bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis, Docking Studies and Biological Evaluation of Triazole‐Thiazolidinedione/Rhodanine Hybrids Targeting Cyclooxygenase‐2, 5‐Lipoxygenase and Peroxisome proliferator‐activated receptor
Authors: Alaaeddine Rana , Elzahhar Perihan , Belal Ahmed , Nassra Rasha , Ibrahim Tamer , Wallner Nadja , Knape Tilo , Von Knethen Andreas , El‐Yazbi Ahmed F.
Published: June 2021

Inflammation is associated with different clinical conditions including rheumatoid disorders and multiple sclerosis. Recently, inflammation has been linked to metabolic diseases, such as diabetes. In addition to their antidiabetic effect, thiazolidinediones (TZDs), through PPARγ transactivation, are also known to negatively control transcriptional pathways that are critical for the synthesis of pro‐inflammatory molecules. Moreover, selective COX‐2 inhibitors serve as effective anti‐inflammatory agents with minimal GIT side effects; however, only blocking COX‐2 shunts the inflammatory pathways towards more activity of LOX, and hence blocking both COX‐2 and LOX would provide safer and more effective anti‐inflammatory activity. In our search for multi‐targeting anti‐inflammatory leads, we adopted a hybrid pharmacophore approach to design and synthesize some novel triazolyl‐thiazolidinedione/rhodanine hybrids as potential simultaneous COX‐2/LOX inhibitors and PPARγ agonists. Ten of these compounds demonstrated significant in vitro COX‐2 and 15‐LOX inhibitory activities, four of which exhibited high in vitro tissue glucose uptake. These drugs revealed comparable nuclear PPARγ translocation to that of pioglitazone and luciferase activity. PMA‐induced macrophage differentiation assay conducted on THP‐1 cells demonstrated similar or exceeding potency than diclofenac. This translated to in‐vivo formalin‐induced rat paw edema bioassay where the selected drugs showed an anti‐inflammatory activity. The pharmacological data were coherent with the in silico results. Accordingly, this series of compounds may represent promising leads to pursue as potential simultaneous COX‐2/LOX inhibitors and PPARγ agonists for the management of diabetes and other inflammatory disorders.

Support or Funding Information

Partially supported by AUB MPP fund #320148

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Genetically‐Encoded Fluorescent Assay to Detect Drug Toxicity and Genetic Stress in Living Cells
Authors: Harlen Kevin M , Hughes Thom , Quinn Anne Marie
Published: June 2021

We have created a genetically‐encoded fluorescent biosensor to detect chemically and genetically induced stress in living cells. Identifying stressed cells long before they enter the apoptotic pathway opens a therapeutic window between cell stress and death. Further, the biosensor can also be used to identify toxic side effects of drugs or lead compounds.

The sensor co‐opts one pathway of the unfolded protein response as the driving mechanism to detect cell stress. Non‐canonical splicing of an intron derived from the XBP1 protein shifts into frame a bright green fluorescent protein, which indicates the onset of cell stress. Importantly, this intron is only spliced upon activation of endoplasmic reticulum stress or the unfolded protein response. Ratioing this signal with a constitutively expressed red fluorescent protein signal allows real‐time monitoring of cells stress and protein production. We have also engineered this biosensor to be reversible, allowing detection of the onset of stress as well as the amelioration of stress. This positions the biosensor to be used for testing compounds that may relieve cell stress.

We applied this assay to examine the toxic side effects of cardiac glycosides and doxorubicin on cardiomyocytes derived from human induced pluripotent stem cells. Drug toxicity was detected within 24 hours of treatment in a dose dependent manner. The cardiac glycosides induced cell stress mediated through either the unfolded protein response or endoplasmic reticulum stress while doxorubicin, which inhibits the XBP1 pathway, mainly displayed cytotoxic effects. We next used this assay to detect cellular stress induced by genetic mutations. Many neurological diseases, such as Retinitis Pigmentosa, Parkinson's and Amyotrophic lateral Sclerosis (ALS), are associated with protein folding or aggregation defects which induce high levels of cellular stress. This live‐cell stress assay is capable of monitoring cellular stress induced by mutations causing Retinitis Pigmentosa (Rhodopsin P23H mutation) and ALS (SOD G86R mutation) in less than 24 hours after mutant gene expression.

Lastly, multiplexing this cell stress assay with other live‐cell assays detecting second messenger signaling revealed cells experiencing either chemically or genetically induced stress had altered basal levels of cytoplasmic Ca2+ and cAMP as well as a blunted Ca2+ signaling response. Together these studies demonstrate the broad utility of this newly developed live‐cell assay for cell stress and toxicity for use in automated screening of drugs and compounds, determination of adverse drug toxicity, and analysis of disease mechanisms.

