bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural insights into RNA recognition and activation by innate immune pattern‐recognition receptor RIG‐I
Authors: Jiang Fuguo , Ramanathan Anand , Miller Matthew T. , Tang Guo-Qing , Gale Michael , Patel Smita S. , Marcotrigiano Joseph
Published: June 2021

RIG‐I (Retinoic acid Inducible Gene‐I) is a cytoplasmic pathogen recognition receptor that differentiates between viral and cellular RNAs to trigger the innate immune response. RIG‐I comprises N‐terminal CAspase Recruitment Domains (CARDs), a DExH/D box helicase/ATPase domain and a C‐terminal Repressor Domain (RD). The helicase and repressor domains (RD) of RIG‐I recognize double‐stranded (ds) RNA and 5′‐triphosphate (ppp) RNA as foreign and activate the RIG‐I CARDs domain for downstream signaling. To understand the synergy between helicase and RD for RNA binding and how ATP hydrolysis contributes to RIG‐I activation, we determined the crystal structure of human RIG‐I helicase‐RD in complex with dsRNA and an ATP‐analog. Helicase‐RD organizes into a ring around dsRNA that caps the blunt‐end with the helicase utilizing previously uncharacterized motifs to recognize dsRNA. Small angle X‐ray scattering (SAXS), limited proteolysis, and differential scanning fluorimetry suggest that RIG‐I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full‐length RIG‐I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG‐I helicase‐RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within DICER and FANCM.
Structural basis for the regulation of endothelin‐1 mRNA stability by glyceraldehyde‐3‐phosphate dehydrogenase
Authors: Neal Jacob P. , Garcin Elsa D.
Published: June 2021

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) plays a key role in vascular homeostasis by modulating levels of the vasoconstrictor endothelin‐1 (ET‐1). Elevated ET‐1 levels are associated with vascular diseases, and its expression is tightly regulated primarily via GAPDH‐mediated destabilization of its mRNA. Recent studies suggest GAPDH binding of adenine‐uridine rich elements in the 3′‐untranslated region of ET‐1 gene leads to decreased mRNA stability. However, structural and mechanistic details underlying the GAPDH‐mediated control of ET‐1 expression are lacking. We seek to probe the structural and mechanistic basis for the GAPDH‐induced destabilization of ET‐1 mRNA via electrophoretic mobility shift assay (EMSA) and x‐ray crystallography. To identify specific GAPDH binding sequences, we constructed short RNA transcripts of the putative mRNA binding region. Utilizing a modified EMSA with biotinylated RNA transcripts, we obtained association and dissociation constants. For transcripts yielding the greatest affinity for GAPDH, we obtained crystals. Ultimately, we aim to solve the crystal structures for these ET‐1 mRNA constructs with GAPDH. These studies will provide the first molecular basis for structure‐based drug design, leading to the development of novel molecules that specifically target ET‐1 mRNA.

Funding: UMBC DRIF SRAIS (EG).
Functional complementation of Hsp90 domains
Authors: Maharaj Kevin Amit , Gill Sabrina , Gewirth Daniel T
Published: June 2021

Hsp90s are essential ATP dependent molecular chaperones required for the maturation of numerous signal transduction proteins. They exist as obligatory homodimers consisting of an N‐, Middle, and C‐domain. In higher eukaryotes there are four hsp90 paralogs: two cytosolic isoforms, Hsp90α and Hsp90β, the ER paralog GRP94 and the mitochondrial paralog TRAP‐1. Despite the high sequence similarity between cytosolic Hsp90 and GRP94, structural and biochemical studies have elucidated different nucleotide induced conformational states which account for a 5–20 fold difference in ATPase rates. To further characterize the similarities and differences between Hsp90 and GRP94, we constructed chimeras between domains of cytosolic Hsp90 and GRP94 and tested the viability of these chimeras when expressed as the sole Hsp90 in yeast. We show for the first time that Grp94 domains can functionally complement yeast Hsp90 domains. In particular, chimeric constructs consisting of Grp94 N or Grp94 MC support growth in yeast while M domain substitutions are detrimental to cell growth. These results indicate the importance of ATP binding and highlight the MC interface as an important structural unit within hsp90s. These observations also suggest a conserved mechanism of action within the hsp90s family. Finally, the GRP N chimera may be used to develop paralog specific inhibitors. Funding: NIH 5F31GM086120, NIH 2R01CA095130
The Apoptosis Related BIM Is An HSP70‐Specific Client Protein
Authors: Rodina Anna , Kang Yanlong , Taldone Tony , Patel Pallav , Patel Hardik , Chiosis Gabriela
Published: June 2021

Heat shock protein chaperones have important roles in regulating the function of cells under pathogenic stress, including cancer. Among them, heat inducible Hsp70 and constitutive Hsc70 are of especial interest because they act upstream of the major onco‐protein regulator chaperone, Hsp90, and additionally, they have potent anti‐apoptotic activities by inhibiting both intrinsic and extrinsic death pathways. Here we use a novel Hsp70/Hsc70 inhibitor, YK5, to study a mechanism of Hsp70‐dependent apoptosis in breast cancer cells. We show that the Hsp70 inhibitor activates caspase 8 – depended apoptotic pathways and induces cleavage of the apoptotic protein Bim. We show through chemical precipitation that Bim physically associates with the Hsp70 protein, demonstrating that it is an Hsp70 client protein. Following Hsp70 inhibitor treatment, we identify the formation of a cleaved Bim product, a Bim‐species previously demonstrated to be more efficient at inducing apoptosis than the full‐length protein. Proteasome and serine proteases are involved in Hsp70 – depended Bim degradation and cleavage, and inhibition of serine proteases correlates with a decrease in the Hsp70 inhibitor – induces apoptosis. The data suggest Bim to be an important player in Hsp70 ‐ regulated apoptosis inhibition in cancer cells, and indicate Hsp70 as a potential inhibitor of the formation of the hyperactive cleaved Bim species.
Time‐dependent inhibitors of Lymphoid Tyrosine Phosphatase
Authors: Ahmed Vanessa , Barrios Amy M
Published: June 2021

