bluuebunny/crossref_metadata_2025 huggingface dataset:
Three‐Dimensional Trunk Kinematics of Humans and Chimpanzees: New Insights on Early Hominin Bipedalism
Authors: Thompson Nathan E , Demes Brigitte , O'Neill Matthew C
Published: November 2023

The human trunk is involved in a variety of locomotor tasks that promote stability and efficiency during locomotion. The trunk and upper body must create a stable base for the head, yet must also move enough to cancel out angular momentum of the swinging lower limbs and to help shift the center of mass (CoM) laterally towards the stance‐side foot. These abilities are likely achieved in part by the derived morphology of the human trunk. Humans possess a tall and narrow trunk compared to the more squat trunk morphology of our closest living relatives (great apes).

Because of some similarities between early hominins and chimpanzees in trunk morphology (thoracic shape in Australopithecus) and lower limb features that may affect trunk motion (potentially wide step widths in Ardipithecus), the role of the upper body during locomotion in our early ancestors remains unclear. Here, we investigated three‐dimensional trunk motion in chimpanzees and humans in order to determine the effects of overall trunk morphology on upper body stability during bipedal locomotion.

The trunk was divided into three anatomical segments (thorax, lumbar region, and pelvis). In the transverse plane, all chimpanzee trunk segments rotated in the same direction at the same time (in phase), whereas in humans, the thorax rotated out of phase with the pelvis. However, relative to the pelvis, both humans and chimpanzees achieved the same amount of thoracic (17.1° and 19.1° respectively, n.s.) and lumbar rotations (6.1° and 6.5°, n.s.). In the frontal plane, chimpanzees laterally flexed (listed) all three trunk segments (13–22°) towards the support side limb, whereas in humans only the thorax listed (3–4°) towards the support side. The greater thoracic listing in chimpanzees may be necessary to compensate for their wider step width, absent a valgus knee. Indeed, in chimpanzees, the angular excursion of the thorax (beyond that which is due to list of the pelvis alone) is correlated with increased lateral displacement of the CoM (P<0.01), which is in turn correlated with increased mediolateral mechanical work (P<0.001).

The results of this research suggest that some important aspects of bipedal locomotion were already present in the trunks of the last common ancestor (LCA) of humans and chimpanzees. Regardless of the precise reconstruction of the trunk, the LCA was likely already capable of using its thorax to compensate for some degree of rotation of the pelvis. However, if early hominins utilized wide step widths (as has been suggested for Ardipithecus ramidus), there would have likely been more active listing of the thorax in order to shift the CoM over the support side foot. This, in turn, would have likely been accompanied by an increase in mediolateral mechanical work.

Support or Funding Information

This research was funded by the NSF BSC 0935321 and The Leakey Foundation.
Identification of a Mitochondrial Oxodicarboxylate Carrier in the Oleaginous Yeast Yarrowia lipolytica
Authors: Trotter Pamela J. , Juco Karen , McGreer Samantha , Orsi Natalie , Pierson Paige N. , Savage Nowlan , Tamayo Lizeth , Nicaud Jean‐Marc
Published: November 2023

In Saccharomyces cerevisiae, the mitochondrial oxodicarboxylate carriers, encoded by the scODC1 and scODC2 genes, are necessary for effective glutamate biosynthesis and fatty acid utilization. Yarrowia lipolytica, an oleaginous yeast, is able to efficiently degrade as well as accumulate large amounts of lipid substrates. The genome of Y. lipolytica encodes one homolog, the YALI0D02629 gene (denoted ylODC1/2), which displays 60–64% identity to the scODC1 and scODC2 genes. The ylODC1/2 coding sequence was amplified from genomic DNA by polymerase chain reaction and cloned into a S. cerevisiae expression vector under control of the Gal1 promoter. When transformed with the ylODC1/2‐containing plasmid, the inability of the odc1Δodc2Δ mutant of S. cerevisiae to grow on fatty acid medium is corrected, indicating that the Y. lipolytica protein possesses a similar function. The gene was disrupted in Y. lipolytica by insertion of the ylURA3 gene via homologous recombination in a strain lacking the heterologous recombination capability normally found in this organism. Initial growth analysis has revealed no fatty acid growth impairment in the new mutant, suggesting the presence of redundant transport functions, as was observed in S. cerevisiae. Examination of the effects of the ylODC1/2 gene disruption on glutamate and fatty acid metabolism will allow further assessment of its cellular function in Y. lipolytica.
The Function of the Iron–Sulfur Cluster Assembly Machinery is involved in Electron Transport Chain Activity and Oxidative Stress in Saccharomyces cerevisiae
Authors: GOMEZ‐GALLARDO MAURICIO , SANCHEZ‐BRIONES LUIS A , DIAZ‐PEREZ ALMA L , LOPEZ‐ALVAREZ ARNOLDO , CORTES‐ROJO CHRISTIAN , AGUILAR‐MENDEZ OSVALDO , SAAVEDRA‐MOLINA ALFREDO , CAMPOS‐GARCIA JESUS
Published: November 2023

