Lipid droplets modulate proteostasis, SQST-1/SQSTM1 dynamics, and lifespan in C. elegans.

In several long-lived Caenorhabditis elegans strains, such as insulin/IGF-1 receptor daf-2 mutants, enhanced proteostatic mechanisms are accompanied by elevated intestinal lipid stores, but their role in longevity is unclear. Here, while determining the regulatory network of the selective autophagy receptor SQST-1/SQSTM1, we uncovered an important role for lipid droplets in proteostasis and longevity. Using genome-wide RNAi screening, we identified several SQST-1 modulators, including lipid droplets-associated and aggregation-prone proteins. Expansion of intestinal lipid droplets by silencing the conserved cytosolic triacylglycerol lipase gene atgl-1/ATGL enhanced autophagy, and extended lifespan. Notably, a substantial amount of ubiquitinated proteins were found on lipid droplets. Reducing lipid droplet levels exacerbated the proteostatic collapse when autophagy or proteasome function was compromised, and significantly reduced the lifespan of long-lived daf-2 animals. Altogether, our study uncovered a key role for lipid droplets in C. elegans as a proteostatic mediator that modulates ubiquitinated protein accumulation, facilitates autophagy, and promotes longevity.

Non-canonical autophagy in aging and age-related diseases.

Autophagy, one of the arms of proteostasis, influences aging and age-related diseases. Recently, the discovery of additional roles of autophagy-related proteins in non-canonical degradation and secretion has revealed alternative fates of autophagic cargo. Some of these non-canonical pathways have been linked to neurodegenerative diseases and improving the understanding of this link is crucial for their potential targetability in aging and age-related diseases. This review discusses recent investigations of the involvement of non-canonical autophagy players and pathways in age-related diseases that are now beginning to be discovered. Unraveling these pathways and their relation to classical autophagy could unearth a fascinating new layer of proteostasis regulation during normal aging and in longevity.

Exportin 1 modulates life span by regulating nucleolar dynamics via the autophagy protein LGG-1/GABARAP.

Altered nucleolar and ribosomal dynamics are key hallmarks of aging, but their regulation remains unclear. Building on the knowledge that the conserved nuclear export receptor Exportin 1 (XPO-1/XPO1) modulates proteostasis and life span, we systematically analyzed the impact of nuclear export on protein metabolism. Using transcriptomic and subcellular proteomic analyses in nematodes, we demonstrate that XPO-1 modulates the nucleocytoplasmic distribution of key proteins involved in nucleolar dynamics and ribosome function, including fibrillarin (FIB-1/FBL) and RPL-11 (RPL11). Silencing xpo-1 led to marked reduction in global translation, which was accompanied by decreased nucleolar size and lower fibrillarin levels. A targeted screen of known proteostatic mediators revealed that the autophagy protein LGG-1/GABARAP modulates nucleolar size by regulating RPL-11 levels, linking specific protein degradation to ribosome metabolism. Together, our study reveals that nucleolar size and life span are regulated by LGG-1/GABARAP via ribosome protein surveillance.

Selective Autophagy Receptor p62/SQSTM1, a Pivotal Player in Stress and Aging.

Efficient proteostasis is crucial for somatic maintenance, and its decline during aging leads to cellular dysfunction and disease. Selective autophagy is a form of autophagy mediated by receptors that target specific cargoes for degradation and is an essential process to maintain proteostasis. The protein Sequestosome 1 (p62/SQSTM1) is a classical selective autophagy receptor, but it also has roles in the ubiquitin-proteasome system, cellular metabolism, signaling, and apoptosis. p62 is best known for its role in clearing protein aggregates via aggrephagy, but it has recently emerged as a receptor for other forms of selective autophagy such as mitophagy and lipophagy. Notably, p62 has context-dependent impacts on organismal aging and turnover of p62 usually reflects active proteostasis. In this review, we highlight recent advances in understanding the role of p62 in coordinating the ubiquitin-proteasome system and autophagy. We also discuss positive and negative effects of p62 on proteostatic status and their implications on aging and neurodegeneration. Finally, we relate the link between defective p62 and diseases of aging and examine the utility of targeting this multifaceted protein to achieve proteostatic benefits.

Sex hormones regulate lipid metabolism in adult Sertoli cells: A genome-wide study of estrogen and androgen receptor binding sites.