Lymphoid tyrosine phosphatase (Lyp) is a 105‐kDa Class I protein tyrosine phosphatase (PTP) encoded by the PTPN22 gene and is expressed exclusively in immune cells. Lyp is known to potently inhibit T cell receptor signaling by dephosphorylating several substrates, including the Src family kinases Lck and Fyn, ZAP70, and TCRzeta. Lyp has recently emerged as a key player in a range of human autoimmune diseases including type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, Graves’ and Addison's disease. The basis for Lyp's involvement in autoimmune disease is a single nucleotide polymorphism in PTPN22 that results in an amino acid change from Arg620 to Trp620 in the non‐catalytic C‐terminal domain of Lyp. The result is an increase in phosphatase activity although the molecular basis is not well understood. To reverse the pathogenic effect of this Lyp‐W620 variant, small molecule inhibitors are desired for therapeutic treatment of autoimmune disorders. The objective of the work presented here is to develop small molecules that can function as Michael acceptors to inactivate Lyp activity in vitro. Kinetic analysis has shown that these inhibitors inhibit Lyp in a time‐ and concentration‐dependent manner under pseudo‐first order conditions, and that Lyp activity does not return after extensive dialysis. Furthermore, the compounds appear to bind in the active site, as inhibition is reduced by co‐incubation of the enzyme with a known competitive inhibitor of PTPs. Further characterization is underway to gain more information on the mechanism of inhibition and selectivity among other members of the PTP family.
Toward development of selective DXP synthase inhibitors
Authors: Freel Meyers Caren L. , Morris Francine
Published: June 2021

The methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most human pathogens, including Mycobacterium tuberculosis and the malaria parasite Plasmodium falciparum. The first enzyme in this pathway, 1‐deoxy‐D‐xylulose 5‐phosphate (DXP) synthase, catalyzes the thiamin diphosphate (ThDP)‐dependent formation of DXP from D‐glyceraldehyde 3‐phosphate (GAP) and pyruvate. Selective inhibition of this ThDP‐dependent transformation is a challenging goal in the development of isoprenoid biosynthesis inhibitors. However, we have determined that DXP synthase follows a random sequential mechanism, distinguishing it mechanistically from other ThDP‐dependent enzymes; thus, we are pursuing it as a potential drug target for the development of new anti‐infective agents. We hypothesize that substrate specificity studies will guide the design of selective inhibitors of DXP synthase. We have utilized nitroso compounds as reactive aldehyde isosteres to investigate substrate specificity and have shown that DXP synthase catalyzes C‐N bond formation using nitroso acceptor substrates. Nitrosonaphthols show remarkably high affinity for DXP synthase as substrates, but low affinity for the related ThDP‐dependent enzyme, PDH. In contrast, these alternative substrates for DXP synthase are weak inhibitors of DXP formation, suggestive of distinct substrate and inhibitor binding modes for this compound class. Our results point to opportunities for development of naphthyl–containing inhibitors that target the unique mechanism of DXP synthase.
Recovery of Activity and Stability for Clinical Mutants of Aspartoacylase
Authors: Zano Stephen Penalosa , Wijayasinghe Yasanandana , Smith Joshua , Maltbie Grace Ann , Viola Ronald
Published: June 2021

Canavan disease is a fatal neurological disorder caused by mutations in the aspA gene, leading to an altered aspartoacylase with diminished catalytic activity. These clinical mutations are found at sites located throughout the enzyme, with majority found far from the catalytically important amino acids. This suggests that destabilization of the protein structure may be responsible for the loss of activity of the enzyme activity. A number of different clinical mutants of aspartoacylase have been cloned, expressed and purified. These purified enzymes were found to have low catalytic activity and diminished thermal and ionic stability as compared to the wild type enzyme. Docking studies have suggested small molecules that could bind at these mutation sites, and potentially stabilize and recover the catalytic activity of these clinical mutants. Screening and optimization of these compounds, guided by kinetic, thermodynamic and structural studies, could lead to pharmacological chaperones that have the potential to reverse the effect of the mutation in Canavan patients.
PCR markers, stress, and healthy aging
Authors: Perry Cynthia , Lu Kristy , Alarcon Tatiana , Hedges Dawson , Nielsen Brent L.
Published: June 2021

The objective of this work is to determine if there is a correlation between genetic markers associated with the dopamine receptor gene (DRD2) (linked to Post‐Traumatic Stress Disorder (PTSD)) and glucocorticoid receptor (GR) (linked with stress level) and the physical activity level of older adults. Saliva samples were collected from participants in the Huntsman World Senior Games and from a second group of older adults living in Utah County, Utah. Surveys about physical and emotional wellness were also administered to all participants. DNA was isolated from the saliva samples and PCR was run with primers specific to each target. Restriction enzymes were used to cut the PCR products, which were then run on agarose gels, and band patterns were assessed based on published findings for each gene. Single sample z‐tests were performed for each gene. For the GR gene, the results show a significant increase in the genotype GG (z score: 2.63) in the Senior Games population. For DRD2, the homozygous genotypes (A1, A2) were more prevalent in the Senior Games population (4, 5.36 respectively). The heterozygous allele (A1A2) was highly prevalent in the Utah County sample population (−8.15). The higher prevalence of alleles correlated with stress in the Senior Games population was unexpected but may indicate an association of these alleles with healthy aging. This work was supported by a grant from the BYU Gerontology Program.
Sustained hypercalcemia primes non‐invasive breast cancer cells for metastasis to high calcium microenvironments
Authors: Sakwe Amos M , Koumangoye Rainelli , Ochieng Josiah
Published: June 2021