Support or Funding Information

Supported by ASEE/NSF Small Business Postdoctoral Research Diversity Fellowship Program NSF Award IIP‐1552305

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular dynamics simulations reveal that tyrosine nitration leads to destabilization of CYP2B6 secondary structure
Authors: Wilderman Ross , Lee Choon‐Myung , Morgan Edward
Published: June 2021

Post‐translational modification of proteins is a major mechanism for the control of cellular machinery. Nitric oxide stimulates ubiquitination and proteosomal degradation of CYP2B6 and CYP51, which is hypothesized to be due to tyrosine nitration. Alanine scanning of CYP2B6 residues indicated that Tyr190Ala, Tyr317Ala, and Tyr380Ala partially protected the enzyme from proteasomal degradation. Alignment of CYP2B sequences from the Cytochrome P450 Homepage revealed that residues 190, 317, and 380 are absolutely conserved tyrosines. Other residues that are tyrosine in CYP2B6 have less conservation across the aligned sequences. Molecular dynamics simulations of CYP2B6 wild‐type, Tyr‐to‐Ala mutants, and Tyr‐to‐3‐nitrotyrosine (NIY) mutants were performed. For most mutants, the protein was stable throughout the simulation period. However, CYP2B6 Tyr235Niy simulations collapsed due to the G‐helix unraveling. Simulation of this mutant was successful if the stabilizing mutations Tyr226His and Lys262Arg from the CYP2B6 crystallization construct were introduced into the protein. In general, mutant proteins behaved similarly to wild‐type CYP2B6 as measured by bond angles and lengths. However, mutating CYP2B6 decreased the solvent accessible surface area and volume while increasing the density of the protein across the simulation. While introduction of alanine did not have large effects on protein secondary structure, NIY caused large changes in protein secondary structural elements close to the residue in space. Furthermore, introduction of NIY at any residue decreased the magnitude of residue‐residue interaction correlations within CYP2B6. Overall, tyrosine nitration leads to secondary structure destabilization and compaction of CYP2B6. These results provide insight into effects of CYP2B6 tyrosine nitration that may lead to proteosomal degradation.

Support or Funding Information

NIEHS Grant # 003619

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deficiency of N‐Acetyltransferase Potentiates Isoniazid‐Endobiotics Interactions and Contributes to Isoniazid Hepatotoxicity
Authors: Wang Pengcheng , Li Feng , Lu Jie , Grant Denis M. , Zhong Xiao‐bo , Ma Xiaochao
Published: June 2021

Isoniazid (INH) is a first‐line anti‐tuberculosis drug, but it causes liver injury frequently. The biochemical basis of INH toxicity in the liver remains elusive. We used an untargeted metabolomic approach to explore the effects of INH on endobiotic homeostasis in mouse liver. We found that overdose of INH resulted in accumulation of multiple endobiotics and INH‐endobiotics conjugates, including long‐chain acylcarnitines, INH‐fatty acid amides, heme, nicotinamide adenine dinucleotide (NAD), and INH‐NAD adduct. Furthermore, we illustrated that overdose of INH depleted vitamin B6 in the liver and blocked vitamin B6‐dependent cystathionine degradation. These results indicate INH can disrupt multiple endobiotics metabolic pathways, which are all critical for hepatocellular functions. Acetylation is the major metabolic pathway of INH and it is mediated by N‐acetyltransferase (NAT). Previous reports showed that slow acetylators have a higher risk of hepatotoxicity than rapid acetylators during INH chemotherapy. We used Nat1/2 (−/−) mice to mimic NAT slow metabolizers. We found that INH acetylation was significantly suppressed in Nat1/2(−/−) mice, whereas INH‐endobiotic interactions were significantly increased. These data demonstrate that deficiency of NAT increases the interactions of INH with endobiotics in the liver, which may in part explains the high risks of INH hepatotoxicity in NAT slow metabolizers.

Support or Funding Information

This work was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases (DK090305), the National Institute of Allergy And Infectious Diseases (R01AI131983), and the National Institute of General Medical Sciences (GM118367).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Physiologically based pharmacokinetic modelling of atomoxetine with regard to CYP2D6 genotypes
Authors: LEE SEOK‐YONG , Kim Se‐Hyung , Byeon Ji‐Young , Lee Choong‐Min , Kim Young‐Hoon , Lee Yun Jeong , Jang Choon‐Gon
Published: June 2021

Atomoxetine is a norepinephrine reuptake inhibitor indicated in the treatment of attention‐deficit/hyperactivity disorder. It is primarily metabolised by CYP2D6 to its equipotent metabolite, 4‐hydroxyatomoxetine, which promptly undergoes further glucuronidation to an inactive 4‐HAT‐O‐glucuronide. Clinical trials have shown that decreased CYP2D6 activity leads to substantially elevated atomoxetine exposure and more adverse reactions. The aims of this study were to analyse the pharmacokinetics of atomoxetine and to develop a pharmacologically based pharmacokinetic (PBPK) model of atomoxetine in different CYP2D6 genotypes. A single 20 mg dose of atomoxetine was given to 19 healthy Korean individuals with CYP2D6*wt/*wt (*wt = *1 or *2) or CYP2D6*10/*10 genotype. Based on the results of this pharmacokinetic study, a PBPK model for CYP2D6*wt/*wt individuals was developed. This model was scaled to those with CYP2D6*10/*10 genotype, as well as CYP2D6 poor metabolisers. We validated this model by comparing the achieved pharmacokinetic parameters with diverse results from the literature. The presented PBPK model describes the pharmacokinetics after single and repeated oral atomoxetine doses with regard to CYP2D6 genotype and phenotype. This model could be utilized for identification of appropriate dosages of atomoxetine in patients with reduced CYP2D6 activity to minimize the adverse events, and to enable personalised medicine.