For their function, many proteins depend on cofactor binding to specific sequence motifs. Fe‐S clusters are conserved and versatile inorganic cofactors that participate in a variety of processes and functions. In eukaryotes, biogenesis and recycling of Fe‐S clusters play important roles in the mechanisms of iron homeostasis involved in mitochondrial function. These Fe‐S clusters are assembled into apoproteins by the iron–sulfur cluster machinery (ISC). The present study aimed to determine the effects of mutations in the ISC genes on mitochondrial functionality and iron homeostasis under oxidative stress in S. cerevisiae BY4741 and its KanMX4 interruption ISC genes mutants: ssq1Δ, grx5Δ and isa1Δ. Intracellular ROS, Fe2+ and mitochondrial membrane potential in yeast cultures were determined using oxidant‐sensitive, cell‐permeant fluorescent probes (DHE, PGFL and Rho123). Fluorescence was quantified by flow cytometry, viewed and colocalized by confocal microscopy. Mitochondria were isolated and permeabilized for spectrophotometric determination of respiratory complexes activities. Oxygen consumption rate (OCR) was measured with a Clark‐type oxygen electrode coupled to a biological oxygen monitor. Electron transport chain (ETC) supercomplexes were identified by BN‐PAGE and Raman spectroscopy was performed to detect mitochondrial Fe‐S content. The results indicate that ROS generation caused by oxidizing agents like menadione is increased by ISC system dysfunction, due to loss of iron homeostasis (i.e. augmented free Fe2+), suggesting that ROS and Fe2+ from Fe‐S cluster proteins react creating a strong oxidative status in mitochondria. This was associated to an impairment on the activity of the complexes II, III and IV from the ETC in the ISC mutants. Indeed, increased oxygen generation instead consumption was observed in SSQ1 and ISA1 mutants suggesting a massive generation of ROS and its detoxification by superoxide dismutase and catalase. In mitochondria from SSQ1 and ISA1 mutants, the content of [Fe–S] centers was decreased along with III2IV2 respiratory supercomplex formation, but not in the iron‐deficient ATX1 (cytosolic copper metallochaperone) and MRS4 (mitochondrial iron transporter) mutants, used as controls. In conclusion these results indicate that the ISC system is important in iron‐homeostasis, ROS stress and in the assembly of the supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.
Free fatty acid activates MMP‐1 and MMP‐3 through PPAR‐γ mitigation
Authors: Tsai Wei‐Ming , Tsai Kun‐Ling
Published: November 2023

Obesity is positively related with the prevalence of coronary arterial disease (CAD). It is well known about the plasma concentrations of free fatty acid (FFA) are up‐regulated in cases with obesity. Oleic acid (OA) is known as the most abundant monounsaturated fatty acid in human circulatory system. Several pro‐atherosclerotic responses of OA had been established. Sirtuin 1 (SIRT1) acts a key role in regulation of the normal physical function in smooth muscle cells (SMCs). SIRT1 activation is thought as a novel approach to delay the progression of atherosclerotic injuries. However, the mechanism is still unclear as to whether OA affects SIRT1 expression and activity in SMCs. This study was designed to validate the hypothesis that OA treatment mitigates the expression level of SITR1 and its down‐stream genes, which is a negative regulator of NF‐κB pathway. We confirmed that OA treatment represses SIRT1 and PPAR‐γ levels in SMCs. Moreover, OA enhances transforming growth factor‐β1 (TGF‐b1) release by activation of NF‐κB. OA causes NO production by inducible nitric oxide synthase (iNOS) overexpression, thereby promoting the secretion of matrix metalloproteinases‐1 (MMP‐1) and matrix metalloproteinases‐3 (MMP‐3). Taken together, we suggested OA enhances MMPs activation by SIRT1 down‐regulation. MMPs activation had been shown to positively link with the atherosclerotic plaque generation, therefore, our findings might provide a novel route for developing new therapeutic treatments for FFAs‐related CAD.

Support or Funding Information

MOST‐103‐2320‐B‐006‐050
Integrated Analysis of Posttranslational Modification Proteomes Reveals Interactions and Cell Cycle Regulatory Patterns in Toxoplasma gondii
Authors: Silmon de Monerri Natalie C , Yakubu Rama R , Weiss Louis M , Kim Kami
Published: November 2023

Toxoplasma gondii is a highly successful protozoan parasite that senses and responds to stimuli by altering morphology at different life cycle stages. It is able to replicate inside nearly any type of nucleated cell, and therefore encounters different nutritional availabilities and environments that it must respond to. Given the role of protein posttranslational modifications (PTM) in signaling and sensing in eukaryotes, PTM are likely to play an important role in regulating how T. gondii responds to stimuli and makes life cycle stage transitions. Recent studies have mapped PTM proteomes in T. gondii, including phosphorylation, succinylation, palmitoylation and acetylation. Here, we aimed to gain a global understanding of the function of PTM in T. gondii, using published datasets and new unpublished datasets for ubiquitination, arginine methylation and O‐GlcNAcylation. By performing enrichment analysis using predefined gene sets, we identified pathways and compartments targeted by PTM in this organism. We show that many PTM in T. gondii occur on cell cycle‐regulated proteins whose expression peaks at specific time points during the cell cycle, representing checkpoint control points. Ubiquitination, phosphorylation and palmitoylation are also enriched in proteins upregulated at a critical time point in the transition between S and M phase. To understand how multiple PTM coregulate proteins in T. gondii, we used hypergeometric testing to score interactions between proteomes and interrogate the nature of their interactions. Surprisingly, we found a pool of proteins that are coregulated by both phosphorylation and arginine methylation, representing a novel interaction between these two modifications. A significant proportion of proteins are also coregulated by ubiquitination and palmitoylation. In current work, using protein interaction network analysis of PTM‐modified proteins, we aim to identify protein complex “hubs” that are regulated by multiple PTMs. Together, these analyses aim to detect pathways and hubs modified by PTM in parasites to sense and respond to stimuli.

