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Autophagy and apoptosis are catabolic pathways essential for organismal homeostasis. Autophagy is normally a cell-survival pathway involving the degradation and recycling of obsolete, damaged, or harmful macromolecular assemblies; however, excess autophagy has been implicated in type II cell death. Apoptosis is the canonical programmed cell death pathway. Autophagy and apoptosis have now been shown to be interconnected by several molecular nodes of crosstalk, enabling the coordinate regulation of degradation by these pathways. Normally, autophagy and apoptosis are both tumor suppressor pathways. Autophagy fulfils this role as it facilitates the degradation of oncogenic molecules, preventing development of cancers, while apoptosis prevents the survival of cancer cells. Consequently, defective or inadequate levels of either autophagy or apoptosis can lead to cancer. However, autophagy appears to have a dual role in cancer, as it has now been shown that autophagy also facilitates the survival of tumor cells in stress conditions such as hypoxic or low-nutrition environments. Here we review the multiple molecular mechanisms of coordination of autophagy and apoptosis and the role of the proteins involved in this crosstalk in cancer. A comprehensive understanding of the interconnectivity of autophagy and apoptosis is essential for the development of effective cancer therapeutics.

Autophagy is a fundamental adaptive response to amino acid starvation orchestrated by conserved gene products, the autophagy (ATG) proteins. However, the cellular cues that activate the function of ATG proteins during amino acid starvation are incompletely understood. Here we show that two related stress-responsive kinases, members of the p38 mitogen-activated protein kinase (MAPK) signaling pathway MAPKAPK2 (MK2) and MAPKAPK3 (MK3), positively regulate starvation-induced autophagy by phosphorylating an essential ATG protein, Beclin 1, at serine 90, and that this phosphorylation site is essential for the tumor suppressor function of Beclin 1. Moreover, MK2/MK3-dependent Beclin 1 phosphorylation (and starvation-induced autophagy) is blocked in vitro and in vivo by BCL2, a negative regulator of Beclin 1. Together, these findings reveal MK2/MK3 as crucial stress-responsive kinases that promote autophagy through Beclin 1 S90 phosphorylation, and identify the blockade of MK2/3-dependent Beclin 1 S90 phosphorylation as a mechanism by which BCL2 inhibits the autophagy function of Beclin 1. Cells keep themselves healthy by breaking down unneeded or damaged internal structures via a process called autophagy. This process also helps a cell to survive if it is starved of nutrients. For example, if a cell does not receive enough amino acids, it cannot make new proteins. Autophagy can break down existing non-essential proteins so that their amino acids can be re-used to build other proteins that the cell needs to survive. Autophagy is performed by a set of proteins that is found in many different species, ranging from yeast to humans and plants. How these proteins are activated when a cell is starved of amino acids is not fully understood. However, evidence suggests that activating one of these proteins, called Beclin 1, by adding phosphate groups to it controls the extent to which autophagy occurs. It is also known from previous work that less autophagy occurs when Beclin 1 binds to another protein called BCL2. Wei, An et al. identified two enzymes that attach a phosphate group to a specific site on Beclin 1 to activate it, and revealed that autophagy is defective in cells that lack these enzymes. Furthermore, Wei, An et al. found the BCL2 protein prevents autophagy by binding to Beclin 1 in such a way that stops these two enzymes from activating Beclin 1. Beclin 1 is also known to prevent the growth of malignant tumors. Wei, An et al. found that to do so, Beclin 1 must have a phosphate group added to the same site that activates the protein during autophagy. This suggests that drugs that enhance the addition of this phosphate group to Beclin 1 could help activate autophagy and have anti-cancer effects.

α 1 -adrenergic receptors (α 1 -ARs) play critical roles in the cardiovascular and nervous systems where they regulate blood pressure, cognition, and metabolism. However, the lack of specific agonists for all α 1 subtypes has limited our understanding of the physiological roles of different α 1 -AR subtypes, and led to the stagnancy in agonist-based drug development for these receptors. Here we report cryo-EM structures of α 1A -AR in complex with heterotrimeric G-proteins and either the endogenous common agonist epinephrine or the α 1A -AR-specific synthetic agonist A61603. These structures provide molecular insights into the mechanisms underlying the discrimination between α 1A -AR and α 1B -AR by A61603. Guided by the structures and corresponding molecular dynamics simulations, we engineer α 1A -AR mutants that are not responsive to A61603, and α 1B -AR mutants that can be potently activated by A61603. Together, these findings advance our understanding of the agonist specificity for α 1 -ARs at the molecular level, opening the possibility of rational design of subtype-specific agonists. α1-adrenergic receptors (α1- AR) play critical roles in the cardiovascular and nervous systems. Here, the authors report molecular insights into the mechanisms underlying the discrimination between α1A-AR and α1B-AR by the agonist A61603.

