bluuebunny/crossref_metadata_2025 huggingface dataset:
Flexible modes of translocation by the eukaryotic CMG helicase facilitate removal or bypass of DNA blocks
Authors: Langston Lance D. , O'Donnell Mike
Published: November 2023

Replicative helicases in all domains of life are hexameric rings that encircle DNA while unwinding it, and studies have sought to distinguish between two different modes of action. In the steric exclusion model the helicase encircles and tracks along one strand and excludes the other strand from the inner channel. The ploughshare model posits that the helicase encircles both strands and a separation pin divides the two strands and directs them into distinct channels. Using purified CMG on model DNA substrates, we find evidence that the CMG helicase switches between both modes. In the ploughshare mode it is capable of stripping streptavidin from biotin on either DNA strand, indicating that it encircles both strands and proceeds with substantial force. In the steric exclusion mode it can simply bypass streptavidin on the lagging strand. The helicase appears to utilize the ploughshare mode by default, facilitating removal of obstacles on both strands at the replication fork, but can switch to the steric exclusion mode depending on the severity of the block. These results point to an unexpected degree of flexibility in the mode of helicase translocation that may also extend to other replicative helicases.

Support or Funding Information

This work was supported by NIH Grant GM38839 and the Howard Hughes Medical Institute
HDAC inhibitors decrease p35 cleavage through increased expression of calpastatin mRNA in primary neuronal cells and MPTP‐treated mice brain
Authors: Seo Jungwon
Published: November 2023

HDAC inhibitors have been reported as potential therapeutic agents for the neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. However, the target molecules of HDAC inhibitors responsible for the therapeutic effects are not yet fully elucidated. In this study, we examined which molecules HDAC inhibitors regulate in primary neuronal cells and evaluated the mechanism in MPTP mouse model of Parkinson's disease. Western blot analysis revealed that HDAC inhibitors such as valproic acid, trichostatin A and FK228 decreased the cleavage of p35 into p25 in response to ionomycin, glutamate, amyloid β and MPP. Previously we reported that Trichostatin A increased mRNA expression of calpastatin, an endogenous inhibitor of calpains, which cleaves p35. The transfection of calpastatin siRNA reversed HDAC inhibitor‐decreased p25 protein level in SH‐SY5Y cells. We also found that the transfection of HDAC2 siRNA increased calpastatin expression. Furthermore, valproic acid increased calpastatin expression and reversed MPTP‐increased p35 cleavage in MPTP‐treated mice cortex, with a concomitant increase of motor behavior in rotarod test. Our results demonstrate that HDAC inhibitors regulate the cleavage of p35 through the increased expression of calpastatin expression via HDAC2 inhibition.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2008‐0062484) and by Basic Science Research Program (NRF‐2013R1A1A3012771)
Analysis of RNA Binding by Evolutionarily Divergent LARP6 Proteins
Authors: Castro Jose Miguel , Salas Eliseo , Peña Eleuterio , Kocian Andrew , Lewis Karen
Published: November 2023

The La‐related protein superfamily is known to bind RNAs, and each subfamily appears to bind to distinct kinds of RNAs. The RNA binding activity of La‐related proteins is exerted by a single RNA binding domain, which consists of a well‐conserved La motif (LAM) and a RNA recognition motif (RRM). However, the ligand specificity of the LARP6 subfamily is unknown. Previous research has shown that Homo sapiens LARP6 interacts with the 5′ untranslated region of the mRNA that encodes for collagen type I (COL1A1), specifically recognizing a 48 nt stem‐loop structure. Collagen is an important structural protein that, in muscle tissue, forms part of the endomysium responsible of covering and protecting each individual muscle fiber. Subsequent work found that LARP6/COL1A1 interaction was important for zebrafish embryonic development. Bony fish like zebrafish and platyfish are widely used as animal models and are well‐suited to explore the function and evolution of LARP6 proteins. Zebrafish (Danio rerio) has two LARP6 paralogs (LARP6a and LARP6b), while platyfish (Xiphophorus maculatus) has only one LARP6, were chosen for biochemical analysis of LARP6 RNA binding activity. The sequence alignment of the fish LARP6 proteins compared to human showed significant differences within the RRMs. This study has three specific aims: (1) to determine if XmLARP6 and DrLARP6a bind the respective COL1A1 RNA; (2) to evaluate how species‐specific the LARP6/COL1A1 interaction is by comparing human and fish RNA ligands; and (3) to test if DrLARP6a and DrLARP6b exhibit different RNA binding activities in either affinity or specificity. All LARP6 proteins were expressed as recombinant proteins in E. coli, and purified to >95% purity by nickel affinity and size exclusion chromatography. RNA binding activity is measured by electrophoretic mobility shift assays (EMSAs) using biotinylated RNAs. The fish LARP6 proteins appear to bind their respective COL1A RNA ligands with comparable affinity as the human system. Human LARP6 shows affinity to the fish RNA ligands as well as the fish for the human ones. Differences in RNA binding activity between DrLARP6a and DrLARP6b are expected to provide insight into the subfunctionalization of these two paralogs.