Breast cancer frequently spreads to calcium‐rich organs/metastatic sites such as skeletal tissues. While patients with metastatic or end‐stage cancer are likely to become hypercalcemic, up to 30% of breast cancer patients develop cancer‐induced hypercalcemia (CIH) in the absence of metastases or bone diseases. The resulting high extracellular ionized calcium (Ca2+) activates the calcium sensing receptor (CaSR) that promotes cell proliferation and the secretion of parathyroid hormone‐related protein by tumor cells. This suggests that CIH promotes both the vicious osteolytic cycle and breast cancer progression. In this study, we examined whether sustained hypercalcemia primes breast cancer cells for metastasis to high Ca2+ microenvironments. We demonstrate that sustained hypercalcemia enables Ca2+‐sensitive non invasive breast cancer cells such as MCF‐7 to become high Ca2+ adapted and that this adaptation is accompanied by increased motility and more aggressive growth. Moreover, breast cancer cells expressing the Q1011E variant (common among African Americans)grow faster at high Ca2+ than those expressing either the wild type or the A986S variant (common among Caucasians)of the CaSR. These data suggest that differences in the adaptation of breast cancer cells to hypercalcemia due in part to the expression of distinct CaSR variants underlie the distinct race‐related breast cancer outcomes.
BRAFV600E‐ and PI3′K‐activated signaling pathways cooperate to regulate phosphorylation of ribosomal protein S6 in human melanoma cells
Authors: Silva Jillian M , McMahon Martin
Published: June 2021

The most common genetic alteration in metastatic melanoma is a T1799A transversion encoding BRAFV600E that results in the constitutive activation of the BRAF→MEK→ERK MAP kinase pathway. In many cases, conversion of BRAFV600E expressing melanocytes to melanoma cells also requires activation of PI3′K signaling, usually through silencing of the tumor suppressor PTEN. To explore the mechanisms of cooperation between the BRAFV600E and PI3′K signaling pathways, we applied pharmacological inhibitors of each pathway to BRAFV600E expressing melanoma‐derived cell lines. Our data indicates that blockade of BRAFV600E→MEK→ERK signaling inhibited p70S6K and ribosomal protein S6 phosphorylation, whereas inhibition of PI3′K suppressed S6 and 4E‐BP1 phosphorylation. In addition, inhibition of BRAFV600E→MEK→ERK or PI3′K blockade also led to the inhibition of melanoma cell proliferation. Moreover, simultaneous inhibition of both the BRAFV600E and PI3′K pathways abrogated the phosphorylation of the cap‐dependent translational regulators, p70S6K, S6, and 4E‐BP1. However, pharmacological inhibition of the PI3′K effector, AKT, had little effect on cell growth and failed to influence p70S6K, S6 or 4E‐BP1 phosphorylation. Thus, these results suggest that the regulation of S6 may serve as a downstream integrator of cooperation between the BRAFV600E and PI3′K pathways independent of AKT activation in melanoma cells.
Trichosanthis Radix inhibits 3T3‐L1 adipogenesis
Authors: Lin Chia-Yu , Shen Yi-Ting , Li Yi-Ying , Huang Yu-Ling , Lai Jui-Sheng
Published: June 2021

There is an increase in the prevalence of obesity and its related health problems. While dietary management and exercise help reduce the accumulation of body fat, recently attention has also been drawn to many medicinal herbs as the possible strategies for fat reduction in addition to medications. In the present study, we examined the effects of extracts from Trichosanthis Radix by ethanol (RE) or water (RW) on the differentiation of 3T3‐L1. Extracts were applied either during differentiation or after induction of cell differentiation for 8 days. We tested fat droplets accumulation by Oil Red O staining and triglyceride assay at the end of study. Treatment of differentiating 3T3‐L1 cells with RE or RW shows a significant decreased fat accumulation (% of reduction: RE 40%~50% and RW 50%~60%). Application of these extracts after the formation of adipocytes reduces fat accumulation to a lesser extent. To study the underlying mechanisms, we analyze the expressions of SRBP‐1, PPARγ and C/EBPα, which are important transcription factors for cell differentiation. Treatment of RW is able to reduce their expressions during early cell differentiation. In conclusion, these results indicate that water extracts of Trichosanthis Radix are more effective in inhibition of fat accumulation which is possible mediated through the decreased expressions of SRBP‐1, PPARγ and C/EBPα.
Substrain‐specific cholesterol‐lowering effects of oats in mice: ‐ Clues for mechanism of action?
Authors: Andersson Kristina , Andersson Ulrika , Xu Jie , Swärd Karl , Ahrné Siv , Molin Göran , Holm Cecilia , Hellstrand Per
Published: June 2021

Two substrains of C57BL/6 mice were recently shown to respond differently to oats with respect to changes in plasma cholesterol. By using this substrain difference we aimed to gain more knowledge about the mechanisms behind the cholesterol‐lowering effect of oats. Mice were fed an atherogenic diet supplemented with oat bran (27 %) or control fibers (cellulose). In C57BL/6NCrl mice plasma cholesterol was reduced by 15 %, the mRNA expression of the rate‐limiting enzyme for bile acid production, CYP7A1, was up‐regulated by fourfold, and the excretion of bile acids was increased by 40 %. In C57BL/6JBomTac mice there was no sustained reduction of plasma cholesterol, no increased excretion of bile acids, and CYP7A1 was down‐regulated with oat bran in the diet. The two substrains also differed regarding diversity and composition of intestinal microbiota. The C57BL/6NCrl mice had higher abundance of the bile acid de‐conjugating bacterial group of Bacteroides fragilis, which was negatively correlated to plasma cholesterol levels and positively correlated to CYP7A1 up‐regulation. No difference was found regarding glucose tolerance. Our findings provide evidence that the cholesterol‐lowering properties of oats involve increased production of bile acids with up‐regulation of CYP7A1. The effects of oats on bile acid excretion may be influenced by the composition of the intestinal microbiota.
Neuroprotection of estrogen on hypoglycemic attack is through AKT/GSK3b pathway
Authors: Alecea Eilliut , Yohanan Darien , Cohen Joshua , Addya Sankar , Chakraborty Sanjoy , Chakraborty Tandra R.
Published: June 2021

