bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationR01DK098672This 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

BackgroundInduced 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 ResultsHuman 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).ConclusionMitophagy 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 InformationNIH R01‐HL126516, NIH P01‐HL060678, NIH 5T32HL007829This 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 InformationThis work was supported by NHLBI Intramural and extramural fundsThis 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.