bluuebunny/crossref_metadata_2025 huggingface dataset:
A Semisynthetic Approach to Understanding PTEN Regulation
Authors: Bolduc David , Gabelli Sandra , Raben Dan , Devreotes Peter , Amzel Mario , Cole Phil
Published: June 2021

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressing, lipid phosphatase that negatively regulates the PI3K/AKT signaling pathway by dephosphorylating the lipid second messenger PIP3. PTEN function is frequently lost in cancer and its normal functions are regulated by a variety of mechanisms within cells. One such mode of regulation acts through the phosphorylation of a cluster of serine and threonine residues on PTEN's C‐terminal tail. Phosphorylation at this cluster has been proposed to induce a conformational change in the PTEN protein which is essential for its tumor suppressive functions. To better understand how phosphorylation at this regulatory cluster affects PTEN's activities we have generated semisynthetic PTEN in its phosphorylated form by expressed protein ligation. An interfacial kinetic analysis reveals that phosphorylation causes an increase in the membrane binding constant Ks, while having no change in the interfacial Km. These results are corroborated using a vesicle binding assay. Using Small Angle X‐ray Scattering and other biochemical techniques we have shown that phosphorylation of PTEN's tail induces an intramolecular conformational change that stabilizes the phosphatase domain of the protein. Using protein semisynthesis we have developed a valuable tool for future studies involving PTEN phosphorylation.

Source of Funding:

NIH Grant: R01CA074305
NEDD4–1‐mediated ubiquitination regulates pAKT nuclear trafficking in IGF‐1 response
Authors: Wang Xinjiang , Fan Chuan‐Dong , Lum Michelle A , Xu Chao , Black Jennifer D
Published: June 2021

AKT is a downstream effector kinase of the PI3K pathway involved in regulation of cell growth, death, differentiation and migration. Regulation of the activated phospho‐AKT (pAKT) translocation to its action sites remains unclear. The objective of this study was to investigate whether AKT is directly regulated by NEDD4–1 E3 ligase through ubiquitination in IGF‐1 response. We found that NEDD4–1 promotes HECT domain‐dependent ubiquitination of exogenous and endogenous AKT in in vivo ubiquitination assays. Co‐imunocpreciptation results indicated that NEDD4–1 physically interacts with AKT. Plasma membrane binding is the key step for AKT ubiquitination by NEDD4–1 in vivo. Ubiquitinated pAKT translocates to perinuclear regions, where it is either released into the cytoplasm, imported into the nucleus or coupled with proteasomal degradation. IGF‐1 signaling stimulates NEDD4–1‐mediated ubiquitination of pAKT but not total AKT. NEDD4–1 catalyzes K63‐type polyubiquitin chain formation on AKT in an in vitro ubiquitination assay. A cancer‐derived plasma membrane‐philic mutant AKT(E17K) is more effectively ubiquitinated by NEDD4–1 and more efficiently trafficked into the nucleus compared with AKT. This study established that AKT is a substrate of NEDD4–1 and NEDD4–1‐mediated ubiquitination plays a role in nuclear trafficking of pAKT that contributes to AKT activation process in IGF‐1 response.
Structure‐function studies of the Golgi Dsc E3 ligase complex required for SREBP activation in yeast
Authors: Espenshade Peter J , Lloyd S. Julie‐Ann , Tong Zongtian , Raychaudhuri Sumana
Published: June 2021

Fungal sterol regulatory element‐binding protein (SREBP) transcription factors are oxygen‐responsive transcription factors required for adaptation to hypoxia. Our recent studies identified five genes (dsc1–5, defective for SREBP cleavage) that code for subunits of the Golgi Dsc E3 ligase complex that is required for fission yeast SREBP activation. Dsc proteins have domains that are predicted to function in the ubiquitin proteasome pathway: Dsc1 is a ubiquitin E3 ligase with a RING domain, Dsc2 has a predicted ubiquitin‐associated (UBA) domain and Dsc5 contains a ubiquitin regulatory X (UBX) domain that binds Cdc48.

To begin to define the mechanism for Sre1 cleavage in fission yeast, we performed structure‐function studies to elucidate the organization of this multi‐subunit complex. Using co‐immunoprecipitation analysis and Blue native PAGE, we found that Dsc1–5 form a stable, detergent solubilized complex with a discrete architecture with distinct subcomplexes. Parallel studies in budding yeast, which lacks SREBP homologs, demonstrate that the Dsc E3 ligase complex is structurally conserved and localizes to the Golgi. Future studies will focus on the identification of additional Dsc E3 ligase substrates and the function of this complex in Golgi protein degradation.

Studies were supported by the National Institutes of Health (HL‐077588).
The 26S proteasome binds and degrades Pih1 via the C‐terminal degron elements in yeast Saccharomyces cerevisiae
Authors: Paci Alexandr , Liu Xiao Hu , Balachandran Banujan , Zhao Rongmin
Published: June 2021