Support or Funding Information

NIH grants AI93220 (L.M.W.), AI092801 (K.K.), and AI087625 (K.K.)
Nm23‐mediated hnRNPA2/B1 to regulate Sp1 expression is associated with lung cancer progression
Authors: HUNG Jan‐Jong
Published: November 2023

Recent studies have indicated that decrease in Sp1 level is benefit to the lung cancer malignancy. In this study, we found that Nm23 not only increased phosphorylation of Sp1 at Thr739 to enhance Sp1 protein stability but also formed a complex with hnRNPA2/B1 that was recruited to the 5′‐UTR of Sp1 mRNA. Knocking down Nm23 or hnRNPA2/B1 decreased Sp1 expression through inhibiting cap‐independent translation manner and also increased the migratory activity of lung cancer cell lines. Furthermore, lung cancer patients with poor prognosis had low levels of Sp1 and Nm23, suggesting association between Nm23/Sp1 levels and survival rate. Studies performed to elucidate the mechanism underlying this relationship indicated that a decrease in Nm23 levels inducing lung cancer cells malignancy and inhibited hnRNPA2/B1 protein stability, and thus subsequently decreased the recruitment of hnRNPA2/B1 to the 5′‐UTR of Sp1 mRNA, repressing Sp1 expression through inhibition of the cap‐independent transcriptional activity. Overexpression of Sp1 abolished hnRNPA2/B1‐induced F‐actin polymerization and upregulation of epithelial‐mesenchymal transition (EMT)‐markers such as N‐cadherin, suggesting that hnRNPA2/B1‐mediated Sp1 expression is critical for lung cancer progression. Taken together, these results suggest that understanding the relationship between Nm23, hnRNPA2/B1, and Sp1 in regulating lung cancer malignancy will be beneficial of lung cancer.
Capacity of chlorogenic acid isomers to modulate oxidative and inflammatory responses in Caco‐2 human enterocyte‐like cells
Authors: Liang Ningjian , Kitts David
Published: November 2023

Chlorogenic acids (CGAs) are esters formed between caffeic and quinic acids, and represent an abundant group of plant polyphenols present in the human diet. CGAs contain different isomers, including the major ones such as 5‐caffeoylqunic acid (5‐CQA), 3‐caffeoylqunic acid, 4‐caffeoylqunic acid, 3,4‐dicaffeoylqunic acid, 3,5‐dicaffeoylquinic acid, and 4,5‐dicaffeoylqunic acid. The antioxidant and anti‐inflammatory properties of 5‐CQA have been established in both cell and animal studies. However, little is known about the ability of other major CGA isomers to modulate oxidative and inflammatory stresses. In this study, we conducted a pre‐treatment experiment in which Caco‐2 cells were exposed to individual physiological relevant concentrations of CGA isomers for 24 hours and once removed were then challenged with oxidative and inflammatory stresses. We found that the pre‐incubation of CGA isomers promoted pro‐inflammatory cytokine, interleukin 8, and activated the nuclear translocation of nuclear factor kappa B (NF‐κB) pathway. The pre‐incubation did not activate nuclear factor‐E2‐related factor 2 (Nrf2) pathway. A co‐incubation experiment was conducted in which Caco‐2 cells were firstly exposed to individual CGA isomers for 24 hours. Then, fresh CGA isomers were added before cells were challenged with oxidative and inflammatory stresses. Herein, we found that the co‐incubation treatment of CGA isomers showed anti‐inflammatory effect by attenuating the secretion of IL‐8. This was accompanied with an increase in NF‐κB signaling. We also found that CGA co‐incubation treatment significantly increased Nrf2 signaling that corresponded to increased ratio of reduced glutathione to oxidized glutathione; both indicating a mitigated oxidative stress. We proposed that CGA isomers exert anti‐inflammatory activity by activating Nrf2 signaling, even though CGA isomer activated NF‐κB pathway at the early stage of response. Our results suggest that Nrf2 is a secondary event that participates in a later negative loop of NF‐kB regulation. Cross‐talk between NF‐κB and Nrf2 seems apparent in this case.

Support or Funding Information

This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant.
A new role for thermosensors in Drosophila melanogaster
Authors: Marquez Gonzalez Keishla M.
Published: November 2023

Temperature is a universal parameter experienced by all animals that can have dramatic effects on the biochemical functions of cells. In Drosophila melanogaster, neuronal receptors of temperature are well known to control behavioral avoidance of thermal extremes. However, whether they have additional roles in helping animals cope with sub‐optimal temperatures is unknown. Preliminary work from Garrity Lab has shown that mutant animals lacking (neuronally‐expressed warmth receptors, Gr28b and TrpA1), become immobilized at normally innocuously warm temperatures (~34°C), suggesting that these thermoreceptors may also play a role in heat tolerance in Drosophila. Based on this model, I focused on identifying the neural circuit that sends thermosensory information to the brain to mediate heat tolerance. Using genetic techniques, I have silenced downstream projection neurons and neuropeptide‐expressing cells and assessed heat tolerance in the resulting animals. The results of this study will lend insight into the cellular and molecular basis of heat tolerance.
A Novel Murine Experimental System for Analyzing Differentiation of Oligodendrocyte Precursor Cells Using Sox10‐Venus Mice
Authors: Suzuki Nobuharu , Sekimoto Kaori , Hayashi Chikako , Mabuchi Yo , Suto Eriko Grace , Nakamura Tetsuya , Akazawa Chihiro
Published: November 2023