Optimal functioning of Sertoli cells is crucial for spermatogenesis which is under tight regulation of sex hormones, estrogen and androgen. Adult rat Sertoli cells expresses estrogen receptor beta (ERß) and androgen receptor (AR), both of which regulate gene transcription by binding to the DNA. The present study is aimed to acquire a genome-wide map of estrogen- and androgen-regulated genes in adult Sertoli cells. ChIP-Seq was performed for ERß and AR in Sertoli cells under physiological conditions. 30,859 peaks in ERß and 9,594 peaks in AR were identified with a fold enrichment >2 fold. Pathway analysis for the genes revealed metabolic pathways to be significantly enriched. Since Sertoli cells have supportive functions and provide energy substrates to germ cells during spermatogenesis, significantly enriched metabolic pathways were explored further. Peaks of the genes involved in lipid metabolism, like fatty acid, glyceride, leucine, and sphingosine metabolism were validated. Motif analysis confirmed the presence of estrogen- and androgen-response elements (EREs and AREs). Moreover, transcript levels of enzymes involved in the lipid metabolic pathways were significantly altered in cultured Sertoli cells treated with estrogen and androgen receptor agonists, demonstrating functional significance of these binding sites. This study elucidates a mechanism by which sex hormones regulate lipid metabolism in Sertoli cells by transcriptionally controlling the expression of these genes, thereby shedding light on the roles of these hormones in male fertility.

Location, location, location: subcellular protein partitioning in proteostasis and aging.

Somatic maintenance and cell survival rely on proper protein homeostasis to ensure reliable functions across the cell and to prevent proteome collapse. Maintaining protein folding and solubility is central to proteostasis and is coordinated by protein synthesis, chaperoning, and degradation capacities. An emerging aspect that influences proteostasis is the dynamic protein partitioning across different subcellular structures and compartments. Here, we review recent literature related to nucleocytoplasmic partitioning of proteins, nuclear and cytoplasmic quality control mechanisms, and their impact on the development of age-related diseases. We also highlight new points of entry to modulate spatially-regulated proteostatic mechanisms to delay aging.

C. elegans to model autophagy-related human disorders.

Autophagy is a highly conserved degradation process that clears damaged intracellular macromolecules and organelles in order to maintain cellular health. Dysfunctional autophagy is fundamentally linked to the development of various human disorders and pathologies. The use of the nematode Caenorhabditis elegans as a model system to study autophagy has improved our understanding of its regulation and function in organismal physiology. Here, we review the genetic, functional, and regulatory conservation of the autophagy pathway in C. elegans and we describe tools to quantify and study the autophagy process in this incredibly useful model organism. We further discuss how these nematodes have been modified to model autophagy-related human diseases and underscore the important insights obtained from such models. Altogether, we highlight the strengths of C. elegans as an exceptional tool to understand the genetic and molecular foundations underlying autophagy-related human diseases.

Genome-wide identification of estrogen receptor binding sites reveals novel estrogen-responsive pathways in adult male germ cells.

Spermatogenesis occurs in the seminiferous epithelium that shows the presence of estrogen receptors alpha (ERα) and beta (ERß), both of which regulate gene transcription by binding to the DNA. Estrogen responsive phases of spermatogenesis are well documented; however, the genes regulated remain inexplicit. To study the regulation of genes by estrogen in male germ cells, we performed chromatin immunoprecipitation (ChIP) sequencing for ERα and ERß under normal physiological conditions. A total of 27 221 DNA binding regions were enriched with ERα and 20 926 binding sites with ERß. Majority of the peaks were present in the intronic regions and located 20 kb upstream or downstream from the transcription start site (TSS). Pathway analysis of the genes enriched by ChIP-Seq showed involvement in several biological pathways. Genes involved in pathways whose role in spermatogenesis is unexplored were validated; these included prolactin, GnRH, and oxytocin signaling. All the selected genes showed the presence of estrogen response elements (EREs) in their binding region and were also found to be significantly enriched by ChIP-qPCR. Functional validation using seminiferous tubule culture after treatment with estrogen receptor subtype-specific agonist and antagonist confirmed the regulation of these genes by estrogen through its receptors. The genes involved in these pathways were also found to be regulated by the respective receptor subtypes at the testicular level in our in vivo estrogen receptor agonist rat models. Our study provides a genome-wide map of ERα and ERß binding sites and identifies the genes regulated by them in the male germ cells under normal physiological conditions.

Autophagy in aging and longevity.

Our understanding of the process of autophagy and its role in health and diseases has grown remarkably in the last two decades. Early work established autophagy as a general bulk recycling process which involves the sequestration and transport of intracellular material to the lysosome for degradation. Currently, autophagy is viewed as a nexus of metabolic and proteostatic signalling that can determine key physiological decisions from cell fate to organismal lifespan. Here, we review the latest literature on the role of autophagy and lysosomes in stress response and longevity. We highlight the connections between autophagy and metabolic processes, the network associated with its regulation, and the links between autophagic dysfunction, neurodegenerative diseases, and aging.

Nuclear Export Inhibition Enhances HLH-30/TFEB Activity, Autophagy, and Lifespan.

