bluuebunny/crossref_metadata_2025 huggingface dataset:
Mapping calmodulin‐induced oxidase domain interactions of neuronal nitric oxide synthase using H/D exchange mass spectrometry
Authors: Barrett Morgan F , Carley Jeffrey R , Underbakke Eric S
Published: November 2023

Nitric oxide (NO) is a short‐lived, freely diffusible gas that serves as an omnidirectional neurotransmitter. As such, NO signaling must be controlled through regulation of localization and activation. Neuronal nitric oxide synthase (nNOS) generates NO at a heme‐containing active site upon Ca2+‐induced activation mediated by calmodulin (CaM). We sought to apply hydrogen/deuterium exchange mass spectrometry (HDX‐MS) to investigate how CaM influences catalysis at the heme domain. HDX‐MS can reveal conformational changes and binding sites by monitoring perturbations in amide proton exchange rate changes. To assess how CaM impacts the electron transport and NO production at the heme domain, we compared inactive nNOS with Ca2+‐CaM‐activated nNOS.

HDX‐MS analysis showed several localized perturbations due to Ca2+/CaM‐binding. Here, we highlight two major effects. First, the CaM‐binding linker exhibited profound decreases in exchange rate, indicating burial and helix formation. Activated nNOS also exhibited exchange rate changes in the oxidase domain suggesting a delivery mechanism for reducing equivalents into the active site. Ultimately, HDX‐MS revealed several important nNOS conformational changes and inter‐domain dynamics responsible for Ca2+/CaM‐gated activation of NO production.
Aspirin: A Regulator of Tumor Angiogenesis in Breast Cancer
Authors: Maity Gargi , Chakraborty Jinia , Banerjee Snigdha , Banerjee Sushanta K
Published: November 2023

Background and Objective

Tumor neovascularization / angiogenesisis a pathophysiological process in which new blood vessels are formed in the primary tumor site or distant organs to supply adequate nutrition to cancer cells for metastatic growth. Therefore, controlling tumor angiogenesis is an attractive target for cancer therapy. Structural abnormalities of vasculature (i.e. leakiness due to abnormal lining of pericytes on the microvessels) is one of the critical features of tumor angiogenesis that sensitizes vascular cells to cytokines and helps circulating tumor cells to metastasize to distant organs. Our long term goal is to repurpose the drugs that may prevent tumor angiogenesis or normalize the vessels by repairing leakiness via recruiting pericytes or both. Our recent studies found that Aspirin (ASA)has the potency to inhibit breast cancer growth and metastasis, as well as reprogram the mesenchymal to epithelial transition (MET). Given the importance of ASA, we tested whether ASA may be able to regulate tumor angiogenesis.

Methods

We investigated the effect of low dose ASA (1mM, which is equivalent to 80mg human dose), ASA‐treated(2.5mM) conditioned media (231‐CMASA) or vehicle‐treated conditioned media (231‐CMVT) of MDA‐MB‐231 cells on different endothelial cell physiology. These include endothelial cells' migration towards serum using modified Boyden chamber assay, in vitro capillary‐like structure formation on Matrigel, cell permeability using in vitro endothelial permeability assay and interactions of pericytes‐endothelial cells. We also determined the effect of ASA on various angiogenic factors associated with tumor angiogenesis. Finally, we determined the effect of ASA on invivo tumor angiogenesis using in vivo Angiogenesis Assay.

Results and Conclusions

We found that 231‐CMASA significantly blocks in vitro migration, the formation of in vitro capillary‐like structures parallel with leakiness via incomplete interaction of pericytes and endothelial cells as compared to 231‐CMASA. The antiangiogenic effect of ASA was also documented in in vivo assays. Mechanistically, ASA treatment blocks several angiogenic factors including VEGF‐A that are associated with these three events, implicating a low dose of Aspirin is potentially therapeutic for breast cancer via blocking and normalizing tumor angiogenesis.

Support or Funding Information

This work was supported by VA Merit Review Grants (S. Banerjee and S.K. Banerjee).
Genetic variants of tumor necrosis factor‐α (rs1800629) gene enhances susceptibility to malaria infection in West Africa
Authors: Snyder Tara , Noble Jenelle , Funwei Roland , Falade Catherine , Ojurongbe Olusola , Thomas Bolaji N
Published: November 2023