In the central nervous system (CNS), myelin surrounding axons are formed by oligodendrocytes. Dysfunction of oligodendrocytes in CNS myelination results in neurological disorders, such as leukodystrophy and multiple sclerosis. However, the detailed mechanism of oligodendrocyte differentiation, including cellular process formation, and of myelination has not been elucidated yet, particularly since purification of oligodendrocyte precursor cells (OPCs) from mice, but not rats, is not efficient. We recently created and established a transgenic mouse line that expresses a fluorescent protein Venus driven by the promoter of the Sox10 gene. Endogenous Sox10 is specifically expressed in OPCs and oligodendrocytes in the CNS. In the present study, we have characterized Venus‐positive (+) cells from the brain of Sox10‐Venus mice for the analysis of oligodendrocyte differentiation. First, we purified Venus (+) cells from the brains at postnatal day 0–2, when OPCs have not differentiated into oligodendrocytes, by flow cytometry. Most Venus (+) cells expressed an OPC marker NG2, but not other glial cell markers including galactocerebroside (GalC), a marker for oligodendrocytes. After induction of differentiation with the serum‐free differentiation medium, an increase of GalC‐positive oligodendrocytes and decrease of NG2‐positive OPCs were observed in the Venus (+) culture. These results suggest that OPCs were efficiently purified and differentiated into oligodendrocytes using the Sox10‐Venus system. In addition, a time‐lapse analysis of the Venus (+) OPC differentiation was performed and showed that Venus (+) OPCs dynamically changed their cell morphology with highly branched cellular processes during their differentiation into oligodendrocytes. We further found that Venus (+) OPCs were differentiated into type II astrocytes with the serum‐containing differentiation medium. In vivo, Venus (+) cells expressed NG2 at P0 and both NG2 and myelin basic protein, an oligodendrocyte marker, at P20, when numerous oligodendrocytes are differentiated from OPCs and form myelin. Moreover, some of S‐100‐positive astrocytes expressed Venus in the ventral cortex. Together, the Sox10‐Venus mouse system is useful for analyzing differentiation and multipotency of murine OPCs.
Arachidonic Acid Cascade Driven Neurotoxicity in Hypobaric Hypoxia: An invivo Study
Authors: Choudhary Richa , Katyal Anju
Published: November 2023

Background

A continuous supply of oxygen to cerebral tissue is required for maintaining its homeostasis. Any interruption in delivery of oxygen leads to cerebral hypoxia related neuronal damage. . The growing bodies of evidences reveal that cerebral hypoxia induced at high altitude promotes the production of excessive ROS resulting in oxidative stress. Cerebral tissue is prone to oxidative stress damage because of high content of PUFA in their cell membrane and weak antioxidant defence system. Arachidonic acid(C20) is the most abundant form of PUFA which is present in brain. Under stress conditions arachidonic acid is metabolized by cyclooxygenase(COX‐2) and 12/15 Lipoxygenase in cerebral tissue which leads to the production of several prooxidant and anti‐inflammatory molecules. Experimental studies suggest that hypoxia induce oxidative burden which initiate the upregulation of arachodonic acid cascade enzyme 12/15LOX and COX‐2.

Method

In present study we aimed to investigate the modulatory role of 12/15 LOX and COX‐2 by using specific inhibitor of 12/15 LOX (baicalein) and COX‐2 (NS398) on neuronal damage induced by hypobaric hypoxia.

Results

Animals subjected to hypobaric hypoxia in a specially designed decompression chamber experience cerebral hypoxia as confirmed by the elevated expression of HIF‐1a protein which is universal transcription factor that senses oxygen balance in cellular milieu. Further COX‐2 and 12/15 lipoxygenase gene and protein expression were significantly upregulated in hypoxia condition. Moreover histopathological study showed marked increase in death of neurons in hypoxia condition and inhibitor of 12/15 LOX and COX‐2 attenuated the neurotoxicity during cerebral hypoxia suggesting the contribution of arachidonic acid cascade in hypoxia condition. In addition to above biochemical studies like LDH assay, MDA assay for lipid peroxidation and nitric oxide levels revealed that oxidative stress mediated neuronal damage caused by hypoxia was attenuated significantly by 12/15 LOX and COX‐2 inhibitors confirming involvement of 12/15 LOX and COX‐2. TUNEL analysis confirmed the apoptotic form of cell death in hypoxia as dUTP labeled DNA fragments were significantly increased further 12/15LOX and COX‐2 enzyme inhibitor rescued the neuronal cells from apoptosis.

Conclusion

The primary study using pharmacological inhibitors provide the strong evidence towards the contribution of arachidonic acid cascade in pathogenesis of cerebral hypoxia injury .

Support or Funding Information

Authors acknowledge Council for Scientific and Industrial Research (CSIR) India for financial assistance.
Chicken Broth AAC1 as a Dietary Supplement Inhibits Pain Transmission and Decreases Serum C‐Reactive Protein Levels In a Model of TMJ Disorder
Authors: Durham Paul , Hawkins Jordan
Published: November 2023

Objective

The goal of this study was to determine if the dietary inclusion of the commercially available chicken broth AAC1 could repress nocifensive behaviors and PKA expression associated with a clinically relevant model of temporomandibular joint (TMJ) disorder caused by prolonged jaw opening.

Background

Routine dental visits for molar extractions or root canals, or to the orthodontist can result in an injury to the TMJ or muscles of mastication if the jaw is held open for long periods of time or is opened to near maximal even for a short period of time. However, the pathophysiological and cellular mediators that underlie the development of chronic orofacial pain associated with TMJ disorder are not well understood. Chicken broth has been often thought of as having beneficial immune effects and may represent an innovative and safe alternative treatment for chronic inflammatory diseases.