Transcriptional modulation of the process of autophagy involves the transcription factor HLH-30/TFEB. In order to systematically determine the regulatory network of HLH-30/TFEB, we performed a genome-wide RNAi screen in C. elegans and found that silencing the nuclear export protein XPO-1/XPO1 enhances autophagy by significantly enriching HLH-30 in the nucleus, which is accompanied by proteostatic benefits and improved longevity. Lifespan extension via xpo-1 silencing requires HLH-30 and autophagy, overlapping mechanistically with several established longevity models. Selective XPO1 inhibitors recapitulated the effect on autophagy and lifespan observed by silencing xpo-1 and protected ALS-afflicted flies from neurodegeneration. XPO1 inhibition in HeLa cells enhanced TFEB nuclear localization, autophagy, and lysosome biogenesis without affecting mTOR activity, revealing a conserved regulatory mechanism for HLH-30/TFEB. Altogether, our study demonstrates that altering the nuclear export of HLH-30/TFEB can regulate autophagy and establishes the rationale of targeting XPO1 to stimulate autophagy in order to prevent neurodegeneration.

Estrogen effects on actin cytoskeletal and endocytic proteins associated with tubulobulbar complex disruption in rat testes.

Tubulobulbar complexes (TBCs), evaginations of mature spermatids, penetrate into the surrounding Sertoli cell cytoplasm of testis seminiferous epithelium during rat spermatogenesis. These structures prepare mature spermatids for their release into the seminiferous tubular lumen via a process called spermiation. Based on their functions of transient attachment and endocytosis, many actin-regulatory and endocytic proteins are associated with TBCs. Previously, exogenous 17ß-estradiol administration to adult male rats showed spermiation failure that was attributed to TBC disruption. To determine the molecular basis of estrogen-induced TBC disruption, we examined the expressions and localizations of actin-regulatory proteins, endocytic proteins, Rho-GTPases, and phosphorylation in TBCs during sperm release. Results demonstrated absence of neural Wiscott Aldrich syndrome protein, cortactin, adaptor-related protein complex 2 sigma-1 subunit, dynamin 2, cell division control protein 42, and phosphocortactin in the concavity of spermatid head where TBCs are present without change in their protein expression levels. Absence of these proteins could have led to collapse of the TBC structure which is involved in its formation and function.

Proteomics in reproductive biology: beacon for unraveling the molecular complexities.

Proteomics, an interface of rapidly evolving advances in physics and biology, is rapidly developing and expanding its potential applications to molecular and cellular biology. Application of proteomics tools has contributed towards identification of relevant protein biomarkers that can potentially change the strategies for early diagnosis and treatment of several diseases. The emergence of powerful mass spectrometry-based proteomics technique has added a new dimension to the field of medical research in liver, heart diseases and certain forms of cancer. Most proteomics tools are also being used to study physiological and pathological events related to reproductive biology. There have been attempts to generate the proteomes of testes, sperm, seminal fluid, epididymis, oocyte, and endometrium from reproductive disease patients. Here, we have reviewed proteomics based investigations in humans over the last decade, which focus on delineating the mechanism underlying various reproductive events such as spermatogenesis, oogenesis, endometriosis, polycystic ovary syndrome, embryo development. The challenge is to harness new technologies like 2-DE, DIGE, MALDI-MS, SELDI-MS, MUDPIT, LC-MS etc., to a greater extent to develop widely applicable clinical tools in understanding molecular aspects of reproduction both in health and disease.

Tubulobulbar complex: cytoskeletal remodeling to release spermatozoa.

Tubulobulbar complexes (TBCs) are actin-based structures that help establish close contact between Sertoli-Sertoli cells or Sertoli-mature germ cells (spermatids) in the seminiferous tubules of the testes. They are actin-rich push-through devices that eliminate excess spermatid cytoplasm and prepare mature spermatids for release into the tubular lumen. Just prior to spermiation, the elongated spermatid interacts with the Sertoli cell via an extensive structure comprising various adhesion molecules called the apical ectoplasmic specialization which is partially replaced by the apical TBC, on the concave surface of the spermatid head. The sperm release process involves extensive restructuring, namely the disassembly and reassembly of junctions at the Sertoli-spermatid interface in the seminiferous epithelium. Based on the presence of different classes of molecules in the TBCs or the defects observed in the absence of TBCs, the main functions attributed to TBCs are elimination of excess spermatid cytoplasm, endocytosis and recycling of junctional molecules, shaping of the spermatid acrosome, and forming transient anchoring devices for mature spermatids before they are released. This review summarizes the recent findings that focus on the role of TBCs in cell cytoskeleton restructuring during sperm release in the testes and the molecular mechanism involved.