Malaria is still a disease that causes untold human death and suffering in many parts of the world. Despite the fact that the parasite genome has been sequenced, much remain to be deciphered, especially on factors that exacerbates or limit susceptibility to infection. We hypothesize that host genetics factors, with particular focus on genetic polymorphisms that affects gene transcription and variability in immune response, are major contributors to host susceptibility, resistance or disease severity. Therefore, we elucidated the genomic diversity and haplotype frequency of tumor nectrosis factor‐a (snp rs1800629) genes among and between malaria‐infected and control groups in West Africa, and extrapolated their role, if any, on parasitemia. To do this, genomic DNA samples obtained from 107 malaria‐infected (microscopy and PCR‐confirmed) patients and 380 uninfected controls from Nigeria, were subjected to a PCR‐RFLP analysis. We report that there is no significant difference in the genotypic (p=0.15; 55.2% versus 50.0% for malaria‐infected and controls respectively) or allelic frequency (p=0.21; 3.8% versus 8.4% for malaria‐infected and controls respectively) of the mutant variant of the vitamin D receptor (rs731236) gene. However, we found a statistically significant difference in the genotypic (p=2.20E‐16) and allelic frequencies (p=2.20E‐16) of the tumor nectrosis factor‐a (snp rs1800629) gene between malaria‐infected and control groups. The highest frequency of the mutant variant (77.6%) was found in the malaria‐infected group with the opposite reported among controls. In addition, haplotype frequencies were different between groups, with the most common haplotypes combining one form of the tumor nectrosis factor‐a mutant variant with a combination of the VDR mutant or wild type variant. Our observation clearly shows that tumor necrosis factor, as a proinflammatory cytokine, necessary to intitiate innate immune response is a major factor in susceptibility to malaria in West Africa. The preponderance of the mutant gene implies these patients cannot mount an effective immune response leaving parasitemia to increase uncontrolled until the initiation of adaptive immune response, thereby worsening disease. This, more than any other, may be the major factor delineating who is susceptible to disease or not, or the degree of infectivity and parasite load post‐infection. Future studies elucidating this genetic data in clearly delineated mild, uncomplicated versus severe disease groups viz a viz serum assays are ongoing.

Support or Funding Information

Funding provided by College of Health Sciences and Technology Faculty Development and Laboratory Support Fund, Rochester Institute of Technology
Effect of the Mitochondrial DNA 4977‐bp “Common Deletion” on Metabolism and Cell Growth in Human Lymphoblast Cell Lines
Authors: Keck Christopher , Gulfo Michelle , O'Donnell Robert , Hoops Harold , Pogozelski Wendy
Published: November 2023

Objective

To determine the effects of elevated levels of the common deletion ΔmtDNA4977 on glucose metabolism, oxidative phosphorylation, and cell growth

Introduction

Human mitochondria (mt) contain circular DNA molecules of 16549 base pairs. Because replication of mtDNA is asynchronous, and because the sequence contains repeats, mispairing can occur during replication, resulting in large deletions. One particular deletion of 4977‐bp is associated with several sporadic and degenerative diseases and is therefore called the “Common Deletion” (ΔmtDNA4977),. The deleted sequence codes for small regions of Complexes I, IV and V in the mitochondrial respiration chain. While resulting in truncated respiratory complexes and reduced cellular lifetimes, mtDNA bearing this deletion are nonetheless tolerated; cells can be functional due to the redundancy of mtDNA copies and the fact that normal and abnormal mtDNAs co‐exist in a heterplasmic state. However, if the level of mtDNA bearing this common deletion (ΔmtDNA4977), rises above a threshold, energy production is compromised enough to result in physiological symptoms. What has not been well‐understood are the details of how metabolism and cell growth are affected.

Methods

We are using lymphoblasts derived from a family with mitochondrial disease. Using real‐time PCR, we previously showed that cells derived from the son of the family (the 848 cell line) had 7% of the mtDNA bearing the common deletion. The boy's sister (849 cell line) had undetectable levels of the deletion and thus her cell line serves as a normal control. We compared the growth and metabolism of these cell lines in media of varying glucose content, measured the rates of glycolysis with a lactate assay, measured oxidative phosphorylation via oxygen consumption, and studied the shapes of the cells via microscopy. We also investigated the ability of succinic acid to rescue metabolically deficient cells.

Results

When compared with normal cells, initial results showed that the cells with elevated ΔmtDNA4977 1) grew more slowly; 2) grew poorly in media with low glucose; 3) showed higher rates of glycolysis; 4) formed spheroids more readily; and 5) were more often polarized in shape. Normal control cells grew well in all media types tested; however, when grown in high glucose, normal control cells were polarized more often. Succinic acid supplementation did not substantially affect growth of either cell type. Initial results suggest that the cell line with the increased levels of the common deletion ΔmtDNA4977 has an increased dependence on glucose metabolism and experiences activation of mechanisms that result in projections and polarization.

Support or Funding Information

Funding is acknowledged from the Geneseo Foundation and Student Foundation
Hydrodynamic Delivery of Isocitrate Dehydrogenase II for the Treatment of Acute Kidney Injury
Authors: Kolb Alexander , Atkinson Simon , Bacallao Robert
Published: November 2023

Mitochondrial Respiration is a critical process for normal kidney function. Kidneys that undergo an Acute Kidney Injury (AKI), specifically those injuries that result from ischemia, lead to changes in mitochondrial function and respiration. These changes in respiration are due to changes in TCA, Electron Transport Chain complexes, and regulation of ROS levels. To study mitochondrial changes in the kidney as a result of an AKI a proteomic screen was conducted on kidneys two weeks after ischemic injury. The screen indicated that in response to an ischemic event there was an upregulation of proteins that may play a pivotal role in kidney recovery and subsequent protection from future ischemic events. One such protein, Isocitrate Dehydrogenase 2 (IDH2) plays an important role in the TCA and ROS clearance. Treatment of AKI is limited to supportive care, which was reported to cost patients approximately 10 billion dollars in 2012. The limitations and cost of treatment lead to the idea of gene therapy as a possible means of treatment. However, delivering genes directly to the kidney and getting sufficient levels of expression has proven difficult. Recently a novel technique, hydrodynamic delivery, has shown promising results as a possible route of delivery for our genes. This technique uses pressure as a means to deliver genes, dyes, and vectors into the kidney. Our data indicates that hydrodynamic delivery of genes, such as IDH2, block creatinine increase, increase mitochondrial membrane potential, respiration, NAD(P)H and ATP levels compared to control or saline only models.

