bluuebunny/crossref_metadata_2025_split huggingface dataset:
Deciphering TRAPP complex function in yeast
Authors: Joiner Aaron , Fromme Chris 
Published: June 2021

All eukaryotes contain organelles that perform specialized functions essential for life. The Golgi complex, an organelle that is important for transport of cellular material, serves as the “Grand Central Station” of the cell by receiving, then sorting proteins and membranes to various locations. Many steps of vesicular trafficking at the Golgi complex are regulated by several families of small GTPases, which modulate between an active and inactive state. One particularly interesting GTPase is Rab1, which also has an established role in autophagy. Rab1 is activated by a guanine nucleotide exchange factor (GEF), a process that is conserved from mammals to yeast. Discordantly, mammals contain two protein complexes named TRAPPII and TRAPPIII (transport protein particle) which activate Rab1; while in yeast, four (TRAPPI ‐ TRAPPIV) have been implicated as GEFs in Ypt1 (Rab1 homolog) activation. Yeast TRAPP complexes share a core set of six subunits. Additional subunits are thought to regulate the complex by adding substrate and compartmental specificity. TRAPPII contains four more subunits, while TRAPPIII and TRAPPIV include one additional subunit. Importantly, the precise functions of the accessory subunits remain unclear. We demonstrate that TRAPPII and TRAPPIII are the only two complexes present in yeast cells, which is consistent with findings in mammalian cells, by quantifying isolated TRAPP complexes and simultaneously monitoring the localization of all TRAPP complexes in vivo. We also find that, using an in vitro fluorescent nucleotide exchange assay, TRAPPIII activates Ypt1 more effectively than the other complexes. Concurrently, we notice that Trs85 localizes to the Golgi complex and that Ypt1 is mislocalized in vivo in the absence of the TRAPPIII‐specific subunit, Trs85. Additionally, using an in vitro membrane binding assay, we find that Trs85 has an affinity for membranes. These findings suggest that there are only two TRAPP complexes in yeast, and that TRAPPIII is involved in trafficking at the Golgi complex in addition to its established role in autophagy. Taken together, our model suggests that the main role of Trs85 in the TRAPPIII complex is to bring the catalytic core close to a membrane surface, localizing the active site to the right compartment in an orientation that facilitates localization and activation of Ypt1.Support or Funding InformationThis material is based upon work supported by the NSF GRFP under Grant DGE‐1144153, by NIH/NIGMS Grant R01GM116942, and by the Alfred P. Sloan Foundation. Any opinions, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF, NIH, the Sloan Foundation or their trustees, officers, or staff.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PML‐Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2‐Nrf2 in the Nucleus of Single Live Cells
Authors: Burroughs Andrea Flores , Eluhu Sylvia , Whalen Diva , Goodwin J. Shawn , Sakwe Amos M. , Arinze Ifeanyi J. 
Published: June 2021

Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a basic leucine‐zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1‐Cullin3/RING box 1 E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2‐Nrf2 in single live cells. Using this approach, we show that the half‐life (t1/2) of Dendra2‐Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG‐132 or induction of oxidative stress with tert‐butylhydroquinone (tBHQ) extended the half‐life of Dendra2‐Nrf2 by 6‐ and 28‐fold, respectively. By inhibiting nuclear export using leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML‐NBs. We further demonstrate that co‐expression of Dendra2‐Nrf2 and Crimson‐PML‐I lacking two PML‐I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2‐Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Altogether, these data suggest that functional PML‐NBs alter the rate of degradation of nuclear Nrf2, which underscores the importance of these structures in Nrf2‐mediated response to oxidative stress.Support or Funding InformationHHS |National Institutes of Health (NIH):, SC1CA143985; HHS |National Institutes of Health (NIH):, SC1CA211030; HHS |National Institutes of Health (NIH):, UL1TR000445; HHS |National Institutes of Health (NIH):, U54MD007593; HHS |National Institutes of Health (NIH):, G12MD007586; HHS |National Institutes of Health (NIH):, U54CA163069; HHS |National Institutes of Health (NIH):, R24DA036420; HHS |National Institutes of Health (NIH):, S10RR0254970; HHS |National Institutes of Health (NIH):, 5R25GM059994; HHS |National Institutes of Health (NIH):, 5T32GM007628‐37This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of GIV‐GRP78 Interaction During Endoplasmic Reticulum Stress
Authors: Limso Clariss , Ngo Jordan , Nguyen Peter , Bhandari Deepali 
Published: June 2021