Support or Funding Information

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, & Future Planning (NRF‐2016R1A2B4007381).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mustard analogs of acetylcholine: irreversible partial agonists at Gq‐linked muscarinic receptors
Authors: Ellis John , Elmslie Gwendolynne
Published: June 2021

Mustard analogs of many receptor ligands have been used to irreversibly label or inactivate receptors. In aqueous solution, mustard groups cyclize to form aziridinium ions that closely mimic quaternary amines. Such a reactive intermediate may therefore bind in the same orientation within the same binding pocket of the receptor that accommodates the related reversible ligand. Once oriented, it is capable of alkylating any of a number of nearby amino acid side chains (including those of cysteine, glutamate, aspartate, and others) and might, in principle, induce irreversible activation of the receptor. However, acetylcholine mustard has been reported by several laboratories to possess agonist activity at muscarinic receptors only until alkylation is achieved, at which point the receptor becomes inactivated. Therefore, we were surprised to find that agonist activity persisted after irreversible binding of acetylcholine mustard and its analog, acetylethylcholine mustard, at Gq‐linked muscarinic receptors (M1, M3, and M5) expressed in CHO cells. The cells were preincubated with cyclized agonist mustards and thoroughly washed prior to assay; the observed responses could be prevented by including atropine in the preincubation phase, but were not inhibited by the presence of atropine in the assay phase. The metabolism of inositol phosphates (IP) was enhanced robustly, though to a lesser degree than that induced by acetylcholine. Acetylethylcholine mustard was less efficacious than acetylcholine mustard, especially at the M5 subtype. For both mustards, the order of efficacy was M1>M3>M5. The most interesting results were obtained measuring the receptor‐stimulated release of labeled arachidonic acid (AA) from intact cells. After preincubation as above, the basal AA release was found to be elevated to a small but significant degree; we have previously shown amiodarone to be a positive allosteric modulator (PAM) at these receptors and inclusion of amiodarone in the assay phase dramatically increased the response of cells that had been preincubated with the agonist mustards. Once again, atropine in the assay itself did not prevent this effect of amiodarone, but atropine in the preincubation phase prevented it completely. At the M1 subtype, the well‐known M1‐specific PAM BQCA enhanced the mustard‐induced activity in a manner similar to that observed for amiodarone. These studies confirm that amiodarone, BQCA, and other ligands investigated act at an allosteric site and suggest that receptors that have been alkylated by acetylcholine mustard and acetylethylcholine mustard do adopt active conformations that can be further enhanced by appropriate allosteric ligands.

Support or Funding Information

Supported AG005214 to JE

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Connexin43 Gap Junctions and the Control of Skeletal Remodeling
Authors: Stains Joseph P
Published: June 2021

Osteoblasts, osteoprogenitors, and osteocytes share and amplify signals through connexin43‐containing gap junctions, coordinating their function in response to local and systemic cues. Thus, connexin43 helps orchestrate bone formation and remodeling to maintain cortical bone integrity. Loss or defects in connexin43 lead to alterations in cortical bone quality and skeletal dysplasia. Using in vitro and in vivo models, our data show how connexin43 regulates the differentiation and activity of osteoblasts and development of skeletal elements through the control of signaling pathways that converge on Runx2, a transcriptional regulator of the osteoblast lineage. In addition, our data show how connexin43 can regulate signal transduction cascades by recruiting signaling complexes to the connexin43 C‐terminus, and how loss of this function can recapitulate many of the skeletal phenotypes caused by loss of connexin43 protein in bone. Work with chimeric connexins composed of connexin43 and connexin45, two connexins with opposing biological functions in bone, underscores that both the connexin channel (e.g., pore function) and connexin C‐terminal tail (e.g., signal complexing docking function) act in concert to regulate downstream signaling. Finally, our preliminary data shows that communication of the second messenger, cAMP, through connexin43 gap junctions spatially coordinates bone formation by linking cell populations across surprisingly large distances.

Support or Funding Information

This work was supported by a grant, R01‐AR063631 (JPS), from the National Institutes of Health/National Institute for Arthritis, Musculoskeletal and Skin Diseases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulators of Inflammatory Responses
Authors: Kanneganti Thirumala‐Devi
Published: June 2021

Inflammasomes are multimeric protein complexes that are formed in a cell to orchestrate host defense mechanisms against infectious agents and physiological aberration. Assembly of the inflammasome complex is initiated by nucleotide‐binding domain and leucine‐rich repeat receptors (NLRs) or absent in melanoma 2 (AIM2)‐like receptors (ALRs). NLRs and ALRs mediate host recognition of pathogen‐associated molecular patterns (PAMPs) released during bacterial, viral, fungal, and protozoan infections, or danger‐associated molecular patterns (DAMPs) released during cellular damage. Activated NLRs and ALRs, in most cases, recruit a bipartite protein known as ASC to engage caspase‐1. NLR‐ and ALR‐mediated caspase‐1 activation drives pyroptosis and are largely beneficial to the host during an infection. However, these cytokines induced by endogenous danger signals trigger sterile inflammation, a risk factor for the development of autoinflammatory and metabolic diseases. Therefore, activation of the inflammasome must be finely controlled to avoid overt tissue damage. These regulatory activities are governed by scaffolding proteins and post‐translational modifications, which together, tightly control and modulate inflammasome activation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sounding the Alarm and Putting Out the Fire: New Mechanistic Insights into Inflammatory Cell Death
Authors: Lieberman Judy
Published: June 2021

