress,” is a key feature of metabolic disorders. Scope of review As ER stress remains a poorly defined phenomenon, this review provides a general guide to understanding the nature, etiology, and consequences of ER stress in metabolic disorders. We define ER stress by its type of stressor, which is driven by proteotoxicity, lipotoxicity, and/or glucotoxicity. We discuss the implications of ER stress in metabolic disorders by reviewing evidence implicating ER phenotypes and organelle communication, protein quality control, calcium homeostasis, lipid and carbohydrate metabolism, and inflammation as key mechanisms in the development of ER stress and metabolic dysfunction. Major conclusions In mammalian biology, ER is a phenotypically and functionally diverse platform for nutrient sensing, which is critical for cell type-specific metabolic control by hepatocytes, adipocytes, muscle cells, and neurons. In these cells, ER stress is a distinct, transient state of functional imbalance, which is usually resolved by the activation of adaptive programs such as the unfolded protein response (UPR), ER-associated protein degradation (ERAD), or autophagy. However, challenges to proteostasis also impact lipid and glucose metabolism and vice versa. In the ER, sensing and adaptive measures are integrated and failure of the ER to adapt leads to aberrant metabolism, organelle dysfunction, insulin resistance, and inflammation. In conclusion, the ER is intricately linked to a wide spectrum of cellular functions and is a critical component in maintaining and restoring metabolic health.

A guide to understanding endoplasmic reticulum stress in metabolic disorders

Hydrogen sulfide (H2S) was once considered to have only toxic properties, until it was discovered to be an endogenous signaling molecule. The effects of H2S are dose dependent, with lower concentrations being beneficial and higher concentrations, cytotoxic. This scenario is especially true for the effects of H2S on mitochondrial function, where higher concentrations of the gasotransmitter inhibit the electron transport chain, and lower concentrations stimulate bioenergetics in multiple ways. Here we review the role of H2S in mitochondrial function and its effects on cellular physiology.

Effects of hydrogen sulfide on mitochondrial function and cellular bioenergetics.

Aging biomarkers are a combination of biological parameters to (i) assess age-related changes, (ii) track the physiological aging process, and (iii) predict the transition into a pathological status. Although a broad spectrum of aging biomarkers has been developed, their potential uses and limitations remain poorly characterized. An immediate goal of biomarkers is to help us answer the following three fundamental questions in aging research: How old are we? Why do we get old? And how can we age slower? This review aims to address this need. Here, we summarize our current knowledge of biomarkers developed for cellular, organ, and organismal levels of aging, comprising six pillars: physiological characteristics, medical imaging, histological features, cellular alterations, molecular changes, and secretory factors. To fulfill all these requisites, we propose that aging biomarkers should qualify for being specific, systemic, and clinically relevant. 

Biomarkers of aging

Hydrogen sulfide (H2S), an important gasotransmitter, regulates cardiovascular functions. Mitochondrial damage induced by the overproduction of reactive oxygen species (ROS) results in myocardial injury with a diabetic state. The purpose of this study was to investigate the effects of exogenous H2S on mitophagy formation in diabetic cardiomyopathy. In this study, we found that exogenous H2S could improve cardiac functions, reduce mitochondrial fragments and ROS levels, enhance mitochondrial respiration chain activities and inhibit mitochondrial apoptosis in the hearts of db/db mice. Our results showed that exogenous H2S facilitated parkin translocation into mitochondria and promoted mitophagy formation in the hearts of db/db mice. Our studies further revealed that the ubiquitination level of cytosolic parkin was increased and the expression of USP8, a deubiquitinating enzyme, was decreased in db/db cardiac tissues. S-sulfhydration is a novel posttranslational modification of specific cysteine residues on target proteins by H2S. Our results showed that the S-sulfhydration level of USP8 was obviously decreased in vivo and in vitro under hyperglycemia and hyperlipidemia, however, exogenous H2S could reverse this effect and promote USP8/parkin interaction. Dithiothreitol, a reducing agent that reverses sulfhydration-mediated covalent modification, increased the ubiquitylation level of parkin, abolished the effects of exogenous H2S on USP8 deubiquitylation and suppressed the interaction of USP8 with parkin in neonatal rat cardiomyocytes treated with high glucose, oleate and palmitate. Our findings suggested that H2S promoted mitophagy formation by increasing S-sulfhydration of USP8, which enhanced deubiquitination of parkin through the recruitment of parkin in mitochondria.

Exogenous H2S Promoted USP8 Sulfhydration to Regulate Mitophagy in the Hearts of db/db Mice.