Support or Funding Information

NIH/NIDDKVA merit
Diabetic Nephropathy Accelerated by Imbalance of Renal Renin‐Angiotensin System Components in db/db Mice with High Fat Diet
Authors: Wang Guan‐Hua , Cho Ching‐Chang , Lin Chung‐I , Wu Hsi‐Tien , Lin Chih‐Sheng
Published: November 2023

Fat accumulation is associated with insulin resistance that results from overactivation of angiotensin converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 receptor (AT1R) axis of renin‐angiotensin system (RAS). Ang II induces macrophage infiltration into adipose tissue and increases pro‐inflammatory cytokines (TNF‐alpha and IL‐6) to inhibit insulin receptor that causes insulin resistance and hyperglycemia.

Several studies support antihypertensive efficacy of RAS blockade in the patients with type 2 diabetes and obesity. Future studies should address the role of kidney‐specific deficiency of RAS components to definitively determine the relevance of renal RAS to the disease progression related with diabetic or obesity‐related nephropathy. Due to BKS db/db mice is similar in human type 2 diabetes which can be spontaneously induced diabetic nephropathy. Therefore, male BKS db/db mice were fed a high‐fat diet (HFD; 60% of fat in diet) from 8 to 12 weeks of age and fed a normal diet (ND) from 8 to (12 or 20) weeks of age then the animals were sacrificed for the further assays in this study. The changes of serum and urine biochemistry, renal MAPK and STAT3 signaling pathways, RAS components, and pathology associated with diabetic nephropathy were evaluated in these mice.

According to the results of body and kidney weight, serum (triglycerides, total cholesterol, glucose, creatinine, blood urea nitrogen, and albumin) and urine (creatinine, blood urea nitrogen, and albumin) biochemical assays, our data confirm the previous reports that db/db mice can be spontaneously induced hyperglycemia at 8 week of age and progressive nephropathy at age of approximate 20 weeks in the mice fed with ND. However, nephropathy and severe diabetic dyslipidemia was occurred early at age of 12 weeks in the db/db mice fed with HFD. Increasing expression levels of renal ACE and chymase would synergistically generate more Ang II and then abnormally flare‐up ACE/AT1R axis that leads local inflammation and promote the progression of nephropathy. Vascular remodeling via MAPK signaling factor, p‐MEK and p‐ERK1/2, an inflammatory signaling factor, p‐STAT3, in the kidney of HFD‐fed db/db mice were significantly higher than those in the ND‐fed db/db mice. The pathological changes also show that diffuse glomerulosclerosis and mesangial matrix fraction in the kidney tissue of HFD‐fed db/db mice were more serious than those in the db/db mice with ND.

In conclusion, HFD treatment accelerate impaired kidney function and progressive diabetic nephropathy by increasing renal ACE and chymase activity of RAS in the db/db mice.
Prohibitin has sex dimorphic role in adipose and immune functions
Authors: Xu Yang Xin Zi , Ande Sudharsana , Mishra Suresh
Published: November 2023

The promoter of a gene that is selectively expressed in just a few cell types provides unique opportunities to study the pleiotropic function of a protein in two different cell types including the cell compartment specific function. This is not possible with a ubiquitous or a highly specific gene promoter. The adipocyte protein‐2 (aP2) is one such gene. It is primarily expressed in adipocytes, but also selectively in monocytic macrophages and dendritic cells among various immune cell types. Thus, the aP2 gene promoter provides an opportunity to simultaneously manipulate adipose and immune functions in a transgenic animal model.

Prohibitin (PHB) is a pleiotropic protein with cell compartment specific functions such as mitochondria and the plasma membrane. New evidence suggests that PHB has roles in adipocyte and immune cells. To discern the cell compartment specific functions of PHB in adipocyte and immune cell, we developed two novel transgenic mice by expressing wild type PHB and a phospho mutant PHB (Y114F or m‐PHB) from the aP2 gene promoter, separately. Both transgenic mice develop obesity in a sex‐neutral manner suggesting that m‐PHB retains mitochondria‐related adipogenic function. However, both transgenic mice develop obesity‐related metabolic dysregulation in a male sex‐specific manner. Interestingly, the male PHB mice spontaneously developed T2D and liver cancer, whereas the male m‐PHB mice developed lymph node tumors, or T1D in a context‐dependent manner. This would imply that plasma membrane‐related phosphorylation of PHB at Tyr‐114 has a role in immune cell functions.

Collectively, our data suggests that PHB has sex dimorphic roles in adipose and immune functions, which are mediated in a cell compartment specific manner. Also, we provide evidence that sex differences in adipose and immune functions contribute to sex differences in diabetes and cancer, which warrant further investigations.