Inflammasomes are multi‐protein signaling scaffolds that assemble in response to invasive pathogens and sterile danger signals to activate inflammatory caspases, which trigger inflammatory death (pyroptosis) and processing and release of pro‐inflammatory cytokines. Inflammasome activation contributes to many human diseases, including sepsis, the often‐fatal response to systemic infection. We recently found that gasdermin D (GSDMD) causes pyroptosis by forming pores in the plasma membrane after it is cleaved by the inflammatory caspases. The N‐terminal fragment binds to acidic phospholipids on the inner leaflet. GSDMD pores also assemble on bacterial membranes to kill the bacteria that trigger pyroptosis. Cryo‐electron microscopy was used to solve the structure of the pore formed by 27 monomers of the related gasdermin, gasdermin A3. The N‐terminal fragment undergoes radical conformational changes after it binds to lipids to assemble into a 108‐stranded b‐barrel that inserts into the membrane to form the pore. A small molecule screen identified several compounds that inhibit pore formation by covalently binding to a reactive cysteine in GSDMD and also inhibit other steps in inflammasome priming and assembly and caspase activation. One of the inhibitors, disulfiram, which is a drug used to treat alcohol dependence, promoted survival and reduced inflammatory cytokine secretion in LPS‐induced sepsis in mice.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Mitochondrial Metabolism by Reversible Phosphorylation
Authors: Pagliarini David
Published: June 2021

Mitochondria are centers of metabolism whose activities need to be calibrated to meet changing cellular needs. General dysfunction of these organelles is implicated in many common human disorders, including Parkinson's, Alzheimer's, various cancers, metabolic syndrome, type 2 diabetes, obesity, non‐alcoholic fatty liver disease, heart failure, and general metabolic inflexibility, most often through unclear means. Defining the pathogenic mitochondrial alterations that contribute to these metabolic disorders and devising new therapeutic strategies to rectify them represent principal challenges in mitochondrial medicine. A potential contributor to this dysfunction is aberrant intra‐mitochondrial protein phosphorylation—a process recognized as critical for pyruvate dehydrogenase regulation for more than 50 years, but relatively unexplored otherwise. Recent efforts from our laboratories and others have now revealed that mitochondrial proteins are replete with dynamic phosphorylation that changes reproducibly between healthy and diseased states, and that phosphorylation can alter the activities of proteins involved in core metabolic pathways. We have also now connected select phosphorylation events to poorly characterized matrix protein phosphatases, thereby beginning to establish a mechanistic framework for understanding mitochondrial protein phosphorylation and its effects on metabolic processes. Given these emerging findings, we hypothesize that reversible phosphorylation may be widely important in calibrating mitochondrial metabolism, and that its mismanagement could contribute to the pathophysiology of mitochondria‐related disorders. To explore this, we combine mammalian physiology, omics‐level analyses, and classical biochemistry to make definitive connections between mitochondrial phosphatases and their substrates, and to establish a broad framework for understanding the role of this post‐translation modification in calibrating mitochondrial activities.

Support or Funding Information

R01DK098672

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cancer Cell Metabolism: Reexamining the Regulation of Anabolic Growth in Health and Disease
Authors: Thompson Craig B
Published: June 2021

Cancer cells engage in cell‐autonomous growth and proliferation through the reprogramming of cellular metabolism. There is a growing appreciation that many oncogenic mutations direct nutrient uptake and the diversion of intermediate metabolites into anabolic pathways. Much of what we know about anabolic metabolism has depended on studies of cells growing in rich medium. In contrast, an emerging feature of cancer cell metabolism is the ability to acquire necessary nutrients from a frequently nutrient‐poor environment and utilize these nutrients to both maintain viability and build new biomass. The alterations in intracellular metabolites that can accompany cancer‐associated metabolic reprogramming have profound effects on gene expression, cellular differentiation and the tumor microenvironment. As recently proposed (Pavlova and Thompson, 2016), cancer‐associated metabolic changes can be grouped into several distinct hallmark features: (1) deregulated uptake of glucose and amino acids, (2) use of opportunistic modes of nutrient acquisition, (3) use of glycolysis/TCA cycle intermediates for biosynthesis and NADPH production, (4) increased demand for nitrogen, (5) alterations in metabolite‐driven gene regulation, and (6) metabolic interactions with the microenvironment. Unraveling how cancer cells reprogram their metabolism to grow and divide in nutrient‐poor environments is shedding new light on the regulation of anabolic metabolism. These insights are identifying not only new approaches to cancer diagnosis and treatment but also mechanisms that support normal tissue regeneration and repair.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing Structural Interactomics and its Application in Cell Biology
Authors: Liu Fan
Published: June 2021