Hydrogen sulfide (H2S) plays physiological roles in vascular tone regulation, cytoprotection, and ATP synthesis. HMG-CoA reductase degradation protein (Hrd1), an E3 ubiquitin ligase, is involved in protein trafficking. H2S may play a role in controlling fatty acid uptake in diabetic cardiomyopathy (DCM) in a manner correlated with modulation of Hrd1 S-sulfhydration; however, this role remains to be elucidated. The aim of the present study was to examine whether H2S can attenuate lipid accumulation and to explain the possible mechanisms involved in the regulation of the H2S-Hrd1/VAMP3 pathway. Db/db mice and neonatal rat cardiomyocytes treated with high glucose, palmitate and oleate were used as animal and cellular models of type 2 diabetes, respectively. The expression of cystathionine-γ-lyase (CSE), Hrd1, CD36 and VAMP3 was detected by Western blot analysis. In addition, Hrd1 was mutated at Cys115, and Hrd1 S-sulfhydration was examined using an S-sulfhydration assay. VAMP3 ubiquitylation was investigated by immunoprecipitation. Lipid droplet formation was tested by TEM, BODIPY 493/503 staining and oil red O staining. The expression of CSE and Hrd1 was decreased in db/db mice compared to control mice, whereas CD36 and VAMP3 expression was increased. NaHS administration reduced droplet formation, and exogenous H2S restored Hrd1 expression, modified S-sulfhydration, and decreased VAMP3 expression in the plasma membrane. Using LC-MS/MS analysis, we identified 85 proteins with decreased ubiquitylation, including 3 vesicle-associated membrane proteins, in the cardiac tissues of model db/db mice compared with NaHS-treated db/db mice. Overexpression of Hrd1 mutated at Cys115 diminished VAMP3 ubiquitylation, whereas it increased CD36 and VAMP3 expression and droplet formation. siRNA-mediated Hrd1 deletion increased the expression of CD36 in the cell membrane. These findings suggested that H2S regulates VAMP3 ubiquitylation via Hrd1 S-sulfhydration at Cys115 to prevent CD36 translocation in diabetes.

Exogenous H2S Induces Hrd1 S-sulfhydration and Prevents CD36 Translocation via VAMP3 Ubiquitylation in Diabetic Hearts.

Background and purpose Hydrogen sulfide (H2 S) plays important roles as a gasotransmitter in pathologies. Increased expression of the E3 ubiquitin ligase, muscle RING finger-1 (MuRF1), may be involved in diabetic cardiomyopathy. Here we have investigated whether and how exogenous H2 S alleviates cardiac muscle degradation through modifications of MuRF1 S-sulfhydration in db/db mice. Experimental approach Neonatal rat cardiomyocytes were treated with high glucose (40 mM), oleate (100 μM), palmitate (400 μM), and NaHS (100 μM) for 72 hr. MuRF1 was silenced with siRNA technology and mutation at Cys44 . Endoplasmic reticulum stress markers, MuRF1 expression, and ubiquitination level were measured. db/db mice were injected with NaHS (39 μmol·kg-1 ) for 20 weeks. Echocardiography, cardiac ultrastructure, cystathionine-γ-lyase, cardiac structure proteins expression, and S-sulfhydration production were measured. Key results H2 S levels and cystathionine-γ-lyase protein expression in myocardium were decreased in db/db mice. Exogenous H2 S reversed endoplasmic reticulum stress, including impairment of the function of cardiomyocytes and structural damage in db/db mice. Exogenous H2 S could suppress the levels of myosin heavy chain 6 and myosin light chain 2 ubiquitination in cardiac tissues of db/db mice, and MuRF1 was modified by S-sulfhydration, following treatment with exogenous H2 S, to reduce the interaction between MuRF1 and myosin heavy chain 6 and myosin light chain 2. Conclusions and implications Our findings suggest that H2 S regulates MuRF1 S-sulfhydration at Cys44 to prevent myocardial degradation in the cardiac tissues of db/db mice. Linked articles This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Hydrogen sulfide regulates muscle RING finger-1 protein S-sulfhydration at Cys 44 to prevent cardiac structural damage in diabetic cardiomyopathy.

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Exogenous H2S Promoted USP8 Sulfhydration to Regulate Mitophagy in the Hearts of db/db Mice.

Exogenous H2S Induces Hrd1 S-sulfhydration and Prevents CD36 Translocation via VAMP3 Ubiquitylation in Diabetic Hearts.

Hydrogen sulfide regulates muscle RING finger-1 protein S-sulfhydration at Cys 44 to prevent cardiac structural damage in diabetic cardiomyopathy.