Glucose is the main metabolic fuel of the brain. and its availability is directly linked to neuronal activity. In the absence of glucose i.e. hypoglycemia, there is loss of neurons, impaired IQ and can ultimately lead to morbidity. In the present study we have reported the neuroprotection of estrogen on hypoglycemic injury in hypothalamic cell line. There was a marked decrease in the cell count and mitochondrial function when exposed to hypoglycemia which increased in presence of estrogen (42.8 vs 98.5, 24 hours, p<0.0001). The attenuation of the death pathways was only seen within the first 24 hours and gradually decreased with time. DNA microarray studies showed AKT, a promoter of cell survival, was down‐regulation in the absence of glucose and in the presence of estrogen an increase in gene expression. RTPCR showed decrease in AKT expression with a slight increase in presence of estrogen. PRAS 40, proline rich substrate for AKT was measured. Results show low expression of PRAS40 in hypoglycemic conditions in 24 hours and 72 hours with a trend to increase in presence of estrogen. Therefore, it seems that one of the mechanism by which estrogen shows its neuroprotection is through the Akt‐GSK3b pathway activation.
AKAP79 Controls β1‐Adrenergic Receptor Trafficking by Multiple Regulatory Mechanisms
Authors: li xin , Bahouth Suleiman
Published: June 2021

Chronic activation of G protein‐coupled receptors promotes their endocytosis from cell membranes into intracellular endosomes. Efficient trafficking of these receptors back into the cell membrane is critical for receptor resensitization. Trafficking of many GPCR is dependent on cis‐acting domains and trans‐acting factors. Examples of cis‐acting domains in the β1‐adrenergic receptor (β1‐AR) are the type‐1 PDZ domain located in the carboxy‐terminus and serine at position 312 in the 3rd intra‐cellular loop of the β1‐AR., which is a substrate for phosphorylation by the cyclic AMP‐dependent protein kinase (PKA). In the β1‐AR, the PDZ type‐1 domain binds to a complex composed of synapse associated protein 97 (SAP97) and A‐kinase anchoring protein‐79 (AKAP79). The c‐terminus of AKAP79 in turn binds PKA, which phosphorylates Ser312 upon the activation of the β1‐AR signaling pathway.

Preliminary studies showed that AKAP79 was involved in trafficking of the β1‐AR, but its precise mechanism was obscure. In this study, we determined that selective down regulation of AKAP79 inhibited the recycling of the WT β1‐AR in HEK‐293 cells and its restoration rescued the recycling of the agonist‐internalized β1‐AR. There are several functional domains in AKAP79 including PKA and PKC binding, membrane targeting/binding, SAP79 and calcineurin binding. We analyzed by confocal microscopy the effect of deleting each of these domains on the ability of the resulting AKAP79 mutant to rescue the recycling of the β1‐AR. Our data revealed that multiple domains of AKAP79 were required, indicating that the effect of AKAP79 on recycling of the β1‐AR occurred by multiple divergent mechanisms. Therefore membrane targeting of AKAP79 and its binding to other accessory protein were all required for AKAP79‐mediated regulation of β1‐AR trafficking. Funded by NIH HL‐085848.
Constitutive K‐RasG12D Activation of ERK2 Specifically Regulates 3D Invasion of Human Pancreatic Cancer Cells via MMP‐1
Authors: Botta Gregory P , Reginato Mauricio J , Rustgi Anil K , Lelkes Peter I
Published: June 2021

Pancreatic ductal adenocarcinomas (PDAC) are highly invasive & metastatic neoplasms unresponsive to current therapies. Overwhelmingly, PDAC harbors constitutive, oncogenic mutations in K‐RasG12D that exist prior to invasion. Histologic & genetic analyses of human PDAC exhibit increased expression of ERK1/2 and pro‐invasive MMPs; indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K‐Ras – ERK1/2 signaling & its influence on MMP‐directed stromal invasion in primary human pancreatic ductal epithelial cells (PDECs) has yet to be elucidated in 3D. Expression of oncogenic K‐RasG12D alone in genetically‐defined PDECs reveals increased invadopodia & epithelial‐to‐mesenchymal transition markers, but only when cultured in a 3D basement membrane (BM) model. Activation of ERK2, but not ERK1, occurs only in K‐RasG12D mutated PDECs cultured in 3D & is necessary for invasion based upon pharmacologic & shRNA inhibition. Increased invasion of K‐RasG12D PDECs through the BM is associated with a specific microarray gene signature & induction of MMPs. Specifically, MMP‐1 RNA, protein, & proteolysis are amplified in K‐RasG12D PDECs when assayed by RT‐PCR, ELISA, & FRET. Importantly, MMP‐1 silencing mimics ERK2 inhibition & disrupts vertical invasion. ERK2‐isoform & MMP‐1 targeting in 3D are shown to be viable strategies to attenuate invasion of K‐RasG12D mutated human pancreatic cancer cells. GPB is funded by an F30 DK088402‐01.
Transmembrane (TMEM)‐176A and 176B Proteins Are Directly Linked to Cancer Pathology
Authors: Valluri Vinod Kumar , Podevin William , Bui Quang , Nguyen Van , Cuajungco Math P
Published: June 2021