Proteins are involved in almost all cellular processes. They are organized through extensive networks of interactions, regulating the working mechanisms of the cell with high fidelity and precision. However, characterization of the protein interactome has been an extremely challenging task due to the lack of highly confident and efficient technology. To address this issue, we have developed a high‐throughput approach for comprehensive protein interactome profiling based on cross‐linking mass spectrometry (XL‐MS). We applied this novel XL‐MS strategy to several highly complex samples, including various cell lysates as well as organelles such as the endoplasmic reticulum the mitochondrion. In particular, in organelle XL‐MS allows covalent capturing of the entire interaction network of all membrane proteins in association with their regulatory partners. Thus, it captures a snapshot of functional protein networks in vivo prior to protein extraction under detergent conditions, enabling directly profiling of the native assembly of macromolecular machineries and their cross‐talk.

We have demonstrated the diverse capabilities of XL‐MS in detail by our recent interactome study of mouse heart mitochondria. The obtained mitochondrial interaction network is comprehensive (more than three thousand cross‐links were identified, covering all major mitochondrial membrane protein complexes), confident (cross‐links are validated on existing high‐resolution structures) and specific (cross‐links are highly specific within each mitochondrial sub‐compartment). Remarkably, through thousands of protein‐protein interactions, we elucidated several organization principles of mitochondrial macromolecular assemblies, discovered previously unrecognized protein interactions, and revealed novel mitochondrial proteins.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Mitofusin 2‐Mediated Mitophagy and Compensatory Mitochondrial Biogenesis in the Differentiation of Human Pluripotent Stem Cells
Authors: Krantz Sarah , Kim Young‐Mee , Toth Peter T , Marsboom Glenn , Rehman Jalees
Published: June 2021

Background

Induced pluripotent stem cells have emerged as a critical new tool to model diseases and generate differentiated cells used for regenerative therapies. Stem cells undergo metabolic reprogramming as they transition from a predominately glycolytic state when they are pluripotent towards enhanced mitochondrial oxidative phosphorylation as they differentiate. The GTPase Mitofusin 2 (Mfn2) is a key mediator of mitochondrial dynamics, regulating mitochondrial fusion as well as the autophagic degradation of dysfunctional mitochondria (mitophagy). However, the role of Mfn2 in regulating the bioenergetics of differentiating cells is not fully understood. We hypothesized that Mfn2‐mediated mitophagy and the compensatory biogenesis of new mitochondria are required for the differentiation of pluripotent stem cells.

Methods and Results

Human induced pluripotent stem cells were differentiated towards an endothelial lineage and were analyzed at day 0 (pluripotent), day 2 (mesodermal), and day 7 (differentiated endothelial cells). In the early stages of differentiation, autophagy increased three‐fold as assessed by the ratiometric fluorescent autophagy sensor Keima (p=0.0074) and increased levels of phospho‐Mfn2, a mediator of mitophagy. When Mfn2 was depleted, autophagy decreased 67%, indicating that this was a predominantly Mfn2‐dependent process. Subsequent to mitophagy, mitochondrial mass increased at days four through seven as measured by immunofluorescence and mitochondrial DNA levels. Mitochondrial biogenesis was assessed by the mitochondrial construct MitoTimer which exhibits time dependent shifts in fluorescence. After cells were fully differentiated, biogenesis decreased to approximately half (p=0.008). To determine if this burst of mitophagy and subsequent mitochondrial biogenesis plays a role in the shift in metabolism towards mitochondrial ATP, we quantified the ATP:ADP ratio using the biosensor Perceval HR. We observed that intracellular ATP levels increased seven‐fold with differentiation (p<0.001). However, Mfn2 depletion markedly attenuated this increase (p=0.0056). We also investigated whether mitophagy would regulate cell differentiation in a feedback manner. The mitochondrial phosphatase PGAM5, which is cleaved during mitophagy and modulates the Wnt pathway, was increased after the induction of mitophagy on day 4 of differentiation (p=.04).

Conclusion

Mitophagy and compensatory mitochondrial biogenesis are Mfn2‐mediated adaptive processes which are required for the metabolic reprogramming of differentiating human pluripotent stem cells and can regulate the differentiation process via a novel feedback mechanism.

Support or Funding Information

NIH R01‐HL126516, NIH P01‐HL060678, NIH 5T32HL007829

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondria, metabolism and aging
Authors: Finkel Toren
Published: June 2021

The molecular pathways controlling lifespan are incompletely understood. One strategy to increase the longevity of multiple diverse organisms is through caloric restriction. Evidence suggests that the mTOR pathway might be an important mediator of the effect of calorie restriction. Here, I will describe two mouse models that my lab has generated. The first is a mouse model of reduced mTOR expression that results in an increase in overall lifespan as well as a segmental increase in healthspan. The molecular basis of these effects, i.e. how a reduction in mTOR signaling promotes longevity is not known. One potential pathway, however, is through augmenting autophaic flux. For the second part of my talk, I will describe a second mouse model that allows for a more accurate assessment of in vivo mitophagy, namely the selective removal of damaged mitochondria. This mouse allowed us to quantify how mitophagy changes as a function of age. These results suggest that a decline in autophagic flux might fuel the aging process.