In yeast Saccharomyces cerevisiae, R2TP complex is required for the box C/D small nucleolar ribonucleoprotein (snoRNP) assembly and ribosomal RNA processing. This complex is composed of Rvb1 and Rvb2, two ATP‐dependent DNA helicases, Tah1, an Hsp90 cofactor, and Pih1, a subunit that acts as a scaffold and likely regulates the Rvb1/Rvb2 helicase activities. We have previously shown that Rvb1 and Rvb2 bind to the N‐terminal fragment of Pih1, while Tah1 binds to the Pih1 C‐terminal fragment. Additionally, the C‐terminus of Pih1 acts as a degron that regulates Pih1 degradation in vivo and controls R2TP complex assembly. To understand the mechanism of Pih1 degradation in vivo, we purified Pih1 protein complex from yeast cells and immunoblotting analysis indicated that Pih1 is able to pull down some 26S proteasome subunits. In vitro binding assays indicated that Pih1 C‐terminal degron elements are likely to interact with 26S proteasome directly. Based on in vitro and in vivo degradation assay, we propose a model of proteasome dependent, but ubiquitin independent degradation of Pih1 in yeast cells. (This work is supported by a Connaught New Staff Matching Fund and an NSERC Discovery Grant to RZ)
Deconvoluting Electrophile Signaling and Stress Response Pathways
Authors: Aye yimon
Published: June 2021

In‐depth understanding of complex biological pathways hinges upon the ability to systematically perturb individual systems within each complex network. However, current experimental conditions to study impacts of reactive oxygen species/carbonyl compounds (ROS/RCC) use far above their physiological levels and are highly non‐specific. As ROS/RCC act indiscriminately, swamping the entire proteome or organelle with a reactive species unavoidably leads to off‐target effects. Specificity in signaling and stress pathways, or response is likely lost in experimental noise. This is the principal reason why so little is known about redox regulation and specific response pathways to oxidative stress. Recent development of optical probes to report levels of H2O2 in specific organelles shows motivation and appreciation toward the need to study individual effects more thoroughly. However, an approach able to perturb individual proteins within redox regulatory pathways has not been achieved. My laboratory has recently developed a biocompatible technology that permits targeted delivery of a given RCC to individual proteins at any locale in mammalian cells. ‘Keap1‐Nrf2 signaling pathway’ serves as a proof‐of‐concept in validating our new methodology that has the potential to comprehend redox regulation and electrophile sensing networks in vivo at a level of biochemical specificity currently not possible.
Novel inhibitors of acetate kinase from Francisella tularensis
Authors: Li Shangqing , Schenkel Matthew , Drake Austin , Liu Dali , Novak Walter R.P.
Published: June 2021

Developed as a bioweapon in the 1940's, Francisella tularensis is the pathogenic bacteria responsible for tularemia, or rabbit fever. In order to develop new treatments for weaponized strains of F. tularensis, potential inhibitors F. tularensis acetate kinase were identified in silico and experimentally tested. Often kinase activity is probed using relatively expensive NADH‐linked assays. Here, we utilized an inexpensive kinetic assay that monitors the production of acetyl phosphate via reaction with hydroxylamine to rapidly characterize potential inhibitors of F. tularensis acetate kinase. The KM values for ATP and acetate were determined to be 1.4 and 15.4 mM, respectively, and the Vmax was determined to be 40.6 μmol min−1 mg−1. Computational screening using DOCK allowed the identification of potential inhibitors from the National Cancer Institute's Developmental Therapeutics Program. Ten compounds were obtained and tested, and two of these reduce the activity of F. tularensis acetate kinase significantly, though at relatively high concentrations. Using these structures as lead compounds, we are mining the database for similar compounds to test as inhibitors. This research was funded by the Wabash College Haines Biochemistry Fund.
Polyisoprenylated methylated protein methyl esterase as a potential novel target in lung cancer therapy
Authors: AMISSAH FELIX , DUVERNA RANDOLPH , AGUILAR BYRON J , POKU ROSEMARY A.
Published: June 2021

Polyisoprenylated monomeric G‐proteins such as Ras, Rho and Rac play important roles in lung carcinogenesis. Targeting the polyisoprenylation pathway enzymes presents therapeutic opportunities but polyisoprenylated methylated protein methyl esterase (PMPMEase) has not been explored. PMPMEase expression and activity were investigated in lung cancer cells, tissues and tissue microarrays (TMAs). PMPMEase activity was significantly elevated in lung cancer cases relative to normal adjacent tissues (p < 0.001), correlating with PMPMEase protein levels. Intense PMPMEase immunoreactivity was observed in 88.3% of over 400 lung cancer cases on TMAs (p < 0.0001). Specific PMPMEase activities in A549 and H460 cells were 1.6‐ and 1.5‐fold higher than in WI‐38 cells, respectively. Exposure of the cells to specific PMPMEase inhibitor altered F‐actin organization and induced apoptosis with EC50 of 3.0 μg/ml for A549 and 9.6 μg/ml for H460 cells. The PMPMEase overexpression and hyperactivity in lung cancer and the apoptosis resulting from its inhibition is a strong indication of its role in lung cancer progression. This implies that PMPMEase would be a strong candidate biomarker for diagnosis and target for therapy in lung cancer.
Probing the role of N152 in the class C beta‐lactamase AmpC
Authors: Wallar Brad , Docter Brianne , Leonard David , Powers Rachel
Published: June 2021

AmpC, a class C β‐lactamase, is a main cause of antibiotic resistance to cephalosporins in many species of bacteria. Although a mechanism involving acylation by S64 followed by hydrolytic cleavage has been generally accepted, the exact roles that some active site residues play in recognition and breakdown of the substrate are not fully understood. Here, we investigate the role of the active site residue asparagine‐152 (N152) in E. coli AmpC by mutating it to a G, S, or T residue and examining the effect that these mutations have on kinetic and structural properties with four different β‐lactam drugs: cefotaxime, cefoxitin, oxacillin, and a derivative of cephalothin (CENTA). We discovered that although the mutations cause higher Km values with all substrates, they result in 50 to 150 times higher kcat values against cefotaxime. In addition, the N152 mutations provided the enzyme the ability to break down oxacillin, which is not a viable substrate for the wild type AmpC. To probe the mechanism behind the observed kinetics changes, crystal structures were obtained of AmpC WT or N125G in acyl‐enzyme complexes with cefotaxime, cefoxitin, and oxacillin. The small structural differences in the active site have been associated with the changes in the Km and kcat kinetic values as a way to uncover the specific role of N152 in the function of AmpC. This work was supported by the Office of Undergraduate Research and Scholarship at GVSU.
Large‐scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis
Authors: Johnson R. Jeremy , Filippova Ekaterina , Weston Leigh , Kuhn Misty , Anderson Wayne
Published: June 2021