Support or Funding Information

Funding provided by the Texas State University Department of Chemistry and Biochemistry; National Science Foundation through the Texas State University CheMIE Summer Research Experience for Undergraduates (NSF 1156579 to A.K.), and the National Institutes of Health through the South Texas Doctoral Bridge Program (GM102783 to J.M.C. & E.S.)
EWSR1 Potentially Functions as an RNA Chaperone in the Stress Response
Authors: Ribadeneyra Clara , Amin Shivas
Published: November 2023

EWSR1 is an RNA‐binding protein first described as part of chromosomal translocations that drive the majority of cases of Ewing sarcoma, a rare childhood and adolescent cancer.1 EWSR1 is interesting in that it is unusually promiscuous in fusion partners, with the N‐terminal half of EWSR1 found fused with the C‐terminal halves of at least 9 different transcription factors in at least 3 different types of cancer. This N‐terminal half of EWSR1 contains a highly disordered activation domain that contains a glycine‐rich region; in fusion proteins, this domain potently and oncogenically activates a transcription factor fusion partner. Previous studies have shown that the deleted C‐terminal domain of EWSR1, which contains an RNA binding motif, is normally responsible for a natural dampening and regulation of the glycine‐rich activation domain2. Surprisingly, however, a normal physiological function has yet to be established for EWSR1 or any of the closest homologs. To solve this problem, we performed simple yet directed bioinformatic analysis to identify distantly related homologs with experimentally validated functions. We specifically identified a family of glycine‐rich RNA binding proteins in Arabidopsis thaliana that act as RNA chaperones in the cold shock response. These RNA chaperones help RNAs to fold properly, as well as aid in their transport, splicing, and translation under stress.3 Interestingly, these plant RNA chaperones show the same self‐associative activity seen in EWS, in which the glycine‐rich region can transiently associate with the RNA‐binding domain to modulate protein activity.4 Based on this similarity, we hypothesize that EWSR1 functions in humans as an RNA chaperone, ensuring correct folding of RNAs, and may have enhanced activity under stress conditions.

Support or Funding Information

Hispanic‐Serving Institutions STEM Grant from the U.S. Department of Education
Substrate Polyubiquitination Increases Proteasomal Processivity
Authors: Reichard Eden L , Dewey William J , Nassif Nicholas D , Kraut Daniel Adam
Published: November 2023

The 26S proteasome in eukaryotes is the macromolecular machine responsible for the degradation of misfolded and damaged polypeptides, as well as signaling and regulatory proteins that accumulate intracellularly. Most proteins are marked for degradation and targeted to the proteasome by the covalent attachment of polyubiquitin chains, which are recognized by receptors on the 19S regulatory particle of the proteasome. After initiating degradation, the polyubiquitin tag is removed and the proteasome moves processively along the protein substrate, unfolding domains as they are encountered and translocating the unfolded polypeptide to the protease active sites in the 20S core for degradation into small peptides. However, the proteasome can stall during this process and rather than successfully unfolding the encountered domain, the proteasome will release the substrate as a partially degraded protein, which can assume a new biological function or form a potentially toxic byproduct. While our understanding of proteasomal processivity remains limited, it has been shown that ubiquitinated substrates can activate the proteasome's ATPase and peptidase activities. We set out to determine if substrate ubiquitination affected the proteasome's ability to unfold its substrates, since the ubiquitin chain is removed early on in the degradation process. To accomplish this, a previously established processivity assay was used, in which the unfolding and subsequent degradation versus the release of a stabilized dihydrofolate reductase (DHFR) domain are quantified. The ratio of degradation to release rates is defined as the unfolding ability U, a measure of the proteasome's processivity. We compared the unfolding abilities of ubiquitin‐independent substrates, as well as substrates ubiquitinated with the E3 ligase Rsp5 (reported to give K63‐linked chains) or the E3 ligase complex Cul3/Rbx1/Keap1 (reported to give primarily K48‐linked chains). We found that significantly less folded DHFR fragment was produced during degradation of the Cul3/Rbx1/Keap1‐ubiquitinated substrate, resulting in a higher unfolding ability, compared to the Rsp5‐ubiquitinated substrate. The unfolding ability for ubiquitin‐independent substrates was much lower than that of either Rsp5‐ or Keap1 ubiquitinated substrates. These results suggest that the presence and chain architecture of polyubiquitin on a substrate can shift the proteasome towards a more active state during engagement, which results in a greater unfolding ability.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under Grant No. 1515229.
The Effects of the H90 and E256 Mutations in the gMDH Interface on the Stability, Function, and Interface Interactions of gMDH
Authors: Stewart Zachary , Garcia Nina Marie , Bell Ellis , Bell Jessica
Published: November 2023