Endoplasmic Reticulum (ER) stress occurs when misfolded proteins accumulate in the ER. Cells facing ER stress initially attempt to restore cellular homeostasis, but if the stress becomes chronic and homeostasis is not achieved within a reasonable timeframe, cells initiate programmed cell death. Cancer cells are able to withstand and survive ER stress better than regular cells. Overexpression of the chaperone Glucose Regulated Protein 78 kDa (GRP78) is one of the key factors that promote cancer cell survival during ER stress. Although it is an ER resident protein, recent studies have shown that when overexpressed, GRP78 can translocate to other cellular locations including the cell surface from where it can activate cytoprotective signals; however, the mechanism by which it does so remains elusive. Our laboratory has recently identified Gα‐Interacting Vesicle associated protein (GIV) ‐ a known enhancer of the pro‐survival Akt signaling ‐ as a novel binding partner of GRP78. We hypothesize that this interaction may be the missing link in understanding the mechanism by which cell surface GRP78 increases cell viability. Because GIV and GRP78 are normally localized in different cellular compartments, we characterized the spatiotemporal dynamics of this interaction by analyzing the sub‐cellular localization of these proteins in HeLa cells treated with Tunicamycin (an ER stress inducer) through differential centrifugation and immunofluorescence microscopy. Our results showed that GIV is present in both membrane and cytosolic fractions during normal as well as ER stress conditions. GRP78, on the other hand, is in the membrane fraction under normal cellular conditions but appears in the cytosolic fraction upon ER stress. GIV and GRP78 co‐localized in a perinuclear region as well as at the cell surface of the ER stressed cells. Finally, we also mapped the interaction between GIV and GRP78 to the carboxyl terminus domains on both proteins by performing pull‐down assays. Taken together, this work has characterized GIV‐GRP78 interaction in the face of ER stress. Our current and future goals include studying the role of this novel interaction in mediating cell survival during chronic ER stress.Support or Funding InformationThis project is supported by the NIH‐NIGMS grant # SC2GM121246 (DB). JN is supported by the NIH MARC program award number: T34GM008074.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Dysfunction in Type 2 Diabetic Skeletal Muscle Cells
Authors: Castaneda Diana L. , Yamazaki Katrina 
Published: June 2021

Type 2 diabetes (T2D) is an epidemic disease characterized by high blood glucose levels and insulin resistance and is usually associated with obesity. Inadequate glucose utilization by the cell leads to an increase in high fat utilization to make cellular energy, which can lead to mitochondrial dysfunction. Typically, autophagy maintains homeostasis by removing damaged portions of the mitochondria and recycling the functional components to form new efficient mitochondria. The main focus of this project is to analyze if there is an impairment of the autophagy process in T2D that leads to an accumulation of dysfunctional mitochondria. We also evaluated mitochondrial dynamics to determine if the rate of mitochondrial fission exceeds that of fusion, as this may also explain the accumulation. Using an in vitro model of T2D, fluorescent microscopy and biochemical analysis of cells treated with high fats were evaluated to look at mitochondrial dynamics and number. Microscopic analysis showed a decrease in mitochondrial number and a decrease in mitochondrial membrane potential in T2D cells versus control. Furthermore, results revealed that in a diabetic environment there was an increase in mitochondrial fragmentation as seen with an increase in DRP‐1 expression. Lastly, results showed that Beclin‐1 increases under diabetic conditions signifying that autophagy is still being activated. In conclusion, our results demonstrate that there is an increase in mitochondrial fission and activation of autophagy in T2D. More work is needed to determine if fission occurs at a rate faster than the normal autophagy response can handle to explain the consequences of T2D.Support or Funding InformationCalifornia State University, Los Angeles Louis Stokes Alliance for Minority Participation (CSULA‐ LSAMP)andCalifornia State University, Los Angeles Minority Opportunities in Research Programs‐ Minority Biomedical Research Support Program‐Research Initiative for Scientific Enhancement (CSULA MORE‐ RISE Undergraduate)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Potential mitochondrial dysfunction in skeletal muscle of mouse models of Osteogenesis imperfecta.
Authors: Gremminger Victoria , Jeong Youngjae , Cunningham Rory , Meers Grace , Rector R. Scott , Phillips Charlotte 
Published: June 2021