Methods

Male Sprague‐Dawley rats were used to investigate the effects AAC1 on behavioral and cellular changes associated with prolonged jaw opening. A surgical retractor was placed around the bottom and top incisors and the jaw held at near maximal opening for 20 minutes. Withdrawal responses to mechanical stimuli were determined following jaw opening for up to 7 days by applying calibrated von Frey filaments to the cutaneous area over the masseter. Some animals received 0.5% solid (w/v) AAC1 dissolved in their drinking water for two weeks prior to jaw opening. After 7 days, animals were euthanized and the upper spinal cord and trigeminal ganglia were removed to study changes in the expression of PKA utilizing immunohistochemistry. Serum samples were collected to determine the levels of C‐reactive protein using an ELISA.

Results

Near maximal jaw opening was induced a sustained increase in the number of nocifensive responses to mechanical stimuli over the masseter area for 7 days. This increased sensitivity correlated with increased levels of PKA expression in both the upper spinal cord and the trigeminal ganglia, and elevated serum levels of C‐reactive protein. Dietary inclusion of AAC1 was able to suppress nocifensive behaviors, PKA expression, and decreased levels of C‐reactive protein associated with prolonged jaw opening.

Conclusion

Our findings provide evidence that dietary inclusion of chicken broth AAC1 inhibits transmission of pain signals and represses levels of inflammatory mediators in serum and nerve cells and thus, may by a useful nutraceutical supplement to help manage inflammatory pain associated with TMJ disorder.

Support or Funding Information

Support provided by International Dehydrated Foods
Dual activation of bile acid receptors FXR and TGR5 plays a protective role in non‐alcoholic fatty liver disease and atherosclerosis
Authors: Jadhav Kavita , Xu Yang , Zhang Yanqiao
Published: November 2023

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of conditions which ranges from fatty liver to non‐alcoholic steatohepatitis (NASH) and liver cirrhosis. The aim of this study is to establish that activation of the bile acid receptors FXR and TGR5 by its semi‐synthetic ligand INT‐767 attenuates the development of NAFLD and atherosclerosis. Administration of the TGR5 specific synthetic ligand INT‐777 led to a significant reduction in liver triglycerides and cholesterol. In addition, treatment with the FXR and TGR5 dual agonist INT‐767 led to a significant reduction in plasma and hepatic triglycerides. In line with this, treatment of high fat diet‐fed C57BL/J6 mice with INT‐767 led to significant decrease in plasma and hepatic triglycerides as well. To further investigate the role of TGR5 and FXR in the development of atherosclerosis, ApoE−/− mice were gavaged with INT‐767 for 60 days. This led to a significant reduction in plasma and liver lipids, and also a marked improvement in the plasma lipoprotein profile. This in turn led to a significant reduction in atherosclerotic plaque formation in INT‐767 treated ApoE−/− mice. These data indicate that dual activation of FXR and TGR5 plays an important role in maintaining hepatic and plasma lipid homeostasis under normal and high fat diet‐fed conditions. These results point to the possibility of targeting both FXR and TGR5 as a potential therapeutic treatment of NAFLD and atherosclerosis.
Palmitoleate Protection Against Palmitate‐induced Cholangiocyte Lipoapoptosis is Sensitive to Pertussis Toxin
Authors: Natarajan Sathish Kumar , Walker Mary Anne , Toews Myron , Mott Justin L
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) patients have elevated plasma saturated free fatty acid (FFA) levels. Our recent work had demonstrated the role of FoxO3 and its downstream target, PUMA in FFA‐induced cholangiocyte lipoapoptosis and provided insights for biliary damage in a subset of NAFLD patients. Palmitoleate has been shown to be protective against FFA‐induced hepatocyte and pancreatic β‐cell lipoapoptosis and has potential as a dietary therapy. Here, we explored the mechanism of palmitoleate protection against cholangiocyte lipoapoptosis.

Methods

Apoptosis was assessed by TUNEL, characteristic nuclear morphology staining with DAPI, transmission electron microscopy, and caspase 3/7 activity assay. Cleaved PARP and cleaved caspase 3 were examined by Immunoblot.

Results

Treatment of rat primary cholangiocytes with palmitate (PA) induced lipoapoptosis as evidenced by a significant increase in TUNEL positive cells, apoptotic nuclear morphology changes and caspase 3/7 activity. We also show that PA treatment dramatically increased the levels of caspase substrate like cleaved PARP and cleaved caspase 3 in normal immortalized cholangiocytes, H69 and cell lines derived from cholangiocarcinoma, KMCH, Mz‐ChA‐1 and HuCCT cells. Treatment with palmitoleate, a mono unsaturated omega‐7 fatty acid mitigated cholangiocyte lipoapoptosis caused due to PA exposure. The protection offered by palmitoleate was abolished in cells treated with pertussis toxin, suggesting an involvement of G‐protein‐coupled receptor (GPCR) in mediating palmitoleate protection of against PA‐induced cholangiocyte lipoapoptosis. The specific GPCR that is responsible for the palmitoleate protection is now under investigation.

In conclusion, palmitoleate protects PA‐induced cholangiocyte lipoapoptosis possibly via activating Gαi protein coupled receptor for cell survival.
Examining Interactions between Bacterioopsin and Carotenoid Biosynthetic Proteins in the Halophilic Archaeon Halobacterium salinarum
Authors: Plesa Alexandru M. , Peck Ronald F.
Published: November 2023

Microbial opsins are complexes consisting of integral membrane proteins covalently bound to the retinal cofactor. These complexes undergo a conformational change in response to light and serve various functions including signaling and energy conservation. First identified in Archaea, microbial opsins have now been found in all three domains of life.