Malate dehydrogenase (MDH) is prevalent in all organisms ranging from simple prokaryotes to higher eukaryotes and plays a central role in energy metabolism.. The protein, with its dimer structure is regulated by citrate, and thought to involve subunit interactions in this regulation. Understanding the mechanism of subunit interactions is central to understanding how it catalyzes its reaction and its regulation. The interface involves both “favorable” and “unfavorable” amino acid interactions which have been explored computationally using HINT. To probe the role of individual sidechains at the interface this project aims to mutate and disrupt unfavorable interactions in the interface by site directed mutagenesis and determine the effects on stability, activity, and/or regulation of enzymes. Two conserved residues, H90 and E256 appear to form a pH dependent interaction across the interface. We have constructed two mutants, H90Q and E256Q, and expressed and purified the resultant proteins. Overall circular dichroism and fluorescence spectroscopy give information on the secondary and tertiary structures of the mutants, while size exclusion chromatography established quaternary structure. The overall stability using thermal denaturation, the kinetic properties including the pH dependence and the impact on Citrate regulation were determined. In addition using fluorescence based thermal shift assays and response to ANS binding we have explored the effects of ligands, including malate, OAA, NAD, NADH, and citrate. Overall the results suggest roles for both residues in several aspects of the stability and regulation of the enzyme and a model is proposed to account for the results obtained.
Establishing a transcription factors protein network for lymphangiogenesis: identification of binding domains, impact of pathological mutations and perturbations by small molecules
Authors: Moustaqil Mehdi , Overman Jeroen , Fontaine Franck , Bailey Timothey , Francois Mathias , Gambin Yann , Sierecki Emma
Published: November 2023

As a major route of cancer metastasis is via the lymphatic vasculature, therapeutic manipulation of neo‐lymphangiogenesis is an important adjunct treatment for cancer. So far only anti‐VEGF signalling molecules have been developed to counteract tumour‐induced de novo vessel formation, yet transcription factors could provide a wealth of potential molecular targets.

Classic reverse genetic approaches in mice have validated individual roles for Sox18, Prox1, Nr2f2 (COUP‐TFII) and Gata2 in lymphangiogenesis. Expression of SOX18, COUP‐TFII and GATA2 coincide only in discrete locations within the vasculature and result in the induction of lymphatic endothelial progenitor cell specification. However, no physical interaction network between those key players has been identified yet.

Here we map the protein‐protein interactions between transcription factors important for lymphatic vascular development to identify key transcription factor interactions as novel potential therapeutic targets. The direct interactions between SOX18, COUP‐TFII, GATA2, MEF2C and components of the Notch pathway (RBPJ, HEY1) have been assessed in vitro using a proximity assay (AlphaScreen) coupled to cell‐free expression of the proteins. These data allowed to reconstitute the network of interactions involved in the control of lymphatic endothelial cell specification.

Using truncations, we established a comprehensive molecular mapping of the dimerization and protein binding domains of SOX18. Further, we defined how those interactions are affected by mutations associated with lymphatic diseases, such as Hypotrichosis‐Lymphedema‐Telangiectasia.

We then used this network as a tool for drug discovery. Perturbations created by small molecules that impact lymphangiogenesis in vivo were measured on the whole network. Inhibition of direct interactions was measured and long‐scale modifications of the network could be inferred. This molecular dissection then provides new information to understand how the physiological effects of drugs are mediated.
Determining histone deacetylase 8 substrates using non‐natural amino acids
Authors: Lopez Jeffrey , Majmudar Jaimeen , Martin Brent , Fierke Carol
Published: November 2023

The Histone deacetylase family is comprised of 18 isozymes that catalyze the removal of an acetyl group from the ɛ‐position of a lysine. Lysine deacetylation is a post‐translational modification that affects a diverse array of different macromolecules and has been implicated in various types of cancers and neurodegenerative diseases. To elucidate and identify HDAC substrates, we have incorporated the photo reactive, non‐natural amino acid ‐ p‐benzonyl‐4‐phenylalanine (pBpa) ‐ into HDAC8, a structurally well characterized isozyme of this family with a largely unknown substrate pool. Photo cross‐linking will trap both substrates and binding partners of HDAC8 and enhance the understanding of how this enzyme is regulated in vivo. Through this system, we have an initial screen of catalytically active peptides toward HDAC8. In addition, this will validate the approach as a feasible system to prove other HDAC isozymes.

Support or Funding Information

NIH – RO1G40602 (C.A.F)NIH ‐‐ 1F31GM116619‐01 (J.E.L)
Mass Spectrometry based Proteomics to Investigate and Characterize the Jumping Translocation Breakpoint (JTB) Protein using Cancer Cell Lines
Authors: Channaveerappa Devika , Li Kangning , Darie Costel C.
Published: November 2023

Human JTB (hJTB) is a gene located on the human chromosome 1 at q21 which is involved in the unbalanced translocation in various types of cancer. It encodes a highly conserved transmembrane protein of 16.4 kDa. JTB protein is ubiquitously present in normal cells and is found to be overexpressed in different types of cancer including prostate and breast cancer. Hence this protein could be a tumor biomarker for different types of malignancies and a potential target for their treatment. However, the biological function and the pathway through which this protein causes increased cell growth and differentiation is not entirely clear. Investigation and comparison of the proteomes of cells with upregulated and downregulated JTB can be a good approach to understand the function of the protein and also its contribution to tumorigenesis.