Osteogenesis imperfecta (OI), also referred to as brittle bone disease, is a heritable connective tissue disorder characterized by short stature, fragile bones, craniofacial deformity and muscle weakness. OI is most often the result of autosomal dominant gene defects in the type I collagen genes: COL1A1 and COL1A2. The remaining rare autosomal recessive forms result from defects in gene products responsible for modification and packaging of type I collagen. Muscle weakness not only impacts a patient's quality of life, but likely negatively affects bone health via altered bone‐muscle crosstalk.The oim/oim mouse models severe human type III OI with reduced body mass, skeletal fragility, and reduced muscle mass and function compared to their wildtype (WT) counterparts. Whereas, the G610C OI mouse models the less severe human type I/IV OI with skeletal weakness, but near normal muscle function. The differential presence of inherent muscle pathology between oim/oim and G610C mice suggest mutation specific mechanisms.To investigate the mechanisms underlying the muscle pathology, we examined citrate synthase activity, a known marker of mitochondrial activity and determined, in the mixed fiber type gastrocnemius muscle, that citrate synthase activity was significantly increased only in oim/oim mice relative to WT mice, despite evidence of reduced muscle function; whereas G610C mice did not have altered citrate synthase activity. The unexpected increase in citrate synthase activity led to the question of whether or not mitochondria play a role in the muscle weakness observed in OI. To assess mitochondrial health, we examined biomarkers of mitogenesis and mitophagy, and found increased mitogenesis in oim/oim mice relative to WT as determined by PGC1α and TFAM protein content in the mixed gastrocnemius muscle. Additionally, measurement of mitochondrial electron transport complex proteins also suggest altered mitochondria in the oim/oim mice. These data suggest that oim/oim mice have increased mitogenesis and altered mitochondrial function accompanying decreased muscle function.Support or Funding InformationSupported by March of Dimes, National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (RO1 AR055907), the Leda J. Sears Trust, the Kansas City Area Life Sciences Institute Patton Trust Research, Inc., University of Missouri Research Board Grant, and in part by the Department of Veterans Affairs (VA‐Merit Grant I01BX003271‐01). A portion of this work was also supported by resources and the use of facilities at the Harry S. Truman Memorial Veterans Hospital in Columbia, MO.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dual Perturbation of Electron Transport Chain (ETC) Complex and ATP Synthase Triggers PINK1/Parkin‐dependent Mitophagy
Authors: Ramirez Adrian Thomas , Liu Xuedong 
Published: June 2021

Neurodegenerative disorders like Parkinson's Disease arise in part due to accumulation of unhealthy mitochondria. PINK1 and Parkin, found to be deficient in some Familial Parkinson's Disease patients, are known to orchestrate mitochondrial quality control responses like mitophagy. Research into PINK1 and Parkin function has been focused primarily on downstream signaling events, but the exact type of damage signals originating from defected mitochondria to trigger PINK1 stabilization and subsequent Parkin recruitment to the outer membrane of damaged mitochondria remains elusive. We propose PINK1 and Parkin act in tandem to monitor the integrity and activity of ETC complexes and ATP synthase to ensure robust energy production. To test this hypothesis, we profiled several small molecule inhibitors with well‐documented inhibitory activities towards ETC complexes for their effects on PINK1 and Parkin activation. We found that while dual inhibition of complex III and V results in robust Parkin recruitment to damaged mitochondria and mitophagy responses, dual inhibition of complex I and V has only modest effect and other combinations have no effect. PINK1 and Parkin activation as a result of dual assault of ETC complexes can be phenocopied with perturbations of these complexes with siRNA. Thus, our study supports the idea that PINK1 and Parkin are guardians of ETC quality and status. Damaged mitochondria as a result of specific defects in oxidative phosphorylation can be cleared by PINK1 and Parkin dependent mitophagy. This mechanism could be relevant in understanding their dysregulation in Parkinson's Disease.Support or Funding InformationNational Institute of General Medical Sciences R01 GM113141, National Institute of General Medical Sciences T32GM08759, S10 RR026680This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding Specificity of Glycosaminoglycan Interactions with Proteins
Authors: Desai Umesh R. , Patel Bhaumik B. 
Published: June 2021