Support or Funding Information

This work was supported by NHLBI Intramural and extramural funds

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thirty Years of Protein Tyrosine Phosphatases – From Housekeeping Enzymes to Therapeutic Targets
Authors: Tonks Nicholas K.
Published: June 2021

Thirty years ago, while working with Eddy Fischer at the UW in Seattle, I succeeded in purifying a protein tyrosine phosphatase (PTP) to homogeneity for the first time. Together with Harry Charbonneau and Ken Walsh, we sequenced the protein, PTP1B, and demonstrated homology with CD45, going on to show that CD45 possessed intrinsic PTP activity. This was important because CD45 is a receptor‐like protein – this observation illustrated the existence of receptor‐like PTPs with the potential to regulate signaling through ligand‐modulated protein tyrosine de‐phosphorylation. Now, we know that PTP1B was the prototype for a large family of such enzymes, comprising ~100 members in humans. Although initially viewed merely as constitutively active housekeeping enzymes, it has now become apparent that the protein phosphatases are critical, specific regulators of signaling in their own right and serve an essential function, in a coordinated manner with the kinases, to determine the response to a physiological stimulus. My lab takes a multidisciplinary approach to characterizing the structure, regulation and function of members of the PTP family of enzymes, with the goal of defining their role in the control of normal and pathophysiological cell function and using them as the basis for identifying novel therapeutic targets and strategies for treatment of major human diseases.

As functional studies have established links to disease, the PTPs have been garnering attention as potential therapeutic targets; however, they remain a largely untapped resource for drug development. As a validated target for diabetes and obesity, as well as HER2‐positive cancer, PTP1B has been the subject of extensive drug discovery efforts. Nevertheless, in following established approaches to develop small molecule drugs that bind to the active site of an enzyme, industry has found PTPs to be challenging therapeutic targets. Although it was possible to generate potent, specific and reversible inhibitors of PTP1B, such molecules were highly charged, due to the chemistry underlying PTP‐mediated catalysis, and thus of limited drug development potential. This led industry to conclude that the members of the PTP family are “undruggable”. Consequently, new approaches are required to exploit this important target effectively and to reinvigorate drug discovery efforts.

In this presentation, I will discuss how a detailed understanding of the structure, regulation and function of PTP1B, generated in an academic setting, is revealing new approaches to the development of small molecule drug candidates. This is also being extended to other members of the PTP family. Continued progress in this area is now leading to the establishment of novel PTP‐based therapeutics for the treatment of major human diseases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mallostery: Ligand‐dependent Misfolding as a Strategy for Protein Regulation
Authors: Wangeline Margaret , Hampton Randolph
Published: June 2021

Protein quality control is an essential set of processes that allow cells to detect, manage, and destroy misfolded and otherwise aberrant proteins. A substantial component of protein quality control involves the selective destruction of misfolded proteins by the ubiquitin proteasome system. Despite the broad range of substrates subject to quality control destruction, these pathways nevertheless show striking specificity for misfolded versions of otherwise stable proteins. This high selectivity can be exploited to regulate the levels of normal proteins through ligand‐mediated, reversible misfolding. This misfolding appears to be a variant of allosteric control that, instead of causing a change in enzyme function, causes a change in enzyme folding to allow recognition and destruction on a contingency basis. This talk will describe an example of such regulated misfolding involved in the control of the sterol synthesizing mevalonate pathway in yeast.

HMG‐CoA Reductase (HMGR) is a rate limiting enzyme of the sterol pathway that undergoes regulated degradation as a mechanism of feedback regulation. In yeast, degradation of the HMGR isozyme Hmg2 is keyed to levels of the 20‐carbon isoprenoid geranylgeranyl pyrophosphate (GGPP). When GGPP levels are high, Hmg2 is efficiently degraded by the HRD pathway, which is a major and highly conserved pathway of quality control in the ER known as ER‐associated degradation (ERAD).

We have shown that GGPP causes reversible misfolding of Hmg2 that promotes HRD dependent degradation. GGPP‐regulated Hmg2 misfolding shows remarkable similarities to allosteric regulation. GGPP action on Hmg2 is reversible and highly potent, occurring in the mid‐nanomolar range. GGPP's effects are highly specific for its structure, and its effects on Hmg2 can be antagonized both in vivo and in vitro by a related molecule. These features point to a high potency, ligand‐mediated effect on Hmg2 structure. Consistent with this, Hmg2 exists as a multimer, and unregulated Hmg2 variants show classic “toxic subunit” behavior on the regulation of wild‐type Hmg2. Finally, these effects are antagonized by chemical chaperones, demonstrating that the transition caused by GGPP is consistent with misfolding. Accordingly we call this type of regulation “mallostery” to include the ideas of misfolding and allostery in a single term.