The immune system plays a role in the fight against cancer, but it has also been found to promote cancer proliferation. Two transmembrane (TMEM) proteins, TMEM176A and 176B, were reported to play a role in the immune cell function. Recent reports showed that TMEM176A mRNA transcripts are highly elevated in liver cancer or kidney proteinuria, while increased TMEM176B mRNA transcripts are detected in tolerated tissue allografts. We set out to determine the functional relationship between TMEM176A and 176B, as well as to provide any direct evidence that one or both proteins are linked to cancer pathology. Real‐time QPCR revealed a diverse mRNA expression pattern for TMEM176A and 176B. Co‐immunoprecipitation showed that both proteins physically interact. TMEM176A and 176B protein expression analysis showed significant elevation in lymphoma compared to normal non‐cancerous tissues. In lung cancer, the protein levels of TMEM176A were also significantly increased. Interestingly, the protein expression ratio of TMEM176A over TMEM176B, revealed significant differences between normal and cancer tissues of the breast, lymph, skin, and liver. Our findings indicate that TMEM176A and 176B proteins physically interact, and that the protein level of TMEM176B is intimately linked to that of TMEM176A protein in distinct cancer tissues.
Trypanosoma brucei Tim50 is a mitochondrial inner membrane protein translocator, which regulates expression and function of VDAC
Authors: Duncan Melanie R , Fullerton Marjorie S , Singha Ujjal K , Chaudhuri Minu
Published: June 2021

A homolog of Tim50 was characterized in Trypanosoma brucei, a parasitic protozoan and the causative agent of human African trypanosomiasis. T. brucei Tim50 (TbTim50) possesses an N‐terminal mitochondrial targeting sequence, a transmembrane domain and a C‐terminal phosphatase domain. Unlike other Tim50 proteins, the transmembrane domain is located near the C‐terminus. TbTim50 separates as a 52‐kDa protein on a denaturing gel, which is larger than its predicted molecular size of 47 kDa, suggesting possible post‐translational modification. TbTim50 is localized in the mitochondrial inner membrane and interacts with TbTim17. It is required for the import of N‐terminal presequence‐containing nuclear encoded proteins into the mitochondria. Induction of TbTim50 RNAi decreased mitochondrial membrane potential, without affecting the levels of many mitochondrial proteins, except for VDAC and TbTim17, which were upregulated. Knockdown or overexpression of TbTim50 reduced cell growth. Unlike its fungal counterpart, TbTim50 possesses phosphatase activity. We hypothesize that TbTim50 regulates phosphorylation of certain mitochondrial proteins such as VDAC. Supported by NIH grants SC1GM081146 and 5T32HL007737‐17.
The actions of SNAREs and Munc18 in neuronal vesicle fusion
Authors: Shen Jingshi
Published: June 2021

SNAREs are believed to constitute the core engine of intracellular membrane fusion. Fusion is initiated when the SNARE motifs of the vesicle‐rooted SNARE (v‐SNARE) and the target membrane‐associated SNAREs (t‐SNAREs) zipper into a four‐helix coiled‐coil bundle (trans‐SNARE complex) between two apposed bilayers. N‐ to C‐terminal zippering of the SNARE core bundle brings two membranes into close proximity to fuse. In neuronal vesicle fusion (neurotransmitter release), mutations of individual SNARE layer residues can strongly inhibit vesicle fusion. It is generally thought that the fusion inhibition is caused by defects in the SNARE bundle assembly. However, our recent biochemical and biophysical data showed that SNARE layer mutations have little effects on the SNARE bundle formation or its fusogenicity. Unexpectedly, we found that mutations of SNARE layer residues selectively block the stimulatory activity of Munc18‐1, a soluble factor belonging to the SM protein family. We will present biochemical, biophysical and genetic evidence to support a model whereby Munc18‐1 acts upon a partially‐zippered SNARE bundle in the fusion reaction. Intriguingly, our data also suggest that Munc18‐1 binding is fully compatible with the association of complexin with the SNAREs, implying that SNAREs, Munc18‐1 and complexin may form a pre‐fusion supracomplex.
Short‐hairpin RNA‐mediated silencing of NKCC1 in COS7 cells and its effect on cell volume
Authors: Alshahrani Saeed Ayed , Di Fulvio Mauricio
Published: June 2021

Na+K+2Cl− cotransporters (NKCCs) effect the electroneutral movement of Na+, K+ and 2Cl− across the plasma membrane. Under physiological conditions NKCCs function as active, Na+‐dependent, Cl− uptake transporters. Two NKCC isoforms, encoded by two different genes have been identified: NKCC1 and NKCC2. NKCC1 is a ubiquitously expressed gene whereas NKCC2 is considered to be exclusive of the kidney. We describe for the first time a viable knock‐down strategy to silence endogenous NKCC1 expression in a widely used, easy to transfect cell line. NKCC1 mRNA expression in COS7 cells was eliminated transfecting these cells with a poly‐cistronic vector encoding for GFP and an shRNA directed against the third exon of human NKCC1. Stable transfectants were selected by continuous incubation in the presence of puromycin. Cells stably expressing GFP and resistant to the drug were selected and cloned. NKCC1 expression was followed by Western bolt, immunofluorescence and reverse transcription (RT) coupled to the polymerase chain reaction (PCR). The impact of NKCC1 depletion on cell volume was followed morphometrically. We found that cells lacking NKCC1 are shrunken suggesting that NKCC1 is the sole regulator of cell volume in these cells. This new cell system will open the possibility to study the functional, pharmacological and molecular properties of candidate genes involved in volume regulation.