Glycosaminoglycans (GAGs) play key roles in virtually all biologic responses through their interaction with proteins. Generally, GAGs are believed to interact non‐specifically with proteins. The primary reason for this belief is the highly electrostatic nature of their interactions and the fact that only one homogeneous GAG sequence has reached the clinic – the anticoagulant heparin pentasaccharide sequence. Are highly specific GAG–protein interactions so rare? Does nature rely primarily on non‐specificity to mediate the biological roles of GAGs? These questions are of major significance and insights into specificity of GAG–protein interactions may lead to many more GAG sequences reaching the clinic. Yet, a major challenge in understanding these interactions is the massive structural complexity of GAGs. Computational approaches are extremely useful in navigating this challenge and, in some cases, the only avenue to gain comprehensive insight. Computationally, most basic and many advanced questions on GAG–protein interactions can be answered including 1) does my protein bind to GAGs?, 2) where does the GAG bind?, 3) does the protein preferentially recognize a particular GAG type?, 4) what is the most optimal GAG chain length?, 5) what is the structure of the most favored GAG sequence?, 6) is the GAG–protein system ‘specific’, ‘non‐specific’, or a combination of both?, etc. Recent advances in the areas of thrombosis and cancer from our laboratories have led to discoveries of GAG sequences that are highly specific for their protein targets. The results raise the promise that many proteins may recognize GAGs with high specificity and clinically relevant GAG sequences are waiting to be identified.Support or Funding InformationP01 HL107152R01 HL090586R25 HL128639This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical Characterization of Missense Mutations in O‐GlcNAc Transferase Found in Patients With X‐Linked Intellectual Disability
Authors: George Stephan Nicholas , Selvan Nithya , Stephen Hannah , Wells Lance 
Published: June 2021

O‐GlcNAc is an abundant post‐translational modification of nucleocytoplasmic proteins that has been implicated in biological processes such as transcription, cell‐cycle regulation, and nutrient‐sensing. O‐GlcNAc transferase (OGT) catalyzes the reversible transfer of the monosaccharide N‐acetylglucosamine (GlcNAc) from the donor substrate UDP‐GlcNAc to serine and threonine residues on nuclear, cytosolic, and mitochondrial substrates, while the enzyme O‐GlcNAcase (OGA) reverses this process. OGT consists of a tetratricopeptide repeat (TPR) region and catalytic domains, both of which are required for substrate glycosylation. Through exome sequencing of multiple patient samples, 5 individual missense mutations in the TPR region of OGT appear causal for X‐linked intellectual disability (XLID). However, the effect of each of these mutations on enzyme characteristics is poorly understood. To elucidate the mechanism through which each mutation causes development of XLID, the recombinant, purified OGT variants are being biochemically characterized. We are currently in the process of defining the differential interactome/O‐GlcNAc proteome of OGT variants using quantitative glycoproteomic approaches and performing functional assays to identify processes linking OGT to XLID.Support or Funding InformationThis work is funded by grants from the Keck Foundation and the NIH (P41GM103490) to Dr. Lance Wells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mapping astrocytes to understand development and function of the hindbrain
Authors: Mostafa Hasnaa R. , Czeisler Catherine , Otero Jose J. , Aljuhani Mona 
Published: June 2021

Astrocytes, the most abundant glial cells in the mammalian brain, are critical regulators of brain development and physiology through dynamic and often bidirectional interactions with neuronal synapses. The embryonic origin of astrocytes is known to play a major role in defining the functional diversity of astrocytes. Astrogliogenesis is known to temporally follow neurogenesis and neural migration in the forebrain. However, recent studies have demonstrated that the differentiation and distribution of astrocytes may vary between different brain regions. The molecular mechanisms by which astrocytes are specified, interact, and grow in order to shape their complex morphologies are still unknown. A first step in understanding this process is to address how astrocytes develop using lineage markers, including PHOX2B, NKX2.2, OLIG3, and ATOH1, in the murine hindbrain. Characterizing the development of astrocytes will significantly improve the understanding astrocytic contributions to neural function. Furthermore, given their multifaceted quality, it is imperative to attribute astrocytic dysfunction to a wide range of disease states, such as neurodegenerative disorders.Support or Funding Information3R01hl132355‐01a1s1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.