In this study, MCF 7 breast cancer cell lines were transfected with the sense and antisense orientation of the JTB cDNA in HA, His and FLAG tagged CMV expression vector. Transiently transfected cells were first selected and were lysed, centrifuged and the proteins were extracted from the cells. Anti‐JTB antibody was used to confirm the expression of JTB by western blotting technique. Immunoprecipitation was also done to study the protein complexes and protein‐protein interactions. The extracted proteins were then separated by SDS‐PAGE. Individual gel bands were cut, washed and digested with trypsin. The resulting peptides were analyzed by a Nano Acquity UPLC coupled with QTOF Ultima Mass Spectrometer and then by a Nano Acquity UPLC coupled with Xevo G2 Mass Spectrometer. The raw data files obtained were processed and converted to peak list files using ProteinLynx Global Server (PLGS 2.4). Further data analysis was done using Mascot server and Scaffold 4.1 software.

We produced and purified two sets of antibodies against the protein JTB. The cells were successfully cultured with both upregulation and downregulation of JTB for proteomics analysis.

Furthermore, stable transfection will also be done for large scale protein production and to evaluate the JTB gene function and regulation mechanism. Two other isoforms of JTB will be analyzed and characterized to study their function and will be compared with the wild type JTB. Validation and functional studies will also be performed. These studies could help us elucidate the mechanism through which JTB induces cell proliferation and test the JTB protein as a potential drug target for malignancies with overexpression of the protein.
Rapid and reproducible proteotype acquisition from biopsy level samples
Authors: Guo Tiannan , Aebersold Ruedi
Published: November 2023

Clinical specimens are each inherently unique, limited and nonrenewable. Small samples such as tissue biopsies are often completely consumed after a limited number of analyses. We have developed an integrative mass spectrometry‐based proteomic methodology that integrates pressure cycling technology‐assisted sample preparation and SWATH‐MS, thereby enabling high‐throughput digitization of the proteome of biopsy‐level samples. Through intelligent data mining based on a prior knowledge of peptide elution and fragmentation, 1000s proteins expressed in 100s of cancer cells and tissues could be precisely determined without any missing value. We define the thus acquired complete acute quantitative proteomic status of a sample as “proteotype”, and demonstrate that proteotype synergizes with genotype and transcriptotype in terms of correlation with clinical phenotypes including sensitivity to drugs in cancer cells (NCI‐60 panel), tissue histomorphology and disease outcome (prostate cancer). The SWATH data maps allow perpetual in silico navigation of the proteome, and uncover signals of non‐canonical peptides such as post‐translational modified peptides and mutated peptides. Examples from clinical samples will be discussed.

Support or Funding Information

The work was supported by the SystemsX.ch project PhosphoNetX (to R.A.), the Swiss National Science Foundation (grant no. 3100A0‐688 107679 to R.A.), and the European Research Council (grant no. ERC‐2008‐AdG 233226 to R.A.).
Achieving enzyme‐like behavior with amino acids and peptides in the outer coordination sphere of hydrogenase mimics
Authors: Shaw Wendy , Borolugodage Nilusha , Dutta Arnab
Published: November 2023

Hydrogenases interconvert H2 and protons at high rates and with high energy efficiencies, providing inspiration for the development of molecular catalysts. Studies designed to determine how the protein scaffold can influence a catalytically active site have led to the synthesis of amino acid derivatives of [Ni(PR2NR′2)2]2+ complexes, [Ni(PCy2NAmino acid2)2]2+ (for H2 oxidation) and [Ni(PPh2NPh‐Amino acid2)2]2+ (for H2 production). These amino acid containing derivatives increase rates in H2 production catalysts, with basic amino acids resulting in the largest enhancements. For H2 oxidation catalysts, the amino acids render the complexes water soluble, and the resulting catalysts have faster rates and significantly lower overpotentials than the unmodified complexes. Reminiscent of enzymes, reversibility is achieved while fast rates are maintained, although acidic conditions are needed for the complex in contrast to the enzyme. These observations demonstrate that outer coordination sphere amino acids work in synergy with the active site and can play an equally important role for synthetic molecular electrocatalysts as the protein scaffold does for redox active enzymes.

Support or Funding Information

Office of Science, Basic Energy Sciences
Mutagenesis and Steady State Kinetic Characterization of a Class B Metallo‐β‐lactamase
Authors: Baker Julia P. , Eberhardt Eric
Published: November 2023

Antibiotic resistance is of growing concern for the medical community worldwide due to the overuse of common antibiotics that has created an opportunity for bacteria to evolve novel mechanisms for resistance to antibiotics. Specifically, bacteria that express enzymes that inactivate β‐lactams prevent the antibiotics from inhibiting cell wall biosynthesis in bacteria.

