Trent D. Evans

Applications and advances of metabolite biosensors for metabolic engineering.

Quantification and regulation of pathway metabolites is crucial for optimization of microbial production bioprocesses. Genetically encoded biosensors provide the means to couple metabolite sensing to several outputs invaluable for metabolic engineering. These include semi-quantification of metabolite concentrations to screen or select strains with desirable metabolite characteristics, and construction of dynamic metabolite-regulated pathways to enhance production. Taking inspiration from naturally occurring systems, biosensor functions are based on highly diverse mechanisms including metabolite responsive transcription factors, two component systems, cellular stress responses, regulatory RNAs, and protein activities. We review recent developments in biosensors in each of these mechanistic classes, with considerations towards how these sensors are engineered, how new sensing mechanisms have led to improved function, and the advantages and disadvantages of each of these sensing mechanisms in relevant applications. We particularly highlight recent examples directly using biosensors to improve microbial production, and the great potential for biosensors to further inform metabolic engineering practices.

Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis

Macrophages specialize in removing lipids and debris present in the atherosclerotic plaque. However, plaque progression renders macrophages unable to degrade exogenous atherogenic material and endogenous cargo including dysfunctional proteins and organelles. Here we show that a decline in the autophagy–lysosome system contributes to this as evidenced by a derangement in key autophagy markers in both mouse and human atherosclerotic plaques. By augmenting macrophage TFEB, the master transcriptional regulator of autophagy–lysosomal biogenesis, we can reverse the autophagy dysfunction of plaques, enhance aggrephagy of p62-enriched protein aggregates and blunt macrophage apoptosis and pro-inflammatory IL-1β levels, leading to reduced atherosclerosis. In order to harness this degradative response therapeutically, we also describe a natural sugar called trehalose as an inducer of macrophage autophagy–lysosomal biogenesis and show trehaloses ability to recapitulate the atheroprotective properties of macrophage TFEB overexpression. Our data support this practical method of enhancing the degradative capacity of macrophages as a therapy for atherosclerotic vascular disease.

Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis

Sequestration of aggregated proteins by p62 prevents macrophages from exacerbating atherosclerosis. Clearing proteins to limit atherosclerosis The release of proinflammatory cytokines, such as IL-1β, by macrophages increases the size and number of atherosclerotic plaques. Macrophages in atherosclerotic plaques have a defect in autophagy, a process that eliminates dysfunctional proteins, and Sergin et al. showed that p62, a chaperone protein involved in autophagy, sequestered polyubiquitinated proteins in cytoplasmic inclusion bodies in macrophages. Macrophages lacking p62 released more IL-1β, and one of the proteins required for the production of IL-1β partially colocalized with these inclusion bodies. In a mouse model of atherosclerosis, p62 deficiency increased macrophage infiltration in atherosclerotic plaques and exacerbated atherosclerosis. Thus, enhancing the function of p62 to promote the sequestration of polyubiquitinated proteins could prevent macrophages from exacerbating atherosclerosis. Autophagy is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. Atherosclerotic plaque formation is enhanced in mice with macrophages deficient for the critical autophagy protein ATG5. We showed that exposure of macrophages to lipids that promote atherosclerosis increased the abundance of the autophagy chaperone p62 and that p62 colocalized with polyubiquitinated proteins in cytoplasmic inclusions, which are characterized by insoluble protein aggregates. ATG5-null macrophages developed further p62 accumulation at the sites of large cytoplasmic ubiquitin-positive inclusion bodies. Aortas from atherosclerotic mice and plaques from human endarterectomy samples showed increased abundance of p62 and polyubiquitinated proteins that colocalized with plaque macrophages, suggesting that p62-enriched protein aggregates were characteristic of atherosclerosis. The formation of the cytoplasmic inclusions depended on p62 because lipid-loaded p62-null macrophages accumulated polyubiquitinated proteins in a diffuse cytoplasmic pattern. Lipid-loaded p62-null macrophages also exhibited increased secretion of interleukin-1β (IL-1β) and had an increased tendency to undergo apoptosis, which depended on the p62 ubiquitin-binding domain and at least partly involved p62-mediated clearance of NLRP3 inflammasomes. Consistent with our in vitro observations, p62-deficient mice formed greater numbers of more complex atherosclerotic plaques, and p62 deficiency further increased atherosclerotic plaque burden in mice with a macrophage-specific ablation of ATG5. Together, these data suggested that sequestration of cytotoxic ubiquitinated proteins by p62 protects against atherogenesis, a condition in which the clearance of protein aggregates is disrupted.