Agonists targeting α 2 -adrenergic receptors (ARs) are used to treat diverse conditions, including hypertension, attention-deficit/hyperactivity disorder, pain, panic disorders, opioid and alcohol withdrawal symptoms, and cigarette cravings. These receptors transduce signals through heterotrimeric Gi proteins. Here, we elucidated cryo-EM structures that depict α 2A -AR in complex with Gi proteins, along with the endogenous agonist epinephrine or the synthetic agonist dexmedetomidine. Molecular dynamics simulations and functional studies reinforce the results of the structural revelations. Our investigation revealed that epinephrine exhibits different conformations when engaging with α-ARs and β-ARs. Furthermore, α 2A -AR and β 1 -AR (primarily coupled to Gs, with secondary associations to Gi) were compared and found to exhibit different interactions with Gi proteins. Notably, the stability of the epinephrine–α 2A -AR–Gi complex is greater than that of the dexmedetomidine–α 2A -AR–Gi complex. These findings substantiate and improve our knowledge on the intricate signaling mechanisms orchestrated by ARs and concurrently shed light on the regulation of α-ARs and β-ARs by epinephrine. α vs. β-ARs: epinephrine signaling mechanisms revealed Our bodies have a system, the sympathetic nervous system, that uses certain chemicals to control heart rate, blood pressure, etc. These chemicals, epinephrine and norepinephrine, work by activating proteins known as adrenergic receptors. Understanding these receptors could help treat diseases like high blood pressure and ADHD. This study used a method called cryo-electron microscopy to see how epinephrine interacts with these receptors. It compared how epinephrine and a similar drug, dexmedetomidine, interact with the receptors. The study found that epinephrine binds to the α and β types of the receptors differently, which could explain their different effects. This helps us understand how drugs that mimic or block epinephrine can treat diseases. This could lead to new, more effective drugs. Future research may use these findings to design better treatments for heart diseases and other conditions. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

The β 1 -adrenergic receptor (β 1 -AR) can activate two families of G proteins. When coupled to Gs, β 1 -AR increases cardiac output, and coupling to Gi leads to decreased responsiveness in myocardial infarction. By comparative structural analysis of turkey β 1 -AR complexed with either Gi or Gs, we investigate how a single G-protein-coupled receptor simultaneously signals through two G proteins. We find that, although the critical receptor-interacting C-terminal α5-helices on Gα i and Gα s interact similarly with β 1 -AR, the overall interacting modes between β 1 -AR and G proteins vary substantially. Functional studies reveal the importance of the differing interactions and provide evidence that the activation efficacy of G proteins by β 1 -AR is determined by the entire three-dimensional interaction surface, including intracellular loops 2 and 4 (ICL2 and ICL4). This study reveals the structural basis for the coupling specificity of one G-protein-coupled receptor, the β 1 -adrenergic receptor, to two different families of G proteins. Although the receptor adopts the same conformation, the G proteins have different interaction modes dictated by the overall structure.

G-protein-coupled receptors (GPCRs) receive signals from ligands with different efficacies, and transduce to heterotrimeric G-proteins to generate different degrees of physiological responses. Previous studies revealed how ligands with different efficacies activate GPCRs. Here, we investigate how a GPCR activates G-proteins upon binding ligands with different efficacies. We report the cryo-EM structures of β 1 -adrenergic receptor (β 1 -AR) in complex with Gs (Gα s Gβ 1 Gγ 2 ) and a partial agonist or a very weak partial agonist, and compare them to the β 1 -AR–Gs structure in complex with a full agonist. Analyses reveal similar overall complex architecture, with local conformational differences. Cellular functional studies with mutations of β 1 -AR residues show effects on the cellular signaling from β 1 -AR to the cAMP response initiated by the three different ligands, with residue-specific functional differences. Biochemical investigations uncover that the intermediate state complex comprising β 1 -AR and nucleotide-free Gs is more stable when binding a full agonist than a partial agonist. Molecular dynamics simulations support the local conformational flexibilities and different stabilities among the three complexes. These data provide insights into the ligand efficacy in the activation of GPCRs and G-proteins. Su et al. report cryo-EM structures of β 1 -adrenergic receptor, Gs and ligands of different efficacies. These complexes have similar overall architecture, but with local conformational differences and different stabilities.

To date, numerous studies have employed single type nitrogen (N) addition methods in reporting influences of N deposition on soil extracellular enzymatic activities (EEA) during litter decomposition in forest ecosystems. As natural atmospheric N deposition is a set of complex compounds including inorganic N and organic N, it is essential for investigating responses of soil EEA to various mixed N fertilization. In a subtropical forest stand in Zijin Mountain, East China, various N fertilizers with different inorganic N and organic N ratios were added to soils monthly from 2008 to 2009. Samples were harvested from N fertilized and control plots every 4 months. Subsequently, six EEA were assayed. A laboratory experiment was also conducted simultaneously. Both field and laboratory experiments showed that various mixed N fertilizations revealed different influences on soil EEA. Acceleration of most soil EEA by mixed N fertilization was greater than that of single N fertilization. The majority of soil extracellular enzymes exhibited the highest activities under mixed N fertilization, with the ratio of inorganic N to organic N at 3:7. These results suggested that N type and ratio of inorganic N and organic N were important factors controlling soil EEA, and the 3:7 ratio of inorganic N and organic N may be the optimum for soil EEA.