When viewed through the lens of mallostery, it appears that a number of highly diverse regulatory situations employ similar molecular tactics. Although the generality remains to be discovered, the broadest possibility is for the design of “mallosteric drugs” that specifically program the destruction of desired targets for clinical or basic scientific benefit.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CAMKK2‐dependent AMPK activation is required to drive cell cycle progression and hepatocyte proliferation after partial hepatectomy in the rat
Authors: Juskeviciute Egle , Antony Anil Noronha , Crumm Sara , Hoek Jan
Published: June 2021

Liver regeneration is a unique repair response to tissue damage where normally quiescent liver cells reenter the cell cycle and undergo cell division, regaining the damaged or lost tissue, while maintaining the differentiated function of the tissue. In previous studies, we and others demonstrated that 70% partial hepatectomy (PHx) in the rat leads to an instantaneous (within 30 sec) release of up to 25% of ATP from the remnant liver. Released ATP served to activate purinergic receptors that enhance cellular stress responses and immediate‐early gene expression. ATP loss was mediated at least in part by a transient opening of connexin hemichannels and activation of stress signals and cell cycle progression was inhibited by hemichannel blocking peptides. The low ATP levels persist through the cell cycle progression and tissue ATP levels did not recover until 24–48 hrs post‐PHx. In the current study, we assessed the intracellular signaling events initiated by ATP loss. The decline in tissue ATP levels was accompanied by a persistent increase in ATP/AMP ratio and a transient activation of AMPK in the remnant liver, which peaked within 1 min after PHx, but then declined to basal levels within 15 min, despite the persistence of a low ATP/AMP. A second, extended phase of AMPK activation occurred, which peaked at 12–24 hrs after 70% PHx without being accompanied by major changes in tissue ATP/AMP ratios. This secondary AMPK activation in the regenerating rat liver accompanied cell cycle progression and DNA synthesis through G1‐S phase. After 30% PHx, a model that exhibits the hepatocyte priming events, but does not display the synchronized cell cycle progression and proliferation response, the initial ATP release and signaling events were comparable, but the secondary peak of AMPK activation is absent in the 30% PHx. Treatment with STO‐609, a selective inhibitor of CAMKK2, suppressed the second AMPK activation peak and inhibited hepatocyte proliferation after 70% PHx, suggesting the importance of calcium signaling in the regenerating liver. We conclude that an AMP‐independent CAMKK2‐mediated activation of AMPK is essential for effective cell cycle progression after PHx.

Support or Funding Information

Supported by AA018873

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Dynamics of D‐Amino Acid Oxidase Using Molecular Dynamics Simulations
Authors: Jonsson Amanda L , Kueffer Lauren , Birdsong Garrick
Published: June 2021

The D‐amino acid oxidase (DAAO) enzyme catalyzes the oxidative deamination of D‐amino acids to give ammonia and an α‐keto acid, coupled to the reduction of the cofactor FAD. In humans DAAO plays a key role in modulating the degradation pathway of the important signaling molecule D‐serine in the brain. We ran all‐atom, explicit‐solvent molecular dynamics simulations of the wild‐type human DAAO, several mutations known to increase DAAO activity or FAD binding (D31H, R279A, and W209R), and mutations known to decrease activity or binding (R199Q and R199W). Theses mutations are spread throughout the protein structure, some far from the FAD‐binding site and active site. We aim to better understand how small changes in the sequence can lead to changes in overall activity and binding affinity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An N‐terminal truncated isoform of the ER‐phagy receptor FAM134B (FAM134B‐2) regulates starvation‐induced ER‐to‐lysosome protein degradation
Authors: Miyazaki Shinobu , Kohno Shohei , Shiozaki Yuji , Keenan Audrey , Miyazaki Makoto
Published: June 2021

Autophagy is a conserved system that adapts to nutrient starvation, after which proteins and organelles are degraded to recycle amino acids in response to starvation. Recently, the endoplasmic reticulum (ER) was added to the list of targets of autophagic degradation. Autophagic degradation of the ER (ER‐phagy) is considered a key mechanism in maintaining ER homeostasis. Four ER‐resident proteins (FAM134B, CCPG1, SEC62, and RTN3) have been identified as ER‐phagy cargo receptors, which contain LC3‐interacting regions. In this study we identified an N‐terminal truncated isoform of FAM134B (FAM134B‐2) that contributes to starvation‐induced ER‐phagy. FAM134B‐2 but not full length FAM134B (FAM134B‐1) is expressed in a fed state. Starvation drastically induces FAM134B‐2 but no other ER‐phagy cargo receptors through transcriptional activation by C/EBPβ. C/EBPβ overexpression increases FAM134B‐2 recruitment into autophagosomes and lysosomal degradation. In addition, FAM134B‐2 is preferentially localized in the smooth ER. This study demonstrates that the C/EBPβ‐FAM134B‐2 axis regulates starvation‐induced smooth ER‐phagy.