Amongst emerging targets for treating diverse bacterial infections are members of the ubiquitous serine hydrolase protein family. Herein, we present the crystal structure and biochemical characterization of FTT258, a novel bacterial carboxylesterase from F. tularensis and a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 in the presence of a mechanistic cyclobutanone ligand reveals an unexpectedly large conformational change where the asymmetric unit for the FTT258 structure contains four open‐ and four closed‐forms of the protein. The large‐scale structural rearrangement in the open form is stabilized by the hydrophobic burial of a repositioned surface loop and is responsible for the exposure of the hydrophobic binding pocket. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. While the same residues in the flexible loop were not essential to membrane binding, FTT258 did show significant association with liposomes designed to mimic bacterial membranes. The observed conformational changes and connection to the catalytic activity suggest a potential dual regulation of catalytic activity and membrane association in FTT258. Funding provided by a Butler University Holcomb research award and NAIAD award HHSN272200700058C
Expression, Purification and Crystallization of the Xanthomonas euvesicatoria Dual Specificity Protein Tyrosine Phosphatase, AvrBs1.1
Authors: Wilt Laura Anne , Potnis Neha , Jones Jeff , Hurlbert Jason
Published: June 2021

Phytopathogenic xanthomonads possess a type III secretion system that allows delivery of proteins, which serve to disrupt normal cellular function in the host. Avirulence proteins (Avr) are type III effector proteins that are recognized by specific R gene products in resistant hosts. A novel Avr protein, AvrBs1.1, from Xanthomonas euvesicatoria was recently found to elicit a delayed hypersensitive response on Capsicum annuum leaves. Amino acid sequence analysis of AvrBs1.1 has revealed the presence of a Dual‐Specificity Protein Tyrosine Phosphatase domain. Screening for novel resistance genes has identified a single binding partner of AvrBs1.1, the R gene product Bs7. Our laboratory has constructed homology models of AvrBs1.1 which have provided some insight into potential differences in activation of Bs7 in C. annuum, a better understanding of the activation mechanism will be gained by x‐ray crystallographic studies of the protein complexes. We have begun purifying recombinant AvrBs1.1 in quantities suitable for crystallization trials. The coding sequence of X. euvesicatoria AvrBs1.1 was cloned into pET21A, which was used to transform E. coli BL21 (DE3) cultures. The recombinant AvrBs1.1 was purified by a combination of metal chelating affinity and gel filtration chromatographic methods. Aliquots of pure protein were used to identify conditions suitable for growth of diffraction quality crystals.
Structural Insights of Outer Membrane Protein Methyltransferases from Rickettsia
Authors: Abeykoon Amila H , Noinaj Nicholas , Chao Chien‐Chung , Wang Guanghui , Chock P.Boon , Gucek Marjan , Ching Wei‐Mei , Buchanan Susan K , Yang David C.H
Published: June 2021

The observation of correlation between hypermethylation of rickettsial outer membrane protein B (OmpB) and the bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. Two protein lysine methyltransferases (rPKMT1 and rPKMT2) were found to methylate recombinant OmpB fragments through the incorporation of radioactively labeled methyl group from S‐adenosylmethionine (SAM). The two proteins share 45 % identity. LC/MS‐MS analyses showed both rPKMT1 and rPKMT2 methylated at numerous lysine residues of OmpB, but rPKMT1 catalyzed mono‐, di‐ and tri‐methylation of lysine, while rPKMT2 catalyzed exclusively trimethylation of lysine. We used X‐ray crystallography to investigate the structure of the lysine methyltransferases. The crystal structures of rPKMT1 and rPKMT2 and the respective complexes with SAM were determined. These are the first reported crystal structures of outer membrane protein methyltransferases. The crystal structures of rPKMT1 and rPKMT2 are superimposable. Both methyltransferases are dimeric, which was confirmed using dynamic light scattering. Each monomer consists of dimerization, SAM binding, middle, and C‐terminal domains. Comparison of the two structures revealed a number of structural insights of the unusual enzymatic methylation of OmpB.
Increased oxygen enhances bone degradation and promotes bone regeneration after digit amputation in mice
Authors: Cammack Alexander James , Mandava Prudhvi , Van Meter Keith , Muneoka Ken , Sammarco Mimi
Published: June 2021

In 2005, approximately 1.6 million persons in the United States were living with the loss of a limb. That number is projected to increase to 3.6 million by 2050. While full limb replacement is not yet a possibility, prosthetic limb replacement is common amongst amputees, and the ability to control bone regeneration is highly favorable for comfortable fit. The capacity to regenerate bone post‐amputation is limited to only the distal 1/3 of the third phalangeal element (P3) of rodents, monkeys, and humans. Regeneration in adult mice is defined as three distinct steps: 1) wound healing and bone degradation, 2) formation of the blastema, and 3) differentiation of the blastema into bone and tissue. Our hypothesis is that P3 regeneration is facilitated by temporally specific fluctuations in oxygen. The objective of this study is to apply hyperbaric oxygen treatment (HBOT) to our well‐characterized P3 regenerative model to evaluate the effects of increased oxygen during P3 regeneration. Our data show that digits treated with HBOT immediately after amputation display elevated levels of bone degradation. These digits are then able to fully regenerate P3, demonstrating an expansion of regenerative capacity to the proximal P3 bone stump. Our findings strongly support that oxygen is able to act as a primary signaling event to influence bone regeneration in vivo. Research funded by grant 1F32HD071763–01A1 from the NIH
Hippocampal place field activity in rats trained to walk forward and backwards
Authors: Ferng Jonathan , Burke Sara N. , Barnes Carol A.
Published: June 2021