This work was supported by funds awarded to MDiF by the American Diabetes Association (Grant JF‐1001730).
The Effect of Phenolic Compounds on the Transport of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine in Caco‐2 Cell Monolayers
Authors: Hong Yun-Jin , Nam Mi-Hyun , Oh Ji-Sun , Oh Jun-Gu , Lee Kwang-Won
Published: June 2021

The goal of this study was to investigate the effect of caffeic acid on the transport of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) in Caco‐2 cell monolayer and the expression of individual efflux transporters. PhIP is formed as a by‐product of Maillard reaction during cooking or frying of protein‐rich foods at high temperatures. PhIP is metabolized in the liver by cytochromes P450 1A1 and 1A2 to its carcinogenic metabolite N‐hydroxy PhIP. The ABC transporters are capable of transporting the food‐borne procarcinogen PhIP back to the intestinal lumen. P‐gp, MRP2 and BCRP in the apical membrane of the enterocyte are involved in the efflux of compounds from the enterocytes. In the present study, MTT assay was performed to confirm the cell viability was not affected by sample concentration and incubation period. The uptake and efflux of PhIP was assessed by determining bidirectional apparent permeability coefficients. Results showed that the uptake of PhIP was reduced in the presence of caffeic acid. The efflux ratio of PhIP in the presence of caffeic acid was also significantly decreased, compared with control (p<0.05). Caco‐2 cells exposed to caffeic acid for 3h also exhibited higher mRNA levels of P‐glycoprotein, BCRP, and Multidrug Resistance Protein 2 than that of control (p<0.05). These results indicated that caffeic acid may reduce uptake and increase efflux of PhIP by up‐regulating efflux transporter mRNA levels.
Expression of human C‐reactive protein in Pichia pastoris
Authors: Smith Eliot T , Thirumalai Avinash , Singh Sanjay K , Agrawal Alok , Johnson David A
Published: June 2021

C‐reactive protein (CRP) is an acute phase plasma protein produced by the liver. The biosynthesis of CRP and its circulating concentration dramatically increase in inflammatory states; however, the in vivo functions of CRP have not been defined. To investigate the functions of CRP employing animal models of inflammatory diseases, there is a need for an in vitro expression system from which CRP can be obtained in large quantity. We developed a eukaryotic expression system for CRP using the yeast Pichia pastoris. The cDNA encoding human CRP was codon‐optimized via gene synthesis for expression in P. pastoris. The protein was directed for secretion by the incorporation of yeast α‐mating factor as an amino‐terminal fusion domain that is removed during secretion. CRP was successfully expressed and could be isolated from the culture media by affinity chromatography based on the calcium‐dependent binding specificity of CRP for phosphocholine. The pentameric structure and the phosphocholinebinding activity of recombinant CRP were not different from that of native CRP. Further characterization, such as the complement‐activating function, of yeast‐expressed recombinant CRP is in progress. The yeast expression system produces endotoxin‐free CRP and is also beneficial for expressing various CRP mutants which can be used to investigate structure‐function relationships of CRP. NIH grants R15HL091770 and R01HL071233
REGULATION OF CANNABINOID RECEPTOR SIGNALING BY RGS (REGULATORS OF G‐PROTEIN SIGNALING) PROTEINS: A NEW PARADIGM FOR MODULATING CB1 AND CB2 RECEPTOR‐MEDIATED SIGNALOSOME
Authors: Mukhopadhyay Somnath , Jha Shalini , Poltoratsky Vladimir , LaRoche Genevieve , GiGuere Patrick , Oestreich Emily , Siderovski David
Published: June 2021

CB1 and CB2 cannabinoid receptors belong to Gi protein‐coupled receptor superfamily. RGS proteins are classically known for turning off Gi and Gq‐coupled receptor signaling. CB1 and CB2 cannabinoid receptors are known to elicit a plethora of cellular responses acting through different G‐proteins. However, till date almost nothing is known about RGS‐mediated regulation of cannabinoid receptor signal transduction. In the current study we attempt to define the role of RGS‐proteins in the regulation of CB1 and CB2 receptor signaling in neuronal and endothelial cells respectively.

In the current study using real‐time PCR we first screened for native expression of individual RGS proteins in CB1 and CB2 receptor expressing neuronal and endothelial cells lines respectively. Once we found the expression profile of RGS proteins in the specific cell lines we selectively knock down (using SiRNA ) individual RGS proteins in these cell lines. We then measured nitric oxide production following CB1 or CB2 receptor activation in these RGS‐knock down cell lines to determine the specificity of RGS proteins for intercepting CB1 vs. CB2 receptor signaling. Our initial findings showed that a) different RGS proteins are coupled to CB1 and CB receptor signaling and b) RGS proteins can differentially regulate ( inhibit or activate) CB1 and/or CB2 receptor‐mediated nitric oxide production. These results suggest that RGS proteins play a pivotal role in the regulation of cannabinoid receptor signalosomes.
Regulation of D5 dopamine receptor signaling in lipid rafts in HEK‐293 Cells
Authors: Zhang Yangron , Li Hewang , Jose Pedro A , Yu Peiying
Published: June 2021

We tested the hypothesis that the D5R is regulated by caveolae in lipid rafts (LRs) using human D5R or D1R heterologously expressed in HEK‐293 cells (n=3). The D5R protein was localized in LRs; the molecular sizes of D5R protein ranged from 45 to 250 kDa. The D5Rs co‐fractionated with caveolin‐2β (cav‐2β), flotillin‐ 1 (flo‐1) and flotillin‐2 (flo‐2), GSα, and several signaling molecules. Disruption of LRs with βCD (2%/1hr) increased basal cAMP accumulation 3‐fold but agonist stimulation with fenoldopam (fen, 5 μM/15 min) decreased cAMP accumulation by 30% (vehicle=10.1 and fen =7.4 pmol/mg protein/min). D5R co‐localized and co‐immunoprecipitated with cav‐2β. Fen also increased the amount of cav‐2β associated with D5R (vehicle=14.6±8.3, fen=53±4.7, density units [DU]), similar to D1R (not shown). However, D1R and D5R differently regulate LR proteins. Fen increased the amount of flo‐1 associated with cav‐2β (fen=36.6±3.6 vs. vehicle=24.7±2.8 DU) (ANOVA, P<0.05) in HEK‐D1R cells while fen increased the amount of flo‐2 associated with cav‐2β (fen=39.7±8.1 vs. vehicle=23.9±3.5 DU) (ANOVA, P<0.05) in HEK‐D5R cells. In addition, fen increased tyrosine phosphorylation in HEK‐D5R cells. Taken together, D5R is mainly distributed in LRs and associated with and regulated by cav‐2β but are differentially associated with flotillin; D1R with flo‐1 and D5R with flo‐2. D5R signaling also involves tyrosine phosphorylation.
The Role of Nro1p in Recognition and Signaling the Presence of Dietary Unsaturated Fatty Acids
Authors: Willey Marshall , Stukey Joseph , McDonough Virginia
Published: June 2021