Support or Funding Information

R01DK096030, R01HL117062, R01HL133545 and R01 HL132318.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nonspecific Electrostatic Interactions between Tau and 14‐3‐3ζ Promote Tau Aggregating
Authors: Xiong Junwen , Gao Meng , Huang Yongqi
Published: June 2021

Aberrant accumulation of microtubule associated protein tau and formation of amyloid fibrils are pathological hallmarks of neurodegenerative tauopathies. Previous studies have suggested that the scaffolding protein 14‐3‐3ζ promotes tau aggregating by interacting specifically with the pSer324 14‐3‐3ζ‐binding motif within the third microtubule binding repeat. Therefore, the tau/14‐3‐3ζ interaction is a promising drug target for Alzheimer's disease treatment. Indeed, inhibitors of 14‐3‐3/tau interaction have been found to reduce binding of PKA‐phosphorylated tau protein to 14‐3‐3ζ. However, 14‐3‐3ζ has also been shown to promote aggregation of unphosphorylated tau protein, suggesting that nonspecific interactions between tau and 14‐3‐3ζ may be also involved in tau aggregating. In this work, the interactions between tau K18 construct and 14‐3‐3ζ were investigated. We compared the aggregating propensity for three tau species (i.e. WT, pS324, and S324A) in the presence or absence of 14‐3‐3ζ. Although phosphorylation at Ser324 increased the binding affinity of tau K18 to 14‐3‐3ζ, phosphorylation only showed marginal effect on 14‐3‐3ζ mediated tau aggregating. Furthermore, WT and S324A tau proteins showed similar aggregating propensity in the presence of 14‐3‐3ζ. Although 14‐3‐3ζ specific inhibitors reduced the binding affinity of pS324 tau with 14‐3‐3ζ, NMR spectroscopy showed that nonspecific interactions between pS324 tau and 14‐3‐3ζ remained when 14‐3‐3ζ specific inhibitors were present. Our results suggest that nonspecific interactions between tau and 14‐3‐3ζ play an important role in the 14‐3‐3ζ mediated tau aggregating process.

Support or Funding Information

This work was supported by National Natural Science Foundation of China (Grant number: 21603121) and Hubei University of Technology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Basis of ClpP Protease Activation by Small Molecules
Authors: Houry Walid A
Published: June 2021

Bacterial ClpP is a highly conserved, self‐compartmentalizing, cylindrical serine protease required for maintaining cellular proteostasis. Acyldepsipeptides (ADEPs) and activators of self‐compartmentalized proteases (ACPs) are small molecules that cause the dysregulation and activation of ClpP, which leads to bacterial cell death, highlighting their potential use as novel antibiotics. However, the molecular basis of ClpP activation by small molecules remains undetermined. Structural changes in ClpP from Neisseria meningitidis and Escherichia coli upon binding to novel ACP and ADEP analogs were probed by X‐ray crystallography, methyl‐TROSY NMR, hydrogen‐deuterium exchange, and small angle X‐ray scattering. ACP and ADEP were found to induce distinct conformational changes in the tertiary structure of ClpP. However, ordered N‐terminal axial loops, reorganization of the hydrogen bonding network at the entrance pore of ClpP, and reduced structural heterogeneity of the ClpP cylinder are common features of activator‐bound complexes. Activated ClpP mutants were obtained that recapitulate these effects. Based on our data, we propose a unified mechanism for compound‐based ClpP activation.

Support or Funding Information

Canadian Institutes of Health Research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular regulation of lysine trimethylation by the P. aeruginosa methyltransferase EftM
Authors: Dey Debayan , Kuiper Emily G , Goldberg Joanna B , Conn Graeme L
Published: June 2021

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of severe life‐threatening lung and other infections in individuals with cystic fibrosis, compromised immune systems or severe burns. P. aeruginosa is also intrinsically resistant to many antibiotics making infections difficult to treat once established. We previously showed that the S‐adenosyl‐L‐methionine‐dependent methyltransferase EftM is responsible for trimethylation of lysine 5 of the translation factor EF‐Tu and that this modification enhances P. aeruginosa adhesion to host epithelial cells. Additionally, methyltransferase activity, and thus EF‐Tu modification, is temperature regulated via the intrinsic thermolability of EftM. Understanding EftM's complete mechanism of action may reveal new strategies for developing effective therapeutics targeting this pathogen. As the next step in this process, we now aim to define the substrate recognition and catalytic mechanisms of EftM. A phylogenetic reconstruction of the larger EftM family was generated to identify conservation patterns of putative key residues involved in interaction with EF‐Tu and facilitating catalysis. This analysis was supported by generation of an EftM homology model and protein‐protein docking studies to propose a EftM:EF‐Tu binding model. This model and phylogentic insights were used to guide site‐directed mutagenesis of residues in EftM and EF‐Tu to test their role in protein‐protein recognition using methyltransferase activity and binding assays. Finally, to begin investigating the role of protein dynamics and potential allosteric effects of SAM cosubstrate binding, other computational methods such as normal mode analysis were used to propose a coarse‐grain model of dynamics of EftM in parallel with experimental hydrogen‐deuterium exchange coupled with mass spectrometry (HDX‐MS) analysis of EftM and its complexes with SAM and EF‐Tu. In conclusion, our results provide the first structural insight into the EftM:EF‐Tu recognition, which paves the way for further understanding EftM's substrate recognition and catalytic mechanisms.

Support or Funding Information

Cystic Fibrosis Foundation Postdoctoral Research Fellowship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.