Class B β‐lactamases, metallo‐β‐lactamases, are of particular concern. These metal dependent enzymes are resistant to almost all classes of β‐lactams, including carbapenems, which have the broadest spectrum of activity. One such enzyme is β‐lactamase 2 [Bc(II)] from Bacillus cereus, a mono‐ or di‐zinc dependent metallo‐β‐lactamase. The amide bond hydrolysis of β‐lactams by Bc(II) is likely facilitated by a zinc(II)‐bound water and the amino acid residue aspartate‐90, which is thought to participate in general acid base catalysis. Bicknell and coworkers suggest that the metal acts as a Lewis Acid and binds a hydroxide ion that carries out a direct nucleophilic attack on the β‐lactam carbonyl. The metal is suggested to stabilize the resulting tetrahedral intermediate, allowing for the carboxylate anion of aspartate‐90 to deprotonate the metal‐bound hydroxide and then protonate the amine nitrogen. To more thoroughly understand the role of aspartate‐90, the steady state kinetics of wildtype, aspartate‐90‐asparagine, and aspartate‐90‐alanine mutant are being evaluated.
Rational engineering of the CDK9/CyclinT complex improves protein stability enabling kinetic analysis of inhibitors by SPR
Authors: Code Erin , Ferguson Andrew , O'Connell Nichole , Doig Peter
Published: November 2023

Inhibition of CDK9, an enzyme that regulates transcriptional elongation, can lead to apoptosis in cancer cells. Thus CDK9 inhibitors are potential new anti‐cancer agents. In order to more fully characterize novel CDK9 inhibitors that we are developing we wanted to be able to perform kinetic analyses including the determination of kinetic on and off rates. The CDK9/CyclinT kinase domain exhibited rapid loss of ligand binding ability when immobilized on a SPR chip, preventing collection of kinetic data. To improve stability, a series of amino acid changes were made based on the crystal structure. These included surface residue changes of both the CDK9 kinase domain and Cyclin T, and combinations of the various changes (up to 7 changes in CDK9 and 5 in Cyclin T). Modified genes were synthesized, a matrix of modified CDK9 and cyclin T combinations defined (18 conditions) and expressed in insect cells. Constructs were evaluated by expression levels, purification yield, ability to concentrate protein to high levels, increased melting temperature, conjugation and stability during SPR. The original CDK9/Cyclin T complex lost activity minutes after conjugation to the SPR chip whereas the “thermostabilized” CDK9 (7 residue modified) Cyclin T (5 residue modified) complex is active for 2.5 days after immobilization allowing for determination of on and off rates of CDK9 Cyclin T inhibitors.
Topoisomerase II Inhibition by BZP Thiosemicarbazones
Authors: Stephens Madison Taylor , Angulo Yocelyne L. , Eatebarian Roya R. , Medawala Wathsala , Morris William , Deweese Joseph E. , Lisic Edward , Jiang Xiaohua
Published: November 2023

Topoisomerase II (TopoII) regulates DNA topology during DNA metabolism and is active in replicating cells. Thus, it is the target of several FDA approved anticancer drugs such as Etoposide. Current topoisomerase II inhibiting drugs do not specifically target the alpha form of the enzyme, affecting normal cells and causing serious health problems. There has been evidence that α‐(N)‐heterocyclic thiosemicarbazone (TSC) compounds have demonstrated antiproliferation effects on tumor lines. Our experiment investigates the inhibition of specific BZP thiosemicarbazone compounds on TopoIIα and stimulation of the cleavage complex of this enzyme. Metal‐free as well as Cu(II) and Pd(II) complexes of BZP derivatives of TSCs were examined against the full TopoII relaxation and its ability to create double strand breaks. The Cu(II) complexes showed the highest inhibitory activity against the enzyme and highest stimulation of the cleavage complexes in the absence of ATP. Among the Cu(II) complexes of BZP derivatives, Cu(BZP‐tBTSC)Cl exhibited a significantly high increase in the cleavage complex of TopoIIα compared with Etoposide. In addition, DTT abolished the effect of BZP TSCs on TopoIIα The mechanism of inhibition is not clear, but seems to be different from that of Etoposide.

Support or Funding Information

Research supported by Tennessee Technological University through Undergraduate Research and Creative Activity (URECA) grants.
Structural and Functional Characterization of the Sco Protein from Thermus thermophilus
Authors: Lopez Liezelle , Hunsicker‐Wang Laura
Published: November 2023