The OLE1 gene in Saccharomyces cerevisiae encodes the sole fatty acyl desaturase in that species. OLE1 gene expression is controlled in part through transcriptional regulators Mga2p and Spt23p. These proteins reside in the ER, and when an insufficient supply of unsaturated fatty acids (UFAs) is detected, they are proteolytically cleaved and translocated into the nucleus, where they activate OLE1 expression. Recently our lab has isolated a mutant that is deficient in regulation of OLE1, called nro1 (no regulation of OLE1). The mechanism for the NRO1 protein's action is unknown. In this study, we report that growth tests using a reporter gene under control of the OLE1 promoter demonstrate that in wild type cells, normal regulation is observed with the UFAs 16:1Δ9 and 18:2Δ9, 12 but not with UFAs 18:1Δ9 or 17:1Δ9. Fatty acid profiles of nro1 mutant cells are similar to wildtype when cells are supplemented with the various UFAs, showing evidence of post‐transciptional regulation. Results examining the effect of nro1 on the proteolytic processing of Mga2p and Spt23p through western analysis are presented. Taken together, there is evidence that the NRO1 system helps to regulate OLE1 expression in response to fatty acids 16:1Δ9 and 18:2Δ9, 12, but not 18:1Δ9 or 17:1Δ9. Supported by a Summer Research Scholar Award from Hope College Biology Department
CT‐SCAN OF THE ENTIRE HEAD OF AN OYSTRICH (STRUTHIO CAMELUS)
Authors: Le Floch-Prigent Patrice Pierre , SENECAIL BERNARD , UHL Jean-François
Published: June 2021

The entire head of an oystrich (Struthio camelus) was frozen. Its main dimensions were: 175mm long, 70mm wide, 75 mm high. The eyes were filled with several ml of formaldehyde. It was then systematically CT‐scanned in the Morvan hospital in Brest (France). 31 cross‐sections in the frontal plane, every 5mm, were kept for the study from the tip of the beak (total length: 115mm) to the cervico‐occipital, last cross‐section. The CT‐scan detailed very well the morphology of the beak which was very flat, and the internal structure of the nasal cavities: a mazzled, tightened structure. The ocular bulbs (28mm) were very large, quite joined on the middle plane. The brain was voluminous, located behind the brain with a maximal transverse diameter of 36mm. The cranial vault was very pneumatized, from the roof to the basis of the cranium, as for the lateral walls.
Investigating the role of a membrane J‐protein in ER quality control and viral trafficking
Authors: Walczak Christopher Paul , Tsai Billy
Published: June 2021

The host‐cell entry pathway of nonenveloped polyomaviruses requires trafficking to the endoplasmic reticulum (ER) with subsequent ER‐to‐cytosol transport for successful infection. We aim to understand how these viruses cross the ER membrane to reach the cytosol and deliver their DNA genome to the nucleus for replication. The ER membrane protein DnaJB14 (B14) was previously identified as a host‐factor involved in the ER‐to‐cytosol transport of polyomaviruses; however, its normal cellular function is unknown. This study has characterized B14 as a type II membrane protein coupled to the cytosolic chaperone Hsp70. Using an siRNA approach, we found that B14 normally functions to stabilize several misfolded proteins, which are substrates of ER‐associated degradation (ERAD). Intriguingly, B14 binds to the highly similar DnaJB12, a protein that instead promotes the degradation of ERAD substrates. Thus, B14 appears to be a multifunctional membrane protein promoting ER‐to‐cytosol transport of polyomaviruses and providing stability to misfolded proteins.
Nitric Oxide in Male Infertility
Authors: Thyagarajan Hiran Maye , Tiwari Kirti K , Rashid Mohammad B
Published: June 2021

Nitric oxide (NO) is differentially formed in almost all types of human cells by two constitutive and one inducible form of nitric oxide synthases (NOS). The possible role of NO in fertility has been proposed by many recent publications. Studies with rats have reported that NO has both normal physiological effect on reproduction and inflammation based infertility. NO seems to be beneficial to sperm viability and motility in both fertile and infertile individuals. However, NO has been implicated in obstructive azoospermia, a common cause of infertility. Like constitutive NOSs, inducible nitric oxide synthase (iNOS) also takes part in protein‐protein interaction that may modulate its function. To further analyze the role of iNOS in NO related infertility we have recently employed a Yeast Two‐hybrid assay where N‐terminus of iNOS was used as the bait since three isoforms varied most in that region. This could possibly identify new putative interacting proteins. We have identified multiple candidate proteins from testis cDNA library in the initial screening and have short listed few of them upon re‐transformation into yeast cells and mating them with bait‐transformed cells. Currently we are in the process of co‐immunoprecipitation using GFP‐tagged bait and prey proteins expressed in mammalian cells to further confirm protein‐protein interaction.
The Influence of dietary lipid composition on skeletal muscle mitochondria from mice following eight months of calorie restriction
Authors: Chen Yana , Hagopian Kevork , Ramsey Jon
Published: June 2021