Support or Funding Information

SM is supported by funds from Natural Sciences and Engineering Research Council of Canada (NSERC) and Research Manitoba.
AGPAT4 is a Mitochondrial Lysophosphatidic Acid Acyltransferase that Regulates Learning and Memory in Mice
Authors: Bradley Ryan , Mardian Emily B , Mitchell Andrew S , Bloemberg Darin , Marvyn Phillip , Bombardier Eric , Moes Katherine , Tupling A. Russell , Quadrilatero Joe , Duncan Robin E
Published: November 2023

We have previously characterized acylglycerophosphate acyltransferase 4 (AGPAT4) as a mitochondrial lysophosphatidic acid acyltransferase. We have also determined that AGPAT4 is important in the regulation of brain levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI), since levels of these glycerophospholipids were decreased significantly by 38%, 32%, and 52%, respectively, in the brains of Agpat4−/− mice. We investigated whether there were any impairments in cognition that may occur due to the significant loss of these three major glycerophospholipid species in the brains of Agpat4−/− mice. We tested the behaviour of Agpat4−/− mice and found that they have impaired spatial learning and memory as compared to wildtype littermates when assessed using the Morris Water Maze (MWM). Mechanisms underlying this effect were studied. No differences in whole‐body physiological parameters including respiratory capacity, food intake, motion and activity, or respiratory exchange ratio were evident. Similarly, we did not detect any changes with loss of Agpat4 in the brain mitochondrial content, or in the function of mitochondrial complexes as assessed in isolated brain mitochondria using high‐resolution respirometry. And, finally, we found that downstream targets of polyphosphorylated PI‐mediated signaling were also unchanged. However, Agpat4−/− mice did have a significantly lower brain content of the NMDA receptor subunits NR2A and NR2B, as well as the AMPA receptor subunit GluR1. Our findings indicate that neuronal membrane phospholipid content and composition are important in regulating NMDA and AMPA receptor content with implications for learning and memory.

Support or Funding Information

This work was supported by grants to RED from the Canada Foundation for Innovation – Leader's Opportunity Fund and Ontario Research Fund (Project#30259), and a Discovery Grant (#418213‐2012) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. EBM is the recipient of an NSERC Master's Scholarship (PGS‐M). RMB, JJAH, ASM, DB, PMM are the recipients of an NSERC Doctoral Scholarship (PGS‐D).
Investigation of a Role for AGPAT4 in Mouse Skeletal Muscle
Authors: Bradley Ryan M , Bloemberg Darin , Mitchell Andrew S , Fajardo Val A. , Henao Juan Jose Aristizabal , Bellissimo Catherine , Mardian Emily B , Paré Marie‐France , Bombardier Eric , Moes Katherine , Tupling A. Russell , Quadrilatero Joe , Stark Ken D , Duncan Robin E
Published: November 2023

Acylglycerophosphate acyltransferases (AGPATs) are a group of homologous proteins that function in the de novo synthesis and remodeling of glycerophospholipids, catalyzing the acyl‐CoA‐dependent acylation of lysophospholipids. We have recently determined that AGPAT4 is mitochondrial, and is a canonical AGPAT, preferentially utilizing lysophosphatidic acid as an acyl acceptor. However, we found that it functions in brain to regulate levels of phosphatidylinositol, phosphatidylcholine (PC), and phosphatdylethanolamine (PE), rather than phosphatidic acid per se. Whether this effect is tissue specific has not yet been investigated. The presence of multiple AGPATs suggests that different homologues may feed phosphatidic acid into unique phospholipid species synthesis pathways, although this remains poorly characterized. Phosphatidic acid (PA) metabolism is of particular interest in skeletal muscle, where levels of this phospholipid are involved in mediating muscle force contractility, muscle size, and initiation of mTOR signaling, among other effects. We have therefore investigated a role for AGPAT4 in skeletal muscle biology. We performed immunohistochemistry on AGPAT4 in cross sections from C57Bl/6J mouse soleus and extensor digitorum longus (EDL) muscle, and found that it is predominantly visualized in type IIa muscle fibres, as compared to type I, IIb, or IIx muscle fibres. We next investigated the impact of Agpat4 gene ablation on muscle physiology and function. Force contraction was significantly reduced in soleus muscle but not EDL from Agpat4−/− mice, in agreement with the higher proportion of type IIa fibres in the former muscle type. Interestingly, however, our studies indicate no difference between Agpat4−/− mice and wildtype littermates in measures of time to exhaustion during treadmill running tests. We found a significantly higher content of PA and PE in the soleus muscles of Agpat4−/− mice, with no difference in TAG content. As well, there were no apparent differences in mitochondrial content or function, suggesting that mitochondrial function is not impaired by Agpat4 deficiency in skeletal muscle. This work is the first to characterize the role of a bona fide AGPAT in skeletal muscle function.