To achieve a better understanding of the hippocampus, a brain structure linked to neurodegenerative diseases such as Alzheimer's, the activity of hippocampal neurons in awake behaving animals was monitored. These cells are known as “place cells” because they show increased firing rates at discrete locations in an environment referred to as the neuron's place field. The composite activity of a population of place cells forms a ‘map’ of an environment. When a rat traverses a track in different directions within the same environment, however, the activity of many place cells is typically uncorrelated; that is, few hippocampal neurons show bi‐directional activity. This could be due to changes in head direction, which is used to align place fields to the environment. This experiment investigated bi‐directionality in the place cells of rats trained to maintain the same head direction when traversing a linear track by walking forwards and then returning to the starting position by walking backwards. Although the rats maintained the same head direction, place field activity was not bi‐directional. The independent place maps observed during the forward and backward running conditions could be due to different cognitive strategies that the rats use during these distinct types of behavior. This research was supported by the Howard Hughes Medical Institute ‘06 Grant (#52005889) and the McKnight Brain Research Foundation.
Apoptosis Associated with Osteoarthritis is Attenuated in Mice Lacking the Receptor for Advanced Glycation End Products (RAGE)
Authors: Wilhelm Spencer K , Larkin D Justin , Doxey Alexander S. , Kartchner Jeffrey Z. , Vanderuff James , Anderson Karl , Draper Christian S. , Chavez Elizabeth , Reynolds Paul R. , Kooyman David L.
Published: June 2021

Objective

It has been previously shown that chondrocyte apoptosis is associated with the progression of osteoarthritis. The objective of this project was to examine the effects of S100, RAGE, Tgf‐β1, HtrA1 and NF κβ on apoptosis in the progression of osteoarthritis (OA).

Methods

Samples of wild type (WT) and RAGE knockout (KO) were assayed for HtrA1, S100, RAGE, TGF‐β1, NF‐kB, TUNEL and evaluated for cartilage degradation four weeks after a knee destabilization procedure.

Results

We observed positive staining for all biomarkers except TGF‐β1examined in both WT and RAGE KO mice with high Mankin score OA. All biomarkers assayed were present, except HtrA1, in samples with low Mankin score OA. We also observed varying degrees of TUNEL assay staining in both WT and RAGE KO mice. Remarkably, despite similar IHC staining profiles, RAGE KO mice exhibited significantly less cartilage degradation and chondrocyte apoptosis, assayed by TUNEL, compared to WT mice.

Conclusion

We conclude that RAGE mediated upregulation of TGF β1 leads to an upregulation of HtrA1 and apoptosis associated with osteoarthritis. This suggests that an inhibition of inflammatory processes involving RAGE may attenuate chondrocyte apoptosis, and slow the progression of OA.
Differential Protein Expression Profiles of Human Lymph and Plasma Mapped by 2D‐DIGE and 1D SDS‐PAGE Coupled with NanoLC‐ESI‐MS/MS Bottom‐Up Proteomics
Authors: Clement Cristina C , Santambrogio Laura
Published: June 2021

In this study a global proteomic approach was used to define the protein content of matched samples of afferent prenodal lymph and plasma derived from healthy volunteers. The analysis was performed using two analytical methodologies coupled with nanoliquid chromatography‐tandem mass spectrometry (nanoLC Orbitrap‐ESI‐MS/MS): one‐dimensional gel electrophoresis (1DEF nanoLC Orbitrap‐ESI‐MS/MS), and two‐dimensional fluorescence difference‐in‐gel electrophoresis (2D‐DIGE nanoLC‐ESI‐MS/MS). The 253 significant identified proteins (p<0.05) obtained from the tandem mass spectrometry data were further analyzed with pathway analysis (IPA) to define the functional signature of prenodal lymph and matched plasma. The common proteome between the two biological fluids (144 out of 253 proteins) was dominated by complement activation and blood coagulation components, transporters and protease inhibitors. The enriched proteome of human lymph (72 proteins) consisted of products derived from the extracellular matrix, apoptosis and cellular catabolism. In contrast, the enriched proteome of human plasma (37 proteins) consisted of soluble molecules of the coagulation system and cell‐cell signaling factors. The functional networks associated with both common and source‐distinctive proteomes highlight the major biological activities of these immunologically relevant body fluids.
3D O2 imaging in the neuronal spheroids
Authors: Dmitriev Ruslan I. , Zhdanov Alexander V. , Nolan Yvonne M. , Papkovsky Dmitri B.
Published: June 2021