Calorie restriction (CR) has been shown to decrease reactive oxygen species (ROS) production, attenuate oxidative damage and retard aging in a variety of species. CR has also been observed to alter membrane composition towards saturation. This has led to a theory that changes in membrane saturation are central to the actions of CR. As a first step towards testing this theory, C57BL/6 mice were assigned to 4 dietary groups (control and 3 CR groups) and fed AIN‐93G diets at 95% (control) or 60% (CR) of ad libitum. To manipulate membrane composition, the primary dietary fats for the CR groups were soybean oil (also used in the control diet), fish oil or lard. The diets were fed for 8 months and then mitochondria from hindlimb skeletal muscle were isolated for measures of mitochondrial lipid composition, proton leak, and H2O2 production. Changes in mitochondrial phospholipid fatty acids in the CR mice reflected the dietary lipid sources. The capacity of mitochondrial complex III to produce ROS was decreased in the CR lard compared to either the CR soybean or fish oil groups. However, diet lipid composition did not alter proton leak kinetics between the CR groups. The results of this study indicate that mitochondrial lipid composition can influence ROS production in the CR regimens. It remains to be determined if lard or other mixtures of dietary oils can maximize the CR‐induced decrease in ROS production. (Supported by NIH RO1 AG028125)
Specific phosphorylation of αA‐crystallin is required for the αA‐crystallin‐induced protection of astrocytes against staurosporine and C2‐ceramide toxicity
Authors: Georg Reiser , Li Rongyu
Published: June 2021

a‐Crystallin is upregulated in neurodegenerative diseases and neurodegenerative conditions. Elevated a‐crystallin apparently plays a self‐protective function. Compared to the studies on the expression and role of aB‐crystallin in the nervous system, there are considerably fewer reports on the role of aA‐crystallin in the brain. We reported that aA‐crystallin and protease‐activated receptor are involved in protection of astrocytes against C2‐ ceramide‐ and staurosporine‐induced cell death (Li et al., J Neurochem 110, 2009, 1433–1444). We investigated the molecular mechanism of aA‐crystallin‐mediated cytoprotection. The phosphorylation of aA‐crystallin at Ser122 and Ser148 is required for protection. We proved that phosphorylation of aB‐crystallin at Ser45 and Ser59 is required for protection. ERK and p38 mediated the protective process by aB‐crystallin.
Structural Biology Knowledgebase: a biologists resource for protein structure and sample information
Authors: Gabanyi Margaret , Chen Li , Westbrook John , Micallef David , Shah Raship , Tao Yi-Ping , Peisach Ezra , Schwede Torsten , Haas Juergen , Bordoli Lorenza , Adams Paul , Gifford Lida , Minor Wladek , Fratczak Zbigniew , Zimmerman Matthew , Berman Helen
Published: June 2021

The Structural Biology Knowledgebase (SBKB, http://sbkb.org) is a scientific search portal that returns comprehensive biological and methodological information about a protein sequence. Information related to 3D protein structures and the sequences targeted by worldwide structural genomics efforts are combined with links to open biological resources to give an integrated view of a protein. It is made in collaboration with the Protein Structure Initiative and the Nature Publishing Group, and features the latest research and technology advances each month to enable researchers in a broad range of biomedical fields. This presentation will introduce the Web portal and its services, and illustrate common user cases of the SBKB.
HEAD OF A LAMA: AN ANATOMICAL STUDY BY CT‐SCAN AND NMR
Authors: Le Floch-Prigent Patrice Pierre , CUBA Juan , FIGUEIRA C. , MARTINOT-LUYO J.
Published: June 2021

The head of a lama (Cuzco, Peru) was studied by CT‐scan and NMR. the head had been taken from a recently died lama. It was 30cm long and weighed 3,24kg. The entire head without any preparation was CT‐scanned and studied by NMR (CEREMA, avenida Javier Prado in Lima) with cross‐sections from the front to the back (vertical plane) every 5 mm with 5mm cross‐section slices and then reconstructed in the two other planes (sagittal and horizontal). The systematical analysis of the views permitted the description of the muscular, osseous and encephalic relationships between them and also their isolated aspect in serial anatomy. The lama is an auchenidae which lives exclusively in south America.
LETM1 is an essential component of mitochondrial calcium uptake and regulation of cellular bioenergetics
Authors: Doonan Patrick John , Mallilankaraman Karthik , Chandramoorthy Harish , Madesh Muniswamy
Published: June 2021

Regulation of mitochondrial Ca2+‐dependent cellular bioenergetics has been implicated in a variety of pathophysiological settings including neurodegeneration, myocardial infarction and immunity. Recently, LETM1 was proposed as a mitochondrial Ca2+/H+ antiporter; however characterization of the functional role of LETM1‐mediated Ca2+ transfer has yet to be studied. Here we demonstrate that LETM1 knockdown significantly impairs mitochondrial Ca2+ uptake and results in bioenergetic collapse. We observed that mitochondrial Ca2+ uptake is impaired in shRNA‐mediated LETM1 knockdown and ΔEF hand LETM1 and D676AD688KLETM1 overexpression cells. Knockdown of LETM1 promotes LC3 positive multilamellar vesicle formation, indicative of autophagy induction. Furthermore, cellular bioenergetic parameters including ATP and oxygen consumption were reduced. In contrast, cellular NADH and mitochondrial membrane potential were unaltered in both control and shRNA LETM1 cells. Interestingly, knockdown of LETM1 by siRNA in primary neonatal cardiomyocytes resulted in a remarkable change in mitochondrial morphology and reduced Ca2+ uptake. We also observed that LETM1 knockdown results in increased ROS production. Our results represent the first time that LETM1 has been linked to cellular bioenergetics through regulation of mitochondrial Ca2+.

Supported by NIH R01 HL086699, HL086699‐01A2S1, 1S10RR027327‐01