Target acquired: Selective autophagy in cardiometabolic disease.

Boosting the autophagic degradation of dysfunctional organelles could be used to treat various cardiometabolic diseases. GLOSS The accumulation of damaged or excess proteins and organelles is a defining feature of cardiometabolic diseases such as diabetes, atherosclerosis, and heart failure. One way in which damaged components such as mitochondria, protein aggregates, or peroxisomes are degraded is through autophagy, a process in which a double-membrane vesicle encapsulates cargo and delivers it to the acidic lysosome for degradation. A diverse array of molecular mechanisms target the autophagic machinery specifically to damaged cargo and spare healthy components in a process called selective autophagy. Disruption of these processes leads to accumulation of unhealthy organelles, oxidative stress, inflammation, and disease progression. Conversely, stimulating the autophagy-lysosomal degradation system in disease states featuring overt accumulation of dysfunctional organelles provides an attractive avenue for therapies. In this Review, which contains 6 figures, 1 table, and 252 citations, we focus on emerging evidence and key questions about the role of selective autophagy in the cell biology and pathophysiology of cardiometabolic diseases. The accumulation of damaged or excess proteins and organelles is a defining feature of metabolic disease in nearly every tissue. Thus, a central challenge in maintaining metabolic homeostasis is the identification, sequestration, and degradation of these cellular components, including protein aggregates, mitochondria, peroxisomes, inflammasomes, and lipid droplets. A primary route through which this challenge is met is selective autophagy, the targeting of specific cellular cargo for autophagic compartmentalization and lysosomal degradation. In addition to its roles in degradation, selective autophagy is emerging as an integral component of inflammatory and metabolic signaling cascades. In this Review, we focus on emerging evidence and key questions about the role of selective autophagy in the cell biology and pathophysiology of metabolic diseases such as obesity, diabetes, atherosclerosis, and steatohepatitis. Essential players in these processes are the selective autophagy receptors, defined broadly as adapter proteins that both recognize cargo and target it to the autophagosome. Additional domains within these receptors may allow integration of information about autophagic flux with critical regulators of cellular metabolism and inflammation. Details regarding the precise receptors involved, such as p62 and NBR1, and their predominant interacting partners are just beginning to be defined. Overall, we anticipate that the continued study of selective autophagy will prove to be informative in understanding the pathogenesis of metabolic diseases and to provide previously unrecognized therapeutic targets.

Degradation and beyond: the macrophage lysosome as a nexus for nutrient sensing and processing in atherosclerosis.