Bacteriorhodopsin (BR), the first microbial opsin described, acts as a light‐driven proton pump in the halophilic Archaeon Halobacterium salinarum. When cellular respiration is inhibited due to low oxygen, BR generates a proton gradient that provides energy for ATP synthesis, active transport, and other cellular activities.

Interestingly, it appears that the BO apoprotein itself plays an important role in regulating the production of the retinal cofactor. When not bound to retinal, BO inhibits the activity of lycopene elongase (Lye), an enzyme that catalyzes the committed step in the synthesis of bacterioruberins. Bacterioruberins are 50‐carbon carotenoids that integrate into the plasma membrane and are believed to increase membrane stability under conditions of osmotic stress. Since bacterioruberins have common precursors with retinal, the inhibition of Lye by BO likely acts to promote retinal synthesis at the expense of bacterioruberin synthesis.

Haloferax volcanii is closely related to H. salinarum and inhabits similarly salty environments. H. volcanii produces bacterioruberins, but does not express any microbial opsins. We had previously determined that, despite a 65% identity between the two Lye homologs, the H. volcanii Lye is not inhibited by BO. This difference suggests that the two proteins have evolutionarily diverged to better suit the needs of the organisms. To identify the motifs in Lye that allow or prevent inhibition by BO, we constructed hybrid genes that included sections of both H. salinarum and H. volcanii lye. These hybrid genes were then placed into H. salinarum strains in the presence or absence of BO expression. BR and bacterioruberin levels were assayed to analyze the activity of Lye. Preliminary evidence suggests that only a small portion of the H. salinarum gene allows inhibition by BO.

We have also begun to explore interactions between microbial opsins and Lye homologs from other organisms. Briefly, opsin and Lye homolog genes were expressed in H. volcanii and bacterioruberin production was monitored. Our initial findings indicate that the opsin from some organisms inhibit the activity of the Lye homolog from the same organism. Overall, our results suggest a widely‐distributed, and previously undescribed, activity for opsins.

Support or Funding Information

Funding was provided by Colby College and the National Institutes of Health (Grant GM094735).
A Functional Relationship Between Plb1‐Mediated Phosphatidylcholine Turnover and Sphingolipid Homeostasis in Saccharomyces cerevisiae
Authors: Patton‐Vogt Jana , Anaokar Sanket , Brant Jacob , Jonik Benjamin , Nesbitt Nicole , Vera Michael
Published: November 2023

Plb1 is a B‐type phospholipase that acts on phosphatidylcholine (PC). Localization studies have found Plb1 to be primarily at the endoplasmic reticulum and secreted from the cell. We have shown previously that Plb1 activity and expression are up‐regulated by loss of Ypk1, the yeast homolog of the human serum‐ and glucocorticoid‐ induced kinase (Sgk1). In addition, Plb1 overexpression is known to rescue the lethality of a ypk1ts ypk2Δ mutant. Ypk1 is a serine/threonine protein kinase that affects several membrane events, including plasma membrane flippase activity and sphingolipid homeostasis. Employing the serine palmitoyltransferse inhibitor, myriocin, and genetic mutants, we report that overexpression of Plb1 compensates for diminished sphingolipid biosynthesis in various strain backgrounds. To probe this result further, we examined the effect of Plb1 overexpression on signaling pathways known to affect, or be affected by, altered sphingolipid metabolism. Overexpression of Plb1 results in up‐regulation of the Unfolded Protein Response (UPR) and up‐regulation of INO1, a gene encoding inositol 3‐phosphate synthase, the rate limiting step in inositol biosynthesis. We are examining both the mechanism by which PC turnover up‐regulates the UPR and INO1 expression, as well as how that up‐regulation affects flux through sphingolipid biosynthesis. Our findings indicate a functional relationship between PC turnover and sphingolipid homeostasis.

Support or Funding Information

NIH GM104876 to JPV
Novel Interactions of Yeast Glycerol‐3‐Phosphate Acyltransferases
Authors: Shabits Brittney , Tavassoli Marjan , Zaremberg Vanina
Published: November 2023

Glycerol‐3‐phosphate acyltransferases (GPATs) catalyze the first committed and rate limiting step in the de novo synthesis of glycerophospholipids and neutral triacylglycerols. GPATs transfer an acyl group from acyl‐coenzyme A to the sn‐1 position of glycerol‐3‐phosphate to produce 1‐acyl glycerol‐3‐phosphate (lysophosphatidic acid). Lysophosphatidic acid can then be further acylated by a second acyltransferase to produce phosphatidic acid, a signalling lipid and key intermediate in lipid biosynthesis. In the model organism yeast Saccharomyces cerevisiae two GPATs, Gpt2 and Sct1, have been identified. Both are integral membrane proteins located to the endoplasmic reticulum (ER), differentially enriched in ER domains. Interestingly, we have shown that Gpt2 (but not Sct1) is responsible for channeling oleate (C18:1) into triacylglycerol that is eventually stored in lipid droplets. Furthermore, our studies on oleate metabolism unveiled novel ER structures that surround lipid droplets and are differentially enriched in Gpt2 while devoid of Sct1. In addition, we also uncovered a dynamic post‐translational regulation of Gpt2 and Sct1 through phosphorylation that responds to growth phase, carbon source and cellular GPAT imbalance. The aim of this work was to understand how GPAT interactions with other proteins contribute to their spatial distribution in the ER and to lipid synthesis partitioning. In order to identify potential protein interactors, Gpt2 and Sct1 were overproduced in cells deleted for endogenous GPATs and then isolated from lysates and microsomes using co‐immuno‐affinity and Ni‐NTA affinity chromatography purification techniques. Potential interactors co‐purifying with Gpt2 or Sct1 were identified through liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Our results suggest that Gpt2 and Sct1 differentially associate with ER sites where inter‐organelle transport of lipids occurs. Of importance are the contact sites between ER‐mitochondria, ER‐lipid droplets and the nuclear‐vacuolar junction.