Support or Funding Information

This work was supported by grants to RED from the Canada Foundation for Innovation – Leader's Opportunity Fund and Ontario Research Fund (Project#30259), and a Discovery Grant (#418213‐2012) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. RMB, JJAH, DB are supported by an NSERC PGS‐D, VAF was supported by a CIHR Doctoral Scholarship, ASM was supported by an NSERC CGS‐D award, EBM was supported by an PGS‐M scholarship.
LplT‐Aas system: The primary mechanism for lysophospholipid remodeling in E. coli
Authors: Zheng Lei , Lin Yibin , Tong Shuilong , Guan Ziqiang , Bogdanov Mikhail
Published: November 2023

Lysophospholipids (LPLs) are metabolic intermediates in phospholipid turnover and are derived from multiple endogenous and exogenous sources in bacterial membrane envelope. Distinct from their diacyl counterparts, these detergent‐like lipid molecules alter the local membrane properties and their accumulation may lead to severe membrane disruption and eventually cell lysis. LplT and Aas form a novel lysophospholipid remodeling system found exclusively in Gram‐negative bacteria. LplT is a member of Major Facilitator Superfamily and facilitates energy‐independent lipid flipping across the bacterial inner membrane. Aas catalyzes acyl transfer to LPL from acyl‐ACP on the cytoplasmic surface. The biological function and molecular mechanism of this lipid remodeling system remain largely unknown. We found that LplT‐Aas effectively facilitate remodeling of all three major bacterial phospholipids, PE, PG and cardiolipin, in E.coli. LplT imports lyso‐PE, lyso‐PG into the cells, which are subsequently acylated by the action of Aas to form their respective diacyl forms. We also identified a novel cardiolipin hydrolysis reaction by venom phospholipase A2 to form diacyl cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives are both translocated by LplT and then remodeled by Aas to form a triacyl form of cardiolipin. These results reveal the first cardiolipin remodeling mechanism in bacteria. LplT cannot transport lyso‐PC or lyso‐PA, suggesting its primary role in phospholipid repairing mechanism in the bacterial membrane envelope. To understand this novel LPL transport mechanism, we found that the substrate fatty acid chain is not required for LplT transport and its substrate binding was not inhibited by either orthophosphate or glycerol 3‐phosphate, suggesting that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Membrane phospholipids could not serve as a competitive inhibitor in vitro. This strict selectivity for lyso lipids allows LplT‐Aas to fulfill their efficient lipid repairing function in a membrane environment. Based on a structural model, we propose a “sideway sliding” mechanism to explain how a conserved membrane‐embedded α‐helical interface excludes membrane from the LplT binding site to facilitate efficient flipping of LPL across the cell membrane. Very recently, we found that LplT‐Aas play a potent role in maintaining membrane stability and integrity in the bacterial envelope and may be involved in the bacterial resistance against secretory phospholipase A2‐mediated host immune attack. These data demonstrate the first evidence for the biological function of this novel LPL remodeling system in E.coli.

Support or Funding Information

NIH grant R01GM098572
Structure‐function Analysis of Yeast Pah1 Phosphatidate Phosphatase
Authors: Park Yeonhee , Han Gil‐Soo , Carman George M
Published: November 2023

Mg2+‐dependent phosphatidate phosphatase (PAP), encoded by PAH1 in Saccharomyces cerevisiae, catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. The diacylglycerol produced in the reaction is utilized for the synthesis of triacylglycerol or the major membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. The PAP enzyme from yeast contains two conserved domains; the NLIP domain is found at the N‐terminus and the haloacid dehalogenase (HAD)‐like domain is found in the middle region of Pah1. The G80R mutation in the NLIP domain of Pah1 causes a 98% reduction in PAP activity along with other phenotypes characteristic of the pah1Δ mutation. The HAD‐like domain of Pah1 contains the DXDX (T/V) motif that is essential for catalytic activity. In this study, we carried out a structure‐function analysis of the conserved and non‐conserved regions of yeast Pah1. For constructs expressed and purified from Escherichia coli, G80R and G80A mutations, as well as deletion of the NLIP domain caused > 99% loss of PAP activity; the deletion of the non‐conserved region at the N‐terminus had little effect on PAP activity; and the deletion of the non‐conserved region at the C‐terminus caused a 3‐fold increase in PAP activity. The effects of the mutations on the in vivo function of Pah1 were examined by the ability to complement cold or warm sensitivities exhibited by the pah1Δ mutant. Cells expressing the deletion of the non‐conserved region at the N‐terminus complemented both the cold and warm sensitive phenotypes of the pah1Δ mutant, whereas the mutations in the conserved NLIP domain and the deletion of the non‐conserved region at the C‐terminus did not rescue the temperature sensitive phenotypes. The effects of the mutations on Pah1 PAP functions such as the synthesis of triacylglycerol and lipid droplet formation are being investigated.