Availability of O2 determines the cell metabolic status and physiological responses to stimulation. The multicellular spheroids made of cultured or primary neuronal cells are commonly used to study neural stem cell proliferation, differentiation and pathological states under conditions that resemble their native micro‐environment. The live cell imaging with cell‐penetrating phosphorescent O2‐sensitive probes allows quantification and high‐resolution mapping of O2 distribution in such structures. Using the Pt‐porphyrin nanoparticle (MM2, NanO2) and peptide‐based icO2 probes, we optimised conditions for internal (core) and surface staining of the rat primary neurospheres, to achieve high phosphorescent signals and low toxicity. Neurosphere oxygenation was analysed on FLIM and 2photon ratiometric‐based imaging systems. The observed gradients of neurosphere oxygenation were correlated with the O2 in the chamber and the localisation of stem cells in the core. Spectral characteristics of icO2 probes were compatible with live‐cell imaging fluorescent markers such as TMRM (mitochondrial membrane potential), BCECF (intracellular pH) or following fixation and immunofluorescence staining. Altogether, 3D O2 imaging represents versatile tool for high‐resolution assessment of metabolic, proliferation and differentiation status of neural stem cell in the spheroid culture. Supported by SFI.
Optimizing environmental conditions and image processing as a means for simplifying BRET imaging
Authors: Finafrock Jeffrey Karl , von Arnim Albrecht G
Published: June 2021

Protein interactions are fundamental to our understanding of biological processes. Bioluminescence Resonance Energy Transfer (BRET) is a method for monitoring protein‐protein interactions (PPIs) and protein conformational changes in living cells in real time. It is based on the well‐established principle of Förster Resonance Energy Transfer (FRET). Unlike the commonly used fluorescence‐based FRET, BRET requires no light input, making it more suitable for monitoring PPIs in light sensitive tissues. Because no phototoxicity occurs, BRET may also lend itself to long‐term time‐resolved measurements. However, producing images with BRET is challenging, as it requires long exposure times, specialized imaging equipment, and extensive image processing. Here, we report BRET imaging results at subcellular resolution that were obtained using an electron‐multiplying camera system. We evaluate various image processing techniques. We also report on two sample preparation techniques that appear to boost luminescence output and thus simplify and shorten the BRET imaging procedures.
Diminished expression of plasminogen activator inhibitor 2 correlates with local tumor aggressiveness in lung adenocarcinoma
Authors: Su Chia Yi , Hsiao Michael
Published: June 2021

Lung cancer is notorious for high rates of local aggressiveness and metastasis. Decreased plasminogen activator inhibitor 2 (PAI2), an inhibitor of urokinase‐type plasminogen activator (uPA), was reported to correlated to lymph node metastasis and poor prognosis. However, the pathomechanism of PAI2 expression is still unknown. Therefore, we performed immunohistochemical staining on 105 non‐small cell lung cancer patients. Higher immunoreactivity was seen in tumors than in normal lung tissues (p<0.001). In adenocarcinoma, lower PAI2 expression correlated with higher T stage (p=0.02) and had trends associated with higher pTNM stage (p=0.06) and shorter overall survival (p=0.24). Besides, the involvement of matrix metalloproteinases (MMP) in the regulation between PAI2 and uPA was identified in bioinformatics database analysis. Here we showed decline of PAI2 level which was regulated by MMP caused overexpression of uPA and further led to local tumor aggressiveness and poor prognosis. In other words, overexpression of PAI2 may prevent the degradation by MMP in turn downregulate uPA to inhibit tumor invasion. In conclusion, our results suggested that PAI2 had more direct impact on local tumor progression than on metastasis. The correlation between diminished PAI2 expression and local tumor aggressiveness indicated that PAI2 may be a promising therapeutic and prognostic marker for lung adenocarcinoma.
Inhibition of cholinergic signaling causes apoptosis in human bronchioalveolar carcinoma
Authors: Brown Kathleen C , Lau Jamie K , Thornhill Brent A , Crabtree Clayton M , Dom Aaron M , Witte Theodore R , Hardman W Elaine , McNees Christopher A , Stover Cody A , Carpenter A Betts , Luo Haitao , Chen Yi Charlie , Shiflett Brandon , Dasgupta Piyali
Published: June 2021

Recent clinical studies show that bronchioalveolar carcinomas (BACs) are correlated with smoking. Nicotine, the major addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChRs). Here we show that human BACs produce acetylcholine (ACh) and contain all of the cholinergic system proteins, including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1), vesicular acetylcholine transporter (VAChT) and nAChRs. Nicotine increases the production of ACh in human BACs. ACh acts as a growth factor for human BACs. We observed that nicotine upregulates ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or IGF‐II‐induced growth of human BAC's. The ablation of VAChT levels by siRNA methodology reversed the apoptotic activity of vesamicol. We also observed that vesamicol caused cell death by inhibiting Akt phosphorylation. The overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our data shows that disruption of nicotine‐induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy.
Defining Trafficking Steps Required for Cardiolipin Remodeling
Authors: Baile Matthew G , Whited Kevin , Claypool Steven M
Published: June 2021

The phospholipid cardiolipin (CL) is required for numerous mitochondrial processes. After its de novo synthesis on the matrix‐facing leaflet of the inner membrane, CL undergoes acyl chain remodeling to achieve its final form. In yeast, this process is completed by the transacylase tafazzin which associates with intermembrane space‐facing membranes via a membrane anchor that does not traverse the lipid bilayer. To gain insight into the topology of CL remodeling, we examined the submitochondrial localization of Cld1p, the lipase that initiates CL remodeling. We show that Cld1p is associated with the matrix‐facing leaflet of the mitochondrial inner membrane. Thus, once generated by Cld1p, monolyso‐CL must be transported to the intermembrane space‐facing leaflets of the inner or outer membrane to gain access to tafazzin, identifying a previously unknown step in the CL remodeling pathway. Additionally, we examined the regulation of CL remodeling. Cld1p expression and function are augmented in growth conditions requiring mitochondrially produced energy. Interestingly, selectively diminishing either the electrical or chemical gradients across the inner membrane with ionophores indicates that function of either the MLCL transporter or tafazzin does not requires an intact proton‐motive force. In contrast, the function of Cld1p is enhanced upon diminution of either aspect of electrochemical gradient. This work was supported by National Institutes of Health grant number R00HL089185 to SMC. MGB is a predoctoral fellow of the American Heart Association.
Protein kinase A (PKA) interferes with the processing and activity of sterol regulatory element binding proteins (SREBPs)
Authors: Esquejo Ryan Magadan , Phelan Peter E. , Roqueta‐Rivera Manuel , Osborne Timothy F.
Published: June 2021