Support or Funding Information

This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to VZ.
The Structure and Function of COMMD1
Authors: Rasmussen Zachary Andrew , Fitzpatrick Kevin , Choi Alice
Published: November 2023

COMMD1 (Copper Metabolism Murr1 Domain 1) is a highly conserved, soluble 21 kD protein which functions in multiple pathways including copper transport. Despite having a variety of known functions, the mechanism of action of COMMD1 along with the structure is still unknown. The purpose of this research is to determine COMMD1's mechanism of action, specifically its ability to bind to phosphatidylinositol(4,5)‐bisphosphate (PTDINS(4,5)P2), a well characterized substrate for cellular signaling proteins (3). COMMD1 is comprised of two domains, the N‐terminal VARiable Domain (VARD), and the C‐terminal (CTD), which contains the COMM domain. COMMD1 readily binds to phosphorylated phosphatidylinositols (PTDINS) with an increased specificity for PTDINS(4,5)P2. Additionally, COMMD1 forms high order oligomeric compounds spontaneously in vitro. The C‐terminus is required for PTDINS binding and performs PTDINS binding and oligomerization in the absence of the N‐terminus. An NMR structure for the non‐binding N‐terminus has been solved, and in silico prediction models of the CTD have been reported. The goals of this study are to identify critical amino acid residues for specific PTDINS(4,5)P2 binding, analyze relative binding affinities for different PTDINS(4,5)P2 lipids, and define structural characteristics of COMMD1 full‐length protein as well as N‐ and C‐terminal domains. The experimental approach uses biochemical and biophysical methods to study recombinant wild type and variant proteins. These studies are expected to inform continued in silico studies and in cell experiments to identify the biochemical mechanism of action that underlies COMMD1's pleiotropic functions.
Fourier Transform Infrared Spectroscopy Detection of Lipid Ion‐Pairing in the Staphylococcus aureus Plasma Membrane
Authors: Duran Andrea T , Marbach Helene , Rasul Bahar , Andrew Chan K. L. , Harvey Richard D
Published: November 2023

Ion‐pairing between lipids in the Staphylococcus aureus plasma membrane has been hypothesized to aid survival of this bacterium at low pH and potentially facilitate antimicrobial drug resistance through increased lipid ordering. Fourier transform infrared spectroscopy (FTIR) is a useful and rapid technique that can potentially characterize the physical properties of phospholipid membranes. The purpose of this study was to determine if FTIR can detect ion‐pairing in natural lipids extracted from bacteria grown under different conditions and also in representative synthetic models. S. aureus reference strain 476 and methicillin‐resistant S. aureus (MRSA) strain A7 were cultured in Brain Heart Infusion broth at pH 7.4 and pH 5.5. The phospholipids were isolated from each of the samples using an organic solvent extraction process. Synthetic lipid models composed of binary mixtures of dipalmitoyl‐3‐aza‐dehydroxy‐lysyl‐phosphatidylglycerol (DP3adLPG) and dipalmitoylphosphatidylglycerol (DPPG) were also created, to mimic the compositions of the S. aureus plasma membrane at pH 7.4 (DP3adLPG/DPPG [30:70]) and pH 5.5 (DP3adLPG/DPPG [60:40]). Humidity chambers containing saturated NaCl solutions were created from T25 flasks, onto which two CaF2 FTIR windows were fixed, onto the inner face of one, the dried lipid sample films were deposited from solution in chloroform. Ion‐pairing was detected by FTIR analysis in both the lipid extracts and synthetic models, by analyzing the ν (PO4)− symmetric and ν (PO4)− asymmetric stretch regions of each FTIR spectrum. The DP3adLPG/DPPG [30:70] model had a ν (PO4)− symmetric (1056.41 cm−1) in between that of the DPPG (1052.93 cm−1) and DP3adLPG (1063.84 cm−1) standards, with a peak closer to that of DPPG. The ν (PO4)− symmetric stretch of the DP3ad‐LPG/DPPG [30:70] model suggests that both ion‐pairing between the two lipids took place, as well as self‐pairing within the headgroup of the DP3adLPG. These results are similar to the ν (PO4)− symmetric stretch observed in Strain A7 at pH 7.4 (1055.34 cm−1), demonstrating that, in terms of the lipid interactions, the synthetic model accurately represents the S. aureus membrane at pH 7.4. The results of the current study established that FTIR can be used to detect ion‐pairing in S. aureus lipid extracts. Furthermore, the similar ν (PO4)− symmetric in the DP3adLPG/DPPG models relevant to the lipid extracts at pH 7.4 and pH 5.5 provides physicochemical evidence that the synthetic DP3adLPG/DPPG models can accurately represent ion‐pairing within the S. aureus membrane.