The Sco family of proteins has been implicated in the assembly of cytochrome c oxidase (Complex IV) of the respiratory electron transport chain, but its function is still under debate. TtSco has been shown to bind to Cu (I) and Cu (II) ions. However, it is also structurally similar to thioredoxins and has been shown to be able to reduce the disulfide bond present in the CuA site of cytochrome c oxidase prior to copper insertion. To provide evidence of Sco's role in the assembly of the CuA‐center, single cysteine to serine mutants, TtSco‐C53S and TtCuA‐C149S, were first produced. TtCuA was conjugated to a chromophore that is released upon reaction with TtSco. The two proteins were reacted and monitored via UV‐Visible spectroscopy in a 1:1 ratio at 412 nm to monitor formation of a mixed disulfide intermediate (MDI) between the two. TtSco and TtCuA have been reacted to form the MDI at different stoichiometric ratios and to probe the kinetics of formation of the MDI. Our results indicate much higher production of MDI with Sco in excess. Our second aim is to crystallize the mixed disulfide intermediate to visualize and better understand the interactions between TtSco and TtCuA. Cation exchange chromatography has shown the most success in separating MDI from unreacted starting materials. A successful condition for crystallization of the MDI has been found and consists of ammonium sulfate and polyethylene glycol 8,000. Lastly, the effects of copper binding on the relative stability of apo and holo forms of TtSco have been studied via chemical denaturation. Interestingly, preliminary data suggests that holo‐TtSco is slightly less stable than apo‐TtSco. Further studies aim to investigate the effect of the copper‐ligating His139 on stability of the protein, both with and without metal bound to gain more insight into TtSco's ability to bind metals and the role metal binding plays in its function.

Support or Funding Information

Welch Foundation
A New Oridonin Analogue Suppresses Triple‐Negative Breast Cancer Cells and Tumor Growth via Inducing the Expression of Death Receptor 5
Authors: Chen Ceshi , Wu Jing , Zhou Jia
Published: November 2023

Triple‐negative breast cancer (TNBC) remains the leading cause of death among women with breast cancer worldwide. Oridonin is a natural anti‐cancer compound isolated from the traditional Chinese herb Rabdosia rubescens. However, the antitumor efficacy of oridonin for TNBC and other cancers is far away from ideal. In this study, we investigated a series of newly designed oridonin analogues against HCC1806 and HCC1937 TNBC cell lines and identified CYD‐6‐28 that significantly inhibits cancer cell proliferation, induces G2/M phase cell cycle arrests and apoptosis. CYD‐6‐28 induces the expression of p21 and the cleavage of caspase‐8, −3, −7 and PARP, and inhibits the expression of Cyclin D1, FLIP and XIAP. CYD‐6‐28 also inhibits the activation of STAT3 and AKT while inducing the activation of ERK. We demonstrated that CYD‐6‐28 induces apoptosis at least partially through inducing the expression of Death Receptor 5 (DR5). Finally, CYD‐6‐28 significantly suppresses HCC1806 xenograft tumor growth in nude mice at 5 mg/kg without significant toxicity. Taken together, CYD‐6‐28 has the potential to be developed as a promising therapeutic agent to treat TNBC.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China (81120108019, U1132605, and 81325016 to Chen, C), and the grants P30 DA028821 and R01 DA038446 from the National Institutes of Health, and Cancer Prevention Research Institute of Texas (CPRIT) award (to Zhou, J).
MAGP2 binding to αvβ3 integrins affects levels of microfibril formation
Authors: Daniels Ryan , Valdivia Michael , Perez Andrew , Miyamoto Alison
Published: November 2023

Matrix metalloproteases (MMPs) are secreted proteins that degrade extracellular matrix (ECM) proteins and are thought to play a major role in cancer cell motility, invasion, and angiogenesis. Activation of αvβ3 integrins has been shown to increase expression or activation of MMPs. Microfibril‐associated glycoprotein 2 (MAGP2) is a small secreted protein that binds to microfibrils in the ECM, and overexpression of MAGP2 in human papillary serous ovarian carcinomas correlates to poor prognosis. MAGP2 has an RGD integrin binding motif in the amino‐terminal half (NT). This motif was shown to be necessary to stimulate cell motility in an an αvβ3 integrin dependent manner. The carboxy‐terminal half of MAGP2 contains a matrix binding domain that is necessary and sufficient for MAGP2 to associate with microfibrils. Direct transfection of a construct expressing the NT‐MAGP2 shows a decrease in extracellular fibers via immunocytochemistry (ICC). This construct can bind to αvβ3 integrins but not microfibrils, suggesting that this effect is mediated by the MAGP2 RGD integrin binding motif. Therefore, we hypothesize that NT‐MAGP2 binding to αvβ3 integrin promotes the activity of MMPs to degrade the ECM in order for the cell to be motile during metastasis. To test the requirement for the RGD motif two mutant constructs were generated in the RGD integrin binding motif of the NT‐MAGP2 construct with a conservative (RGE) and non‐conservative (RGA) mutation to assess levels of cell‐associated MAGP2 and microfibril levels via ICC. As expected, the wild type NT‐MAGP2 construct had the most cell association and greatest loss of microfibrils, followed by the RGE mutant, and lastly the RGA mutant. These preliminary results suggest that there is an inverse relationship between amount of cell associated MAGP2 and amount of microfibrils present. Current studies are testing whether the NT‐MAGP2 treatment increases MMP activity via zymography. These studies could provide insights for possible anti‐MAGP2 treatments of ovarian cancer.