Purpose of review The ability of macrophage lysosomes to degrade both exogenous and internally derived cargo is paramount to handling the overabundance of lipid and cytotoxic material present in the atherosclerotic plaque. We will discuss recent insights in both classical and novel functions of the lysosomal apparatus, as it pertains to the pathophysiology of atherosclerosis. Recent findings Lipid-mediated dysfunction in macrophage lysosomes appears to be a critical event in plaque progression. Consequences include enhanced inflammatory signalling [particularly the inflammasome/interleukin-1&bgr; axis] and an inability to interface with autophagy leading to a proatherogenic accumulation of dysfunctional organelles and protein aggregates. Aside from degradation, several novel functions have recently been ascribed to lysosomes, including involvement in macrophage polarization, generation of lipid signalling intermediates and serving as a nutrient depot for mechanistic target of rapamycin activation, each of which can have profound implications in atherosclerosis. Finally, the discovery of the transcription factor transcription factor EB as a mechanism of inducing lysosomal biogenesis can have therapeutic value by reversing lysosomal dysfunction in macrophages. Summary Lysosomes are a central organelle in the processing of exogenous and intracellular biomolecules. Together with recent data that implicate the degradation products of lysosomes in modulation of signalling pathways, these organelles truly do lay at a nexus in nutrient sensing and processing. Dissecting the full repertoire of lysosome function and ensuing dysfunction in plaque macrophages is pivotal to our understanding of atherogenesis.

Transcriptional factor EB regulates macrophage polarization in the tumor microenvironment

ABSTRACT Tumor microenvironment (TME) contains a variety of infiltrating immune cells. Among them, tumor-associated macrophages (TAMs) and their alternative activation contribute greatly to the progression of tumors. The mechanisms governing macrophage polarization in the TME are unclear. Here, we show that in TAMs or macrophages under tumor-conditioned medium treatment, the expression of transcription factor EB (TFEB) is reduced and more of the TFEB protein is in an inactive cytosolic form. Transforming growth factor (TGF)-β is identified as a main driving force for the reduced TFEB expression and activity in TAMs via activating ERK signaling. TFEB interference in macrophages significantly enhanced their alternative activation, with reduced expression of MHC-II and co-stimulatory molecule CD80, decreased ability to activate T cells, and increased ability to attract tumor cells. When co-inoculated with tumor cells, macrophages with TFEB knockdown significantly enhanced tumor growth with increased infiltration of M2-like macrophages, reduced infiltration of CD8+ T cells, and enhanced angiogenesis in the tumors. Mechanistic studies revealed that TFEB downregulation resulted in macrophage M2 polarization through reducing SOCS3 production and enhancing STAT3 activation. We further demonstrate that the activation of TFEB by hydroxypropyl-β-cyclodextrin in macrophages suppressed their M2 polarization and tumor-promoting capacity, and that macrophage-specific TFEB overexpression inhibited breast tumor growth in mice. Therefore, our data suggest that TFEB plays critical roles in macrophage polarization, and the downregulation of TFEB expression and activation is an integral part of tumor-induced immune editing in the TME. This study provides a rationale for a new cancer treatment strategy by modulating macrophage polarization through activating TFEB.

Hypoxia in Plaque Macrophages A New Danger Signal for Interleukin-1β Activation?

The recruitment of inflammatory cells to the arterial wall and their critical role in increasing plaque size and complexity is now dogma in the field of atherosclerosis.1 Macrophages compose the majority of the inflammatory burden in plaques and incite many of the deleterious responses that exacerbate disease. Thus, the mechanisms by which macrophages are activated to secrete cytokines and other inflammatory mediators are of intense interest. The atherosclerotic milieu is replete with cellular stressors such as modified apolipoprotein B–containing lipoproteins (eg, oxidized low-density lipoprotein) and reactive oxygen species, which can act as triggers of the inflammatory response. These danger signals compose a repertoire of triggers of so-called sterile inflammation that characterizes atherosclerosis. Article, see p 875 The prototypical mediator of sterile inflammation is the proinflammatory cytokine interleukin (IL)-1β.2 Because of its potent downstream effects, the production of mature, biologically active IL-1β is tightly regulated at 2 distinct steps.3 In the priming phase, activation of signaling pathways such as nuclear factor-κB by Toll-like receptors, other cytokines, or IL-1β itself, leads to the transcriptional induction of precursor IL-1β (pro–IL-1β). Generation of the mature form is then governed by activation of a complex of proteins known as the inflammasome. Although several distinct complexes with unique triggers have been described, the NLRP3 inflammasome composed of nucleotide-binding oligomerization domain-like receptor family member (NLRP3), apoptosis-associated speck-like protein (ASC), and pro–caspase-1 is the most relevant to metabolic diseases such as atherosclerosis. A variety of pathogen- or endogenously derived danger signals (known as pathogen- or damage-associated molecular patterns) can activate the NLRP3 inflammasome, leading to proteolytic activation of caspase-1 and consequent cleavage of pro–IL-1β to the mature form.3 The process of inflammasome activation and its contribution to atherogenesis has been the focus of significant recent investigation in the field. Cholesterol crystals, which were previously …