Several kinases are known to modulate the activity of the sterol regulatory element binding proteins (SREBPs), which are transcription factors that activate lipid biosynthesis. The inactive SREBP precursor forms are bound to the endoplasmic reticulum (ER) and require the SREBP cleavage activating protein (SCAP) to shuttle SREBPs to the Golgi, where proteases cleave and release the active SREBPs, which can then enter the nucleus and activate transcription. SREBPs are known to be down‐regulated by conditions where PKA activity is higher such as by glucagon action in the liver; however, a mechanism to explain this effect is lacking. Here, we show that PKA can block processing of SREBPs in various cells types. We used forskolin to induce PKA activity and found lower levels of mature SREBP‐1 in BCR‐ABL transformed bone marrow. We also used H‐89, a PKA inhibitor, and found higher levels of mature SREBPs in the BCR‐ABL transformed bone marrow, HeLa cells, and mouse primary hepatocytes. Preliminary results showed that H‐89 injection into mice can blunt the negative effect of glucagon on mature SREBP accumulation in the liver. In preliminary studies, we found higher amounts of SCAP in the Golgi when H‐89 is added to Chinese hamster ovary (CHO) cells that stably express GFP‐SCAP. There is a conserved putative PKA phosphorylation motif in SCAP and many phosphoproteomic studies show the site is phosphorylated in many different settings. Thus, our recent discovery points to a novel role for PKA in SREBP trafficking and activation, possibly via direct PKA phosphorylation of SCAP.
Inositol biosynthesis is fine‐tuned by inositol pyrophosphates in Saccharomyces cerevisiae
Authors: Ye Cunqi , Greenberg Miriam L
Published: June 2021

In yeast, inositol biosynthesis is coordinated with the synthesis of phospholipids by controlling the expression of enzymes in the biosynthetic pathways. Inositol pyrophosphates, most recently characterized in the inositide family, have diverse roles in phosphate signaling and other important cellular processes. However, little is known about the function of inositol pyrophosphates in the biosynthesis of inositol and phospholipids. The objective of this study is to understand the mechanistic links between inositol metabolism and inositol pyrophosphates. Our results indicated that inositol pyrophosphate deficient mutants kcs1Δ and vip1Δ exhibit decreased inositol biosynthesis due to decreased derepression of INO1, which encodes the rate‐limiting enzyme for inositol biosynthesis. Further studies revealed that the inositol pyrophosphate kinase activity and the basic leucine zipper (bZIP) domains in KCS1 are both required for optimal INO1 expression. Interestingly, the levels of Kcs1p correlate with INO1 expression. We conclude that conversion of IP5 to inositol pyrophosphates modulates INO1 transcription.

This work is funded by grant R01 DK081367 from NIH and a travel grant from Wayne State University.
Exploring vesicle size as a variable in antimicrobial peptide‐lipid vesicle interactions
Authors: Shortt Emily M. , Elmore Donald E.
Published: June 2021

Antimicrobial peptides (AMPs) selectively kill bacteria, fungi, and some cancer cells, either by membrane permeabilization or by translocation across the membrane and interference with intracellular processes. Lipid vesicles provide an effective model of cell membranes that can be prepared in various sizes to examine the effect of local membrane curvature on AMP activity. Magainin and buforin II are well‐characterized permeabilizing and translocating peptides, respectively, and DesHDAP1, DesHDAP3, and DesHDAP3 P13A are designed histone‐derived peptides that are translocating (DesHDAP1) or permeabilizing (DesHDAP3 and DesHDAP3 P13A). We performed fluorescence assays to measure the membrane permeabilization and translocation of these peptides with different sized lipid vesicles. Magainin and DesHDAP3 P13A permeabilized smaller vesicles more effectively than large ones. Buforin II and DesHDAP1 translocated more readily with increasing vesicle size. We also found that DesHDAP3 showed translocating activity with larger vesicles but not smaller vesicles, highlighting the potential effect of membrane curvature on antimicrobial mechanisms.
Structural and Functional Analysis of a Bacterial DNA Translocation Channel
Authors: Berkmen Melanie Barker , Swerdlow Kyle J , Morton Georgeanna M , Broulidakis Matthew P , Mancuso Gianna R , Laurer Stephanie J , Leonetti Cori T , Hamada Matthew A
Published: June 2021