α -adrenergic receptors (α -ARs) play critical roles in the cardiovascular and nervous systems where they regulate blood pressure, cognition, and metabolism. However, the lack of specific agonists for all α subtypes has limited our understanding of the physiological roles of different α -AR subtypes, and led to the stagnancy in agonist-based drug development for these receptors. Here we report cryo-EM structures of α -AR in complex with heterotrimeric G-proteins and either the endogenous common agonist epinephrine or the α -AR-specific synthetic agonist A61603. These structures provide molecular insights into the mechanisms underlying the discrimination between α -AR and α -AR by A61603. Guided by the structures and corresponding molecular dynamics simulations, we engineer α -AR mutants that are not responsive to A61603, and α -AR mutants that can be potently activated by A61603. Together, these findings advance our understanding of the agonist specificity for α -ARs at the molecular level, opening the possibility of rational design of subtype-specific agonists.

G-protein-coupled receptors (GPCRs) receive signals from ligands with different efficacies, and transduce to heterotrimeric G-proteins to generate different degrees of physiological responses. Previous studies revealed how ligands with different efficacies activate GPCRs. Here, we investigate how a GPCR activates G-proteins upon binding ligands with different efficacies. We report the cryo-EM structures of β -adrenergic receptor (β -AR) in complex with Gs (Gα Gβ Gγ ) and a partial agonist or a very weak partial agonist, and compare them to the β -AR-Gs structure in complex with a full agonist. Analyses reveal similar overall complex architecture, with local conformational differences. Cellular functional studies with mutations of β -AR residues show effects on the cellular signaling from β -AR to the cAMP response initiated by the three different ligands, with residue-specific functional differences. Biochemical investigations uncover that the intermediate state complex comprising β -AR and nucleotide-free Gs is more stable when binding a full agonist than a partial agonist. Molecular dynamics simulations support the local conformational flexibilities and different stabilities among the three complexes. These data provide insights into the ligand efficacy in the activation of GPCRs and G-proteins.

Autophagy and apoptosis are catabolic pathways essential for homeostasis in all eukaryotes. While autophagy usually promotes cell-survival by enabling degradation and recycling of damaged macromolecules, apoptosis is the canonical programmed cell death pathway. Dysfunction of autophagy and apoptosis is implicated in diseases like cancers, cardiac disease, and infectious disease. Beclin homologs are key for autophagy as they are core components of class III phosphatidylinositol 3-kinase autophagosome nucleation and maturation complexes. Anti-apoptotic Bcl-2s inhibit apoptosis through binding and antagonizing pro-apoptotic Bcl-2s. Anti-apoptotic Bcl-2s also down-regulate autophagy by binding to the Bcl-2 homology domain 3 (BH3D) of Beclin homologs, thereby enabling crosstalk between apoptosis and autophagy. The central goal of my doctoral research is to understand the structure-based mechanism of selected Bcl-2s and Beclin homologs in the regulation of autophagy and apoptosis. γ-Herpesviruses are common human pathogens that encode viral Bcl-2s to facilitate viral reactivation and oncogenic transformation. M11, a viral Bcl-2, and Bcl-XL, a cellular Bcl-2, bind with comparable affinities to the Beclin 1 BH3D. By assessing the impact of different Beclin 1 BH3D mutations on binding to M11 and Bcl-XL, we developed a cell-permeable inhibitory peptide that targets M11, but not Bcl-XL. The mechanism by which this peptide specifically binds to M11 was elucidated by determining the X-ray crystal structure of the peptide:M11 complex. Our attempts to investigate the role of other Bcl-2s in these pathways were unsuccessful. In one project, we were unable to express and purify BALF1, another viral Bcl-2. In another, no interaction was detected between purified samples of the anti-apoptotic Bcl-2 homolog Mcl-1, and the autophagy protein Atg12, that had previously been shown to bind. We also delineated the domain architecture of Beclin 2, a novel Beclin homolog and attempted to express and purify different Beclin 2 constructs for structural studies. We successfully purified and solved the X-ray crystal structure of the Beclin 2 coiled-coil domain (CCD), showing it is a curved, anti-parallel, meta-stable coiled-coil homodimer with seven pairs of non-ideal packing residues. In general, mutating the non-ideal packing residues to leucines enhanced Beclin 2 CCD homodimerization, but also weakened its binding to Atg14 CCD.