Support or Funding Information

Supported by NIH grant GM028140
Lantibiotic synthetase C‐like protein 2 (LanCL2) is necessary for 3T3‐L1 Differentiation to Adipocytes
Authors: Dutta Debapriya , Chen Jie , van der Donk Wilfred
Published: November 2023

Adipocyte differentiation is a highly regulated process, necessary for metabolic homeostasis and nutrient sensing. The expression of peroxisome proliferator‐activated receptor gamma (PPARγ) and the subsequent transactivation of adipogenic genes is critical for adipogenesis, and is controlled through the coordinated efforts of CCAAT‐enhancer‐binding (C/EBP) proteins and a PPARγ ligand. In this study, we identified lanthionine synthetase C‐like protein 2 (LanCL2), as a positive regulator of adipogenesis in 3T3‐L1 cells. Knockdown of LanCL2, but not LanCL1, inhibited adipocyte differentiation, and this effect was not mediated through cAMP or Akt signaling pathways. The expression of early adipogenic markers C/EBPβ and C/EBPδ remained intact in LanCL2 knockdown cells, and an arrest in differentiation was observed at the stage of PPARγ activation. The addition of a PPARγ ligand, troglitazone rescued differentiation in LanCL2 knockdown cells and restored expression of late adipogenic markers. Furthermore, addition of troglitazone partially restored the transactivation potential of PPARγ. Taken together, our study reveals a novel role of LanCL2 in adipogenesis, acting through the ligand‐mediated activation and/or propagation of PPARγ signaling.

Support or Funding Information

The work was supported by the Howard Hughes Medical Institute (HHMI)
Use of the auxin‐induced degradation system to dissect factors important for exocyst assembly and localization
Authors: Heard Rachael D , Lepore Dante , Munson Mary
Published: November 2023

Exocytosis is the delivery of cargo molecules from the Golgi to the plasma membrane. In the budding yeast, S. cerevisiae, the proteins regulating the process of exocytosis are found predominantly at the bud tips in G1 phase and mother‐bud necks in G2/M phase. The exocyst complex regulates localization and fusion of secretory vesicles at these sites. Exocyst is a multi‐subunit tethering complex that is required for post‐Golgi vesicle tethering at the plasma membrane. This hetero‐octameric protein complex is composed of the following subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. Our lab recently showed that each subunit has equal stoichiometry within the complex which is predominately soluble, and all but Sec3 are essential in maintaining the structure of the complex. Using electron microscopy, the assembly and architecture of exocyst shows that this complex forms an elongated conformation consisting of two modules of four helical rods each.

We were interested in understanding the roles of different subunits in controlling the polarized localization of exocyst. Specifically, we asked what happens to the structure, stability and localization of exocyst when other subunits are degraded. An auxin induced degradation system (AID) will be used with the subunits Sec6, Sec3, and Exo70 to determine if the structure of the complex is affected by this degradation. The AID system allows for fast depletion of targeted proteins in response to the hormone auxin. We take advantage of the GFP tag on Sec8 in order to determine what subunits are still attached to Sec8 when degradation occurs, by pulling down the complex onto GFP‐bound nanobody beads. Next, we are determining where Sec8‐GFP is localized in the cell after Sec6, Sec3, and Exo70 degradation. These results will help elucidate the requirements for exocyst complex assembly, localization and secretory vesicle trafficking.

Support or Funding Information

This work supported by NIH R01GM068803 parent grant (to MM) and an R01GM068803‐11S1 Diversity Supplement (to RDH).
Mapping the Atg9 Binding Site on Atg11, a Central Organizer of the Selective Autophagy Initiation Complex
Authors: Meyer Mitchell , Winzeler Jasmine , Sheppard Damien , Backues Steven K
Published: November 2023

Autophagy is a dynamic process by which cytosolic material is sequestered into double membrane vesicles called autophagosomes and then sent to the vacuole / lysosome for degradation. Selective autophagy, which targets specific unwanted components such as a damaged mitochondria, protein aggregates and pathogens, plays central roles in innate and adaptive immunity, regulation of inflammatory responses, pathogen clearance and protection against Parkinson's and Alzheimer's diseases. Autophagy is performed by dozens of autophagy‐related proteins (Atg) that assemble in a hierarchal fashion to form the pre‐autophagosomal structure (PAS). Atg11, a protein crucial for selective autophagy in the yeast Saccharomyces Cerevisiae, contains two coiled‐coil domains, CC2 and CC3, which are the primary binding sites used to organize the autophagy initiation complex and recruit Atg9‐containing vesicles to the PAS. It is not known whether Atg11 performs both of these processes simultaneously, or whether there is competition between its binding partners that would suggest a sequential model of action. Here we use a random mutagenesis / yeast‐2‐hybrid screen to determine the specific amino acid residues in the Atg11 CC2 region that are necessary for the interaction with Atg9 in order to more precisely map its binding site. Knowledge of the specific binding sites of all of Atg11's partners will help us to determine how Atg11 organizes these proteins to promote the creation of the autophagosome and accomplish selective autophagy.

Support or Funding Information

NSF RUI Grant #1613653 “Scaffold or Assembly Line: How Does Atg11 Organize its Binding Partners for the Initiation of Selective Autophagy?” to S. K. Backues
The Effect of Cellular Autophagy on Replication and Dissemination of Theiler's Murine Encephalomyelitis Virus
Authors: Benner Lars
Published: November 2023