Mating, or conjugation, is the process by which a cell transfers DNA to another cell through a multi‐protein DNA translocation channel (Type IV Secretion System) at the membrane. Our research focuses on identifying and characterizing the components of the mating machinery of ICEBs1, a conjugative element of Bacillus subtilis. Our previous work indicates that the ICEBs1 protein ConE is a critical energetics component of the channel. ConE is essential for mating, localizes to the cell membrane, and belongs to the HerA/FtsK superfamily of ATPases. Here, we show that ConE likely functions as an oligomer. His6‐ConE forms dimers, trimers, and higher order oligomers in vitro and ConE self‐interacts in vivo. We hypothesize that the ICEBs1 proteins ConB and ConD interact with and recruit ConE to the cell membrane, as ConE‐GFP is dispersed throughout the cytoplasm in strains lacking these proteins. Since ConB and ConD are conserved membrane proteins and are required for DNA transfer, we hypothesize that they encode critical components of the ICEBs1 DNA translocation channel. This research was supported by an NSF‐RUI grant.
Interactions between Arf guanine nucleotide‐exchange factors, BIG1 and BIG2
Authors: Li Chun‐Chun , Le Kang , Moss Joel , Vaughan Martha
Published: June 2021

ADP‐ribosylation factors (Arfs), a family of ~20‐kDa GTPases that regulate intracellular membrane traffic, are activated by the Sec7 domains of guanine nucleotide‐exchange factors (GEFs) that catalyze replacement of Arf‐bound GDP by GTP. Two ca. 200‐kDa brefeldin A‐inhibited Arf GEFs, BIG1 and BIG2, purified together from bovine brain in ca. 670‐kDa multiprotein complexes, are 90% identical in Sec7 domains. Novel functions of their other molecular sequences continue to be identified. Interactions of N‐terminal regions of BIG1 and BIG2 called DCB and HUS that produce homodimers have been reported by Ramaen et al. (JBC, 282, 28834–28842, 2007). We explored additional associations of BIG1 and BIG2 using yeast two‐hybrid assays and in vitro‐synthesized recombinant proteins to show that BIG1 N‐terminal region interacted with structure in the C‐terminal part of BIG2 and vice versa. Interactions between N‐ and C‐termini of BIG1 or BIG2 to form multimers were also seen. These newly demonstrated interactions of the BIG Arf GEFs may offer clues to a better understanding of their function in multimolecular complexes to produce appropriate cellular response. (Funding: Intramural Research Program, NHLBI/NIH).
TLR4 Signaling Regulates Lysosome Exocytosis to a Novel Extracellular Compartment
Authors: Singh Rajesh K , Haka Abigail S , Chin Harvey F , Grosheva Inna , Maxfield Frederick R
Published: June 2021

We have characterized a novel form of endocytosis in which macrophages use lysosome exocytosis to degrade large species, such as aggregated lipoproteins. Exocytosis is targeted to an extracellular, acidic, hydrolytic compartment (a lysosomal synapse) and its formation is dependent on TLR4 and its adaptor MyD88. Macrophage degradation of aggregated lipoproteins using the lysosomal synapse transforms them into foam cells, a key pathological feature of atherosclerotic plaques.

Compartment formation is characterized by local actin polymerization, targeted secretion of lysosomal contents and generation of an acidic environment. These activities require carefully coordinated signal transduction processes. Using macrophages from knockout mice, siRNA silencing or pharmacological inhibitors, data identify phosphoinositide 3‐kinase, phosphoinositide‐dependent kinase 1, protein kinase B (Akt), Bruton's tyrosine kinase, phospholipase C, protein kinase C, soluble NSF attachment receptor (SNARE) proteins and calcium mobilization as important components downstream of TLR4/MyD88 leading to lysosome exocytosis to the lysosomal synapse.

Efforts are currently underway to examine the relevance of this pathway to several other diseases, such as Alzheimer's, diabetes and cancer.
Intensities of recognition factors at two combining sites of R. solanacearum lectin (RSL) for adopting LFucα1→, DManα1→ and Galβ1→3/4GlcNAc glycotopes
Authors: Wu Albert M. , Liu Jia‐Hua , Herp Anthony , Sudakevitz Dvora , Gilboa‐Garber Nechama
Published: June 2021

Ralstonia solanacearum is one of the most destructive phytopathogenic bacteria on earth and causes great agricultural and economical losses. The differences among interaction intensities of RSL toward four mammalian glycotopes (LFucα1→, DManα1→ and Galβ1→3/4GlcNAcβ1→, Iβ/IIβ) and their recognition mechanisms at two RSL combining sites for their accommodation were defined by both enzyme‐linked lectinosorbent and inhibition assays. From the results, it is concluded that optimal high LFucα1→ complex density enhanced their recognition intensities at the LFucα1→ site; polyvalent DManα1→ was essential for its binding at the DManα1→ site, and polyvalent Iβ/IIβ was required for accommodation at both sites. Four glycotope recognition systems at LFuc and DMan combining sites of R. solanacearum lectin (RSL) provides one of the most comprehensive knowledges about the combining sites of RSL, which creates a better understanding of the recognition mechanism of R. solanacearum at the molecular and submolecular levels. More lectins are being explored to upgrade the classical concept of lectin recognition sites.
The role of N‐Linked glycosylation during Drosophila development
Authors: McCague Allison , Selva Erica M.
Published: June 2021

N‐linked glycosylation is a key post‐translational modification for many secretory pathway targeted proteins. Endoplasmic reticulum glycosyltranferases construct a 14‐sugar precursor on a dolichol carrier, which is transferred to protein consensus sites. alg9 and alg10 N‐glycosylation mutants were used to examine the role of this modification during Drosophila development. alg10 encodes the enzyme catalyzing terminal glucose addition to the sugar‐precursor prior to transfer, while alg9 encodes an enzyme acting five steps earlier. In embryos, loss of alg9 and alg10 caused severe and pleotrophic defects. Central nervous system (CNS) neurons were specified in both alg9 and alg10 embryos. alg10 loss disrupted axon pathfinding, while alg9 embryos lacked mature neurons. Drosophila eye development in the absence of alg9 and alg10 yielded small rough adult eyes, but alg9 eyes were more severe. Examination of molecular markers in alg9 and alg10 late 3rd instar eye imaginal discs suggested adult small rough adult eyes might be due to neuronal apoptosis. These eye discs also showed defects in axon pathfinding, as shown by the loss of Bolwig's nerve and disrupted axon tracks. These results suggest loss of alg10 may disrupt the maturation of a subset of N‐glycoproteins, since alg9, which acts earlier, has more severe developmental defects. Funding sources: UD Undergrad Research Program, NSF and HHMI
NKCC1 localization and intracellular trafficking: Role of N‐glycosylation
Authors: Singh Richa , Di Fulvio Mauricio
Published: June 2021