Support or Funding Information

Howard Hughes Medical School Two‐Year Undergraduate Research Scholars Programs (RD), California Institute of Regenerative Medicine Bridges to Stem Cell Research Program (MV, AP), Department of Biological Science, CSUF (AM).
Small fungal molecule metabolites targeting mosquito midgut FREP1 block malaria transmission
Authors: Li Jun
Published: November 2023

Malaria transmission depends on sexual stage Plasmodium parasites successfully invading Anopheline mosquito midguts following a blood meal. However, the molecular mechanisms of Plasmodium invasion of mosquito midguts have not been fully elucidated. Previously, we showed that genetic polymorphisms in the fibrinogen‐related protein 1 (FREP1) gene are significantly associated with P. falciparum infection in An. gambiae, and FREP1 is important for P. berghei infection of mosquitoes. Here we identify that the FREP1 protein is secreted from the mosquito midgut epithelium and integrated as tetramers into the peritrophic matrix, a chitinous matrix formed inside the midgut lumen after a bloodmeal feeding. Moreover, we show that the FREP1 can directly bind Plasmodia sexual stage gametocytes and ookinetes. Notably, ablating FREP1 expression or targeting FREP1 with antibodies significantly decreases P. falciparum infection in mosquito midguts. Our data support that the mosquito‐expressed FREP1 mediates mosquito midgut invasion by multiple species of Plasmodium parasites via anchoring ookinetes to the peritrophic matrix and enabling parasites to penetrate the peritrophic matrix and the epithelium. Thus, targeting FREP1 can limit malaria transmission. To test the hypothesis that small molecules that disrupt this interaction will prevent parasites from infecting mosquitoes, we developed an ELISA‐based method to screen a fungal extract library. We obtained a candidate fungal extract of Aspergillus niger that inhibited the interaction between FREP1 and P. falciparum infected cells by about 92%. The inhibition specificity was confirmed by immunofluorescence assays. Notably, feeding mosquitoes with the candidate fungal extract significantly inhibited P. falciparum infection in the midgut without cytotoxicity or inhibition of the development of P. falciparum gametocytes or ookinetes. A bioactive natural product that prevents FREP1 from binding to gametocytes or ookinetes was isolated and identified as P‐orlandin. Importantly, the nontoxic orlandin significantly reduced P. falciparum infection intensity in mosquitoes. Therefore, disruption of the interaction between FREP1 and parasites effectively reduces Plasmodium infection in mosquitoes. Targeting FREP1 with small molecules is thus an effective novel approach to block malaria transmission.

Support or Funding Information

Oklahoma Center for Advancement of Science & Technology (HR13‐055), National Institute of Health (1R21AI11517801A1), and National Science Foundation Career Award (1453287)
Towards Understanding Cell Cycle Regulation in the Pathogenic Cryptococcus neoformans and Saccharomyces cerevisiae
Authors: Kawamoto Susumu , Virtudazo Eric , Ohkusu Misako , Suganami Akiko , Tamura Yutaka
Published: November 2023

We have reported that the cell cycle behavior of the pathogenic yeast Cryptococcus neoformans (C. neoformans) is different from the cell cycle control exhibited by the model yeast Saccharomyces cerevisiae (S. cerevisiae), and also have reported the molecular characterization and physiological roles of the two main eukaryotic cell cycle genes, C. neoformans cyclin dependent kinase 1 (CnCdk1) and cyclin homologues. Only a single Cdk1‐related G1 and G1/S cyclin homologue was found in the genome sequence of C. neoformans and was designated CnCln1. Surprisingly, CnCln1 was not only able to complement the function of the G1 cyclins of S. cerevisiae, such as ScCln3, but also the G1/S cyclins of S. cerevisiae, such as ScCln1 and ScCln2. Our in silico analysis demonstrated that the CnCln1/ScCdk1 complex was more stable than any of S. cerevisiae cyclins (ScCln1, ScCln2, ScCln3) and ScCdk1 complexes. These results are consistent with in vitro analysis that has revealed the flexible functional capacity of CnCln1 as a Cdk1‐related G1 and G1/S cyclin of S. cerevisiae.

On the other hand, Cln1 and Cln2, G1/S cyclins of S. cerevisiae, oscillate during the cell cycle, rising in late G1 and falling in early S phase. We have been tried to elucidate the structure basis of the functional distinction between Cln1 and Cln2. We performed in silico simulations: construction and evaluation of three dimensional structures of Cln1‐Cdc28 and Cln2‐Cdc28 complexes. Our in silico simulations suggested that the interaction of Cln1 and Cln2 with Cdc28 were in the two distinct situations, designated as flip and flop conformation, at the extra amino acid region in the cyclin box of Cln1 and Cln2. We speculated the trigger of this flip‐flop conversion of the extra amino acid region in the cyclin box of Cln1‐Cdc28 and Cln2‐Cdc28 might be regulated by the ubiquitination of the sequences rich in Pro (P), Glu (E), Ser (S) and Thr (T), so‐called PEST motifs, in Cln1 and Cln2. Furthermore, we presumed that the functional superiority between Cln1 and Cln2 in the G1/S phase of S. cerevisiae might be controlled by flip‐flop conversion and ubiquitin‐proteasome pathway.