Autophagy is a catabolic pathway that enables the cell to sequester and degrade cytoplasmic contents and also serves as an innate response against intracellular pathogens. Many viruses have evolved to subvert this fundamental pathway for their own replication and spread. For example, poliovirus uses autophagy for replication and nonlytic dissemination. However, the replication and spread of rhinovirus type 2 has been shown to be unaffected by autophagic induction and inhibition. Theiler's Murine Encephalomyelitis Virus (TMEV), a related picornavirus, causes a demyelinating disease that serves as a model for multiple sclerosis. The effect of autophagy on cell viability, viral replication, and viral spread in TMEV infected cells has not been elucidated. By immunofluorescence staining and confocal microscopy, we have demonstrated that the 3D polymerase colocalizes with the autophagosomal marker microtubule‐associated protein light chain 3 (LC3). Furthermore, western blotting shows significant LC3 upregulation during the course of TMEV infection. Therefore, TMEV likely regulates the autophagic pathway for its replication and spread. Further understanding of how the virus utilizes autophagy may lead to novel insights regarding TMEV‐induced demyelinating disease.
Examining the Role of the AMP‐activated Protein Kinase in Stress‐Dependent O‐GlcNAc Signaling
Authors: Maduka Austin , Fahie Kamau , Zachara Natasha
Published: November 2023

Acute enhancement of O‐linked‐beta‐N‐acetylglucosamine (O‐GlcNAc) modification on intracellular proteins activates an endogenous protective program in numerous models of injury, including cardiac ischemia reperfusion injury (IR/I). The O‐GlcNAc transferase (OGT) and the O‐GlcNAcase (OGA) enzymes are responsible for adding and removing the O‐GlcNAc moiety from proteins, respectively. This project aims to understand how cells regulate OGT and OGA in response to oxidative stress to promote cell survival and cardioprotection. AMP‐activated protein kinase (AMPK) integrates cellular stress signals with energy balance to affect cellular function, and thus plays a critical role in the cellular stress response. Our data and other studies suggest a complex interplay between OGT, OGA, AMPK, and downstream kinases that may impact stress‐induced O‐GlcNAcylation. These data include: 1) mTOR interacts with OGA in response to oxidative stress; 2) OGT and AMPK interact in response to oxidative stress, and AMPK is O‐ GlcNAc modified; and 3) ULK‐1 interacts with OGT in response to oxidative stress. To explore the significance of these interactions, we are taking two approaches. Firstly, we are examining stress‐induced glycosylation in AMPK knockout MEFs. Secondly, we are generating tagged constructs of OGT and AMPK to test the impact of the interaction between these enzymes on their function, as well as the impact of O‐GlcNAcylation on AMPK function. These experiments will further elucidate the molecular mechanisms by which OGT is regulated during injury.
Identifying C‐mannosylated Peptides Involved In Hsc70 Mediated Inflammation
Authors: Kegley Nicholas Ryan
Published: November 2023

Glycosylation functions in a variety of ways including ligand recognition and to ensure only proteins which are properly folded are transferred to the Golgi apparatus. Glycoproteins are proteins in which a carbohydrate binds a specific motif of amino acid residues located on the protein. There are three binding types, including O‐linked, N‐linked, and the less commonly detected C‐linked. C‐mannosylation is a form of glycosylation in which mannose is attached via a carbon‐carbon bond with the first tryptophan in trp‐x‐x‐trp sequences. Many proteins that are instrumental in the process of inflammation have been found to possess C‐mannosyltryptophan. The primary antibody 1B5, IgG class has been shown to recognize Hsc70, a heat shock family protein that interacts with mannosylated peptides. Our objective is to use the 1B5 antibody to isolate Hsc70 ligands from cell culture. We have applied this technique to probe Hsc70 from lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophages. LPS stimulates the production of Hsc70 in macrophage cultures, allowing for analysis of ligands that associate with Hsc70. After isolating Hsc70, identification of mannosylated ligands can occur. Both LPS and non‐LPS treated cells are grown in parallel and collected at 0, 2, 24, and 48 hour time periods. GNL‐pulldowns are then used to isolate C‐mannosylated proteins and anti‐Hsc70 immunoprecipitation was utilized to isolate all Hsc70‐binding proteins. An interaction between C‐mannosylated proteins and heat shock protein 70 (Hsp70) has been found to increase the production of TNF‐α in macrophages, which can result in a strong inflammatory response, potentially leading to septicemia if unregulated. Our goal is to identify what C‐mannosylated proteins are interacting with Hsc70 by probing Hsc70 ligands and C‐mannosylated proteins.
Understanding the Effect of Community‐Based Health and Nutrition Program on Uptake of Iron and Folic Acid Supplementation and Breastfeeding Practices in a Pastoralist Community in Rural Ethiopia
Authors: Kulkarni Shibani , Frongillo Edward A , Girma Mamo
Published: November 2023