TFEB and trehalose drive the macrophage autophagy-lysosome system to protect against atherosclerosis

ABSTRACT In the atherosclerotic plaque, macrophages are the key catabolic workhorse responsible for clearing lipid and dead cell debris. To survive the highly proinflammatory and lipotoxic plaque environment, macrophages must adopt strategies for maintaining tight homeostasis and self-renewal. Macroautophagy/autophagy is a pro-survival cellular pathway wherein damaged or excess cellular cargoes are encapsulated by a double-membrane compartment and delivered to the lysosome for hydrolysis. Previously, macrophage-specific autophagy deficiency has been shown to be atherogenic through several complementary mechanisms including hyperactivation of the inflammasome, defective efferocytosis, accumulation of cytotoxic protein aggregates, and impaired lipid degradation. Conversely, in a recent study we hypothesized that enhancing the macrophage autophagy-lysosomal system through genetic or pharmacological means could protect against atherosclerosis. We demonstrated that TFEB, a transcription factor master regulator of autophagy and lysosome biogenesis, coordinately enhances the function of this system to reduce atherosclerotic plaque burden. Further, we characterized the disaccharide trehalose as a novel inducer of TFEB with similar atheroprotective effects. Overall, these findings mechanistically interrogate the importance and therapeutic promise of a functional autophagy-lysosome degradation system in plaque macrophage biology.

Modulating Oxysterol Sensing to Control Macrophage Apoptosis and Atherosclerosis.

Atherosclerosis is the silent underlying cause of acute myocardial infarction and stroke, some of the most common causes of mortality worldwide. Aside from managing risk factors, such as hyperlipidemia, few therapies can be said to directly target atherosclerotic plaque progression partially because of remaining gaps in basic knowledge of the disease. Atherosclerosis is initiated by endothelial injury and deposition of lipids in the subendothelial layer called the intima. Circulating monocytes traffic to the site of injury, transmigrate through the endothelium, and differentiate to macrophages in an attempt to repair damage and remove lipids, much of which include deleterious oxidatively modified forms. Subsequently, plaque macrophages become central players in the progression of atherosclerosis because of a feedforward cycle of rampant lipid accumulation, proinflammatory cytokine release, cell death, and further monocyte recruitment. Article, see p 1296 A major area of focus in the field is in understanding why macrophages cannot efficiently clear lipid and cell debris deposits. Lipid species in the plaque, including oxidized low-density lipoprotein (oxLDL), are known to induce apoptotic cell death, though the precise lipid species and signal transduction mechanisms involved are unclear. This apoptotic response may actually be adaptive in the early stages of plaque development because secondary macrophages can adeptly clear apoptotic cells through efferocytosis before cell-intrinsic damage becomes overwhelming (Figure, top left). Indeed, many features of apoptotic cell death, including expression of eat-me surface signals, are purposed toward efferocytic clearance. In contrast, secondary necrosis occurs when apoptotic cells are left unengulfed, resulting in uncontrolled disintegration, release of cytosolic components, and damage to local tissue.1 Advanced plaques lack efficient efferocytosis such that many apoptotic cells progress to secondary necrosis, adding to plaque size and complexity (Figure, top right). Thus, understanding mechanisms of apoptotic cell death in atherosclerotic plaque macrophages and the impact on overall …