N‐glycosylation plays a vital role in protein folding, maturation, assembly and exit from endoplamic reticulum (ER). Trimming of core N‐glycan structures is required for ER exit and traffic of proteins to other organelles. In the cis‐, medial‐ and trans‐Golgi complex plasma membrane (PM) proteins undergo posttranslational modifications to become complex N‐glycosylated. The Na+K+2Cl− cotransporter 1 (NKCC1) is a complex N‐glycosylated membrane protein. It has been postulated that N‐glycosylation is required for PM targeting of NKCC1. To understand the role of N‐glycosylation on localization or trafficking of endogenous NKCC1 we used COS7 cells, a mammalian model with a characterized secretory pathway. We show that NKCC1 is expressed in COS7 as a complex N‐glycosylated protein. However, most of NKCC1 locates in ER and recycling endosomes but minimally at the PM. NKCC1 was not found in lysosomes or the cis‐Golgi stacks suggesting that complex N‐glycosylation of NKCC1 is a fast process. Treatment of COS7 cells with tunicamycin or swainsonine, inhibitors of the first and last enzymatic steps in N‐glycan biosynthesis, respectively, resulted in ER‐retention, degradation and prevention of NKCC1 PM localization. Together, our results suggest that core N‐glycosylation impacts NKCC1 ER exiting whereas complex N‐glycosylation of the transporter participates in PM targeting.
RGS6 Functions as a Novel Suppressor of Tumor Initiation and Progression in a Mouse Model of Breast Carcinogenesis
Authors: Maity Biswanath , Stewart Adele , OMalley Yunxia , Askeland Ryan W , Sugg Sonia L , Fisher Rory A
Published: June 2021

Breast cancer is the most frequently diagnosed malignancy in women worldwide. Here, we utilized RGS6−/− mice to interrogate the role of RGS6 as a novel tumor suppressor. RGS6−/− mice exhibit accelerated DMBA‐induced tumor initiation and progression as well as decreased survival. Analysis of carcinogenic aberrations revealed a failure of the DNA damage response (DDR) concurrent with augmented oncogenesis in mammary glands from DMBA‐treated RGS6−/− mice. RGS6 also suppressed cell growth induced by either HER2 or ER activation in MCF‐7 cells and mammary epithelial cells (MECs). Isolated RGS6−/− MECs showed a defect in DMBA‐induced ATM/p53 activation, ROS generation, and apoptosis demonstrating that RGS6 is required for effective activation of the DDR in these cells. The ability of RGS6 to simultaneously enhance DNA damage‐induced apoptotic signaling and suppress oncogenic growth likely underlies the accelerated tumorigenesis and cellular transformation observed in DMBA‐treated RGS6−/− mice and isolated MECs. Unsurprisingly, spontaneous tumor formation was also seen in aged female RGS6−/− mice. Our finding that RGS6 is down‐regulated in all human breast cancer subtypes independent of their molecular classification indicates that obtaining a means to restore the growth suppressive and pro‐apoptotic actions of RGS6 in breast might be a viable means to treat a large spectrum of breast tumors. (NIH CA 161882).
Sphingosine‐1‐phosphate (S1P) Signaling for Breast Cell Invasion
Authors: Moon Aree , Kim Eun‐Sook , Kim Jong‐Sook , Kim Sang Geon , Hwang Sejin , Lee Chang Ho
Published: June 2021

Recent evidence suggests that inflammation is involved in malignant progression of breast cancer. Sphingosine 1‐phosphate (S1P), acting on the G‐protein‐coupled receptors, is known as a potent inflammatory mediator. In this study, the effect of the inflammatory lipid S1P on the regulation of invasive/migratory phenotypes of MCF10A human breast epithelial cells was investigated to elucidate a causal relationship between inflammation and the control of invasiveness of breast cells. We show that S1P causes induction of matrix metalloproteinase‐9 (MMP‐9) in vitro and in vivo, and thus enhances invasion and migration. We also show that fos plays a crucial role in the transcriptional activation of MMP‐9 by S1P. In addition, activation of extracellular‐signal‐regulated kinases 1 and 2 (ERK1/2), p38 and alpha serine/threonine‐protein kinase (Akt) are involved in the process of S1P‐mediated induction of MMP‐9 expression and invasion. Activation of the S1P receptor S1P3 and Gαq are required for S1P‐induced invasive/migratory responses, suggesting that the enhancement of S1P‐mediated invasiveness is triggered by the specific coupling of S1P3 to the eterotrimeric Gαq subunit. Activation of phospholipase C‐β4 and intracellular Ca2+ release are required for S1P‐induced MMP‐9 upregulation. Taken together, this study demonstrated that S1P regulates MMP‐9 induction and invasiveness through coupling of S1P3 and Gαq in MCF10A cells, thus providing a molecular basis for the crucial role of S1P in promoting breast cell invasion.