Maternal and child health and nutrition are important issues to address in Ethiopia. Poor socioeconomic context and inadequate healthcare affects availability, access, and utilization of health services. The Community‐Based Maternal and Neonatal Health and Nutrition (CBMNH‐N) project by the Micronutrient Initiative aimed at strengthening the health system capacity to improve access and quality of maternal and newborn health and nutrition services by increasing the knowledge and skill of community health workers, traditional birth attendants, and health care providers. We undertook a qualitative inquiry from the endline data from four intervention and six comparison woredas to understand the effect of CBMNH‐N project on iron and folic acid supplementation and breastfeeding practices in the Afar community in Ethiopia. We used open coding to identifying themes and analyzed 46 key informant interviews from fathers, health extension workers, midwives, traditional birth attendants, and clan leaders, and 11 focus‐group discussions with pregnant and lactating mothers. Overall, mothers, health workers, and community members in the intervention woredas were more knowledgeable about the use of iron and folic acid supplementation and showed improvement in early initiation and exclusively breastfeeding practices as compared to the comparison woredas. Mothers noted that health extension workers were instrumental in increasing knowledge and awareness of iron and folic acid supplements, and trained traditional birth attendants helped with follow‐up and compliance in taking the supplements. Program efforts helped the community, including clan leaders to be more aware and supportive of the importance of iron and folic acid supplementation during pregnancy and the post‐partum period. Community members and clan leaders also noted the importance of early initiation and exclusive breastfeeding. Most respondents also discussed the change from traditional feeding practices such as providing the baby with goat milk to putting the baby to the breast immediately after birth and exclusively breastfeeding. Mothers, health extension workers, and traditional birth attendants were aware of the health benefits of exclusively breastfeeding. Several respondents attributed their knowledge of infant feeding to health education provided by the health extension workers and traditional birth attendants. Our findings suggest that community‐based programs can build capacity, increase knowledge, and improve health practices that can ultimately promote maternal and child health and nutrition.

Support or Funding Information

Funding: Micronutrient Initiative
An Integrated Approach to Harmonize Nutrition Knowledge across Sectors in the Upper Manya Krobo District of Ghana
Authors: Birks Katherine , Marquis Grace , Matey Stephen , Owusu Raymond , Aidam Bridget , Aryeetey Richmond , Thompson‐Colón Theresa
Published: November 2023

Determinants of nutrition are addressed by multiple service delivery platforms, including government sectors such as agriculture, education, finance, and health, in addition to community members and volunteers. Consistent messages must be integrated across these platforms to make education efforts more effective, particularly among vulnerable communities. As part of the Building Capacity for Sustainable Livelihoods and Health in Ghana (Nutrition Links) project, a needs assessment was conducted to identify knowledge gaps among service providers in the Upper Manya Krobo District. An integrated, multi‐level approach was subsequently developed to address these needs: (i) training workshops (e.g., Community‐Based Growth Promotion, Mother‐to‐Mother Support Group) for health sector staff and community volunteers; (ii) workshops to harmonize nutrition promotion messages (e.g., Essential Nutrition Actions) for non‐health sector (agriculture and finance institutions) staff; and (iii) Lots Quality Assurance Sampling and data analysis workshops for staff from all government sectors (health, agriculture, education, social development, environmental health, and governance) and finance institutions. These training sessions allowed participants to interact with multiple sectors and use local data to examine specific district‐wide nutrition concerns. Each session had on average 26 people in attendance and ranged from 4 to 7 days in duration. A questionnaire was administered before and after each training session and the results were compared to determine if participant knowledge had improved. All pre‐ and post‐training comparisons demonstrated an increase in participants' knowledge (p<0.0001). Training sessions with the greatest improvement included Mother‐to‐Mother Support Group for community members (mean increase = 70 ± 11 %), Essential Nutrition Actions for staff from the agriculture sector and finance institutions (mean increase = 60 ± 15 %), and Lots Quality Assurance Sampling (mean increase = 50 ± 14 %). Integrated, multi‐sector training sessions can be useful in harmonizing nutrition knowledge and may provide opportunities for improving communication and coordination of services across sectors.

Support or Funding Information

Global Affairs Canada, McGill University, World Vision
Integrated DHS and National Micronutrient Survey: A Case Study from Malawi
Authors: Rhodes Elizabeth , Tripp Katie , Mshali Glory , Nyirenda Eunice , Suchdev Parminder
Published: November 2023

Objective

To document lessons learned from an integrated Micronutrient Survey (MNS) and Demographic and Health Survey (DHS) in Malawi from 2015–2016.

Methods

We conducted semi‐structured, in‐depth interviews with 26 individuals who planned and implemented the integrated MNS‐DHS. We used purposive recruitment to ensure that we captured a diversity of views and interviewed individuals from each of the partner organizations. Interviews were conducted in‐person or by telephone and audio recorded. We analyzed the data using thematic analysis to identify key themes related to the benefits, drivers of success, and challenges of an integrated MNS‐DHS.

Results

Preliminary findings indicate that an integrated approach increased data collection efficiency, reduced time burden for respondents, and enabled research inquiries into the socio‐demographic determinants of micronutrient deficiencies using DHS data. The perceived drivers of success for this integration included investment in coordination activities and regular communication among the sub‐teams carrying out the DHS and MNS. A primary challenge was overcoming implementation obstacles (e.g., delays in procuring equipment, lengthy decision‐making processes due to the command chain structure) and ensuring the DHS and MNS began and ended simultaneously.

Conclusions

An integrated MNS‐DHS was feasible, efficient, and acceptable to respondents in Malawi. Further, the integration of the surveys can help from a research perspective; it provides the opportunity to examine the determinants of micronutrient deficiencies using DHS data. The lessons learned in Malawi can inform other countries on how to best integrate MNS and DHS, which can provide nationally representative nutrition and inflammation biomarkers in the same cycle as DHS.

Support or Funding Information

Emory University