Support or Funding Information

Supported in part by grant MHIRT 2T37MD001368 from the National Institute on Minority Health and Health Disparities, National Institute of Health
Transmembrane (TMEM)‐163 protein is a novel zinc transporter
Authors: Cantrell Quinlan W , Silva Joshua , Nguyen Cathleen , Hildebrand Leanne D , Rivas Tiffany , Shoemaker Ryan , Rojas America , Cuajungco Math P
Published: November 2023

A mutation or deletion of the MCOLN1 gene which codes for the Mucolipin‐1 (TRPML1) ion channel protein causes the human lysosomal storage disorder called Mucolipidosis IV (MLIV). An elevation of chelatable zinc, particularly in lysosomes, is observed in cells affected by MLIV compared to normal cells. The mechanism behind the zinc buildup in MLIV cells remains elusive; however, evidence suggests that it is due to the failure of non‐functional TRPML1 to mediate the release of zinc from the lysosomes or failure in lysosomal exocytosis. We recently identified TMEM163 as an interacting partner of TRPML1. We hypothesize that TMEM163 is a transporter that facilitates zinc flux in normal cells, but that the loss of TRPML1 function in MLIV cells leads to abnormal TRPML1‐TMEM163 protein interaction such that the cells uncontrollably accumulate zinc. To infer function as a zinc transporter, we first studied the membrane topology of heterologously expressed TMEM163 protein. Contrary to its predicted orientation, we found that both the N‐ and C‐terminus regions of TMEM163 lie at the extracellular rather than intracellular milieu. To test functional similarities between TMEM163 and several known zinc transporters (ZNTs), we cloned the cDNA of TMEM163 and specific ZNTs into the pBI‐CMV dual‐expression construct (containing a minimal CMV promoter vector). Single cDNA constructs were created as controls. The single gene and dual gene constructs were then heterologously expressed in human embryonic kidney (HEK)‐293 cells. Twenty‐four hours post‐transfection, treated and control HEK‐293 cells were then exposed to zinc chloride (ZnCl2, 100 mM) for 3 and 5 hours. Spectrofluorometric end‐point assay using the zinc‐specific Fluozin‐3 AM dye showed that the single TMEM163 expression in cells produces intracellular zinc accumulation upon 5 hours of ZnCl2 exposure, while co‐expression of TMEM163 with ZNT4, but not other ZNTs, showed a significant synergistic effect on intracellular zinc levels at both time points tested. Overall, these results suggest that TMEM163 may serve a similar role as those of the ZNTs in transporting zinc, but that it is an influxer. Future studies relating to TMEM163‐TRPML1 protein interaction should provide new research opportunities to further understand the involvement of zinc in the underlying neuropathology and progressive cell degeneration observed in MLIV disease.

Support or Funding Information

NIH R15‐NS070774, NSF MCB‐0920127, and NIH T34‐GM008612‐20
Heterodimerization of SLC30A10/ZnT10 and SLC30A3/ZnT3 Modulates ERK1/2 Activity
Authors: Zhao Yitong , Feresin Rafaela G. , Falcon‐Perez Juan Manuel , Salazar Gloria
Published: November 2023

Zinc is an essential micronutrient required for metabolic function, but high concentration of zinc is toxic. The solute carrier 30A (SLC30A) family of zinc exporters transports zinc into the lumen of intracellular organelles in order to prevent zinc toxicity. We reported that the formation of tyrosine dimers is required for zinc transporter 3 (ZnT3) activity and targeting to synaptic‐like micro‐vesicles (SLMVs) in PC12 cells, as well as the formation of ZnT3/ZnT10 heterodimers. Here, we focus on ZnT10 to determine the role of heterodimerization in the sorting of ZnTs in the endosomal/lysosomal pathway. Using cell fractionation, immunoprecipitation and immunofluorescence approaches, we found that ZnT10 resides in transferrin receptor (TfR) and Ras‐related protein 5 (Rab5) positive endosomes and forms covalent heterodimers and oligomers with ZnT3. The interaction of ZnT10 with ZnT3 is mediated by dityrosine bonds and was required to increase the phosphorylation of extracellular signal regulated kinase (ERK1/2), but not Akt, in response to epidermal growth factor (EGF), a regulator of cell growth, proliferation, and differentiation. Further, mutation of tyrosine 4 in ZnT10 reduced ZnT3/ZnT10 dityrosine‐mediated heterodimerization, and ERK1/2 phosphorylation by EGF. We propose that ZnT10 and zinc play a role in signal transduction, which is mediated by homo and heterodimerization with other ZnTs, and that ZnT3/ZnT10 heterodimer could be a potential therapeutic target to regulate cell proliferation during tumorigenesis.
Structural Determination of the BBSome Core Complex
Authors: Aoba Takuma
Published: November 2023

Bardet‐Biedl syndrome (BBS) stems from failed assembly of BBS proteins into a functional protein complex called the BBSome. The BBSome consists of eight BBS proteins (1–2, 4–5, 7–9 and 18) and constitutes a key component of intraflagellar transport system in eukaryotic primary cilia. Lack of a functional BBSome results in cilium‐related symptoms such as retinal degradation, polydactyly, obesity, kidney dysfunction, and other disorders. To better understand mechanisms of BBSome assembly and function, we have analyzed the structure of a BBSome assembly intermediate, the BBSome core complex (BBS2, 7, and 9), by a combination of cryo‐electron microscopy and crosslinking coupled with mass spectrometry (XL‐MS). The structure of the BBSome core complex revealed a parallel arrangement of BBS2 and 7 through a coiled‐coil interaction and a perpendicular arrangement of BBS2 and 9 through interactions in their α‐helical domains. Unnatural amino acid cross‐linking and mutagenesis identified specific residues at the binding interfaces between the core complex components as well as BBS‐linked mutations that disrupt the complex. Thus, these structural studies not only visualize the BBSome core complex and reveal its assembly process, but they also provide insight into the molecular basis of Bardet‐Biedl syndrome.

Support or Funding Information

Barry M. Willardson