Support or Funding Information

This work was partially supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and Cooperative Research Program of Medical Mycology Research Center, Chiba University.
Tellimagrandin I Decreases Cyclin B1 Protein Levels through SP1 in MDA‐MB‐231 Cells
Authors: White Brandon , Luis Tiani , Rodriguez y Baena Alessandra , Wang Laura , Shah Savita
Published: November 2023

Consumption of walnuts has been shown to inhibit MDA‐MB‐231 breast cancer xenograft growth in nude mice. However, identification of compounds and mechanism of action have not been identified. We recently reported that a walnut extract treatment of the triple‐negative breast cancer cell type, MDA‐MB‐231 cells, could a) arrest the cells in G2/M and decreased cyclin B1 levels, b) decrease mitochondrial membrane potential and release cytochrome c from the mitochondria, and c) increase phosphotidyl serine flipping in the cell membrane, and that these events were associated with cell growth inhibition and apoptotic death. Over‐expression of Cyclin B1 has been shown in breast cancers that lack estrogen receptor, progesterone receptor, and HER2 (i.e. the triple‐negative breast cancer) and is a potential emerging biomarker as well as therapeutic target. In an attempt to conduct detailed mechanistic studies with active compounds from walnut extracts, we were able to identify numerous ellagitannins which induced cell death in breast cancer cells. Of the ellagitannins isolated, tellimagrandin I (TMI) showed the strongest cell death. We have gone on further to characterize the mechanism of action of TMI. We have performed time and dose response analysis of TMI on MDA‐MB‐231 cells for cell death and show that TMI has an IC50 of 60 μM. TMI is also able to reduce Cyclin B1 mRNA and protein levels similar to a walnut extract. We see that mRNA levels are reduced relatively quickly (within 1 hour) while protein levels begin to be reduced at a much later time suggesting that the effects are transcriptional. We have identified other cell cycle specific mRNAs that are also reduced by TMI. A careful examination of all mRNAs that are reduced show that they all have SP1 binding sites. Interestingly, we show that SP1 mRNA levels are also reduced upon TMI treatment in a similar fashion as Cyclin B1. Taken together our results suggest that TMI is able to reduce Cyclin B1 through inhibition of SP1 in MDA‐MB‐231 cells.

Support or Funding Information

This work was supported by a grant from the California Walnut Commission to BW.
Curcumin Inhibits Development and Cell Adhesion in Dictyostelium discoideum: Implications for YakA Signaling and GST Enzyme Function
Authors: Garige Mamatha , Walters Eric
Published: November 2023

The molecular basis for nutraceutical properties of the polyphenol curcumin (Curcuma longa, Turmeric) is complex, affecting multiple factors that regulate cell signaling and homeostasis. Here, we report the effect of curcumin on cellular and developmental mechanisms in the eukaryotic model, Dictyostelium discoideum. Dictyostelium proliferation was inhibited in the presence of curcumin, which also suppressed the prestarvation marker, discoidin I, members of the yakA‐mediated developmental signaling pathway, and expression of the extracellular matrix/cell adhesion proteins (DdCAD and csA). This resulted in delayed chemotaxis, adhesion, and development of the organism. In contrast to the inhibitory effects on developmental genes, curcumin induced gstA gene expression, overall GST activity, and generated production of reactive oxygen species. These studies expand our knowledge of developmental and biochemical signaling influenced by curcumin, and lends greater consideration of GST enzyme function in eukaryotic cell signaling, development, and differentiation.

Support or Funding Information

Department of Biochemistry and Molecular Biology, Howard University College of Medicine, Washington D.C.
Alcohol and HIV Protease Inhibitor‐induced inflammasome activation and liver lipotoxicity
Authors: Hinton Michael
Published: November 2023

The development of highly‐active‐antiretroviral therapy(HAART) has allowed management of HIV and extended the lives of those infected. Alcohol abuse, which is very common in HIV‐1 infected patients, is one of the most important co‐morbid risk factors for liver injury and has been associated with the occurrence of serious metabolic syndrome and subsequent discontinuation of HAART in HIV patients. The importance of the NLRP3 inflammasome in liver diseases has been recognized. Kupffer cells( KC) are the major resident macrophages and play a critical role in regulating hepatic inflammatory response. We have shown that HIV protease inhibitors (PI) increase the production of mature IL‐1β production in macrophages suggesting the activation of NLRP3. The aim of this study is to examine the effect of alcohol and HIV PIs on NLRP3 activation in KCs and characterize the impact of infllammasome activation in KCs on alcohol and HIV PI‐induced hepatic lipotoxicity.

Methods

Primary mouse KCs will be isolated and treated with individual HIV PIs with or without alcohol. The mRNA levels of inflammasome component genes including NLRP3, ASC, and pro‐caspase will be measured by real‐time RT‐PCR. The cell culture medium will be collected for ELISA analysis of IL‐1β and IL‐18.

Results

We find both alcohol and HIV PI‐induced activation of NLRP3 in KCs. The expression of mature IL‐1β and IL‐18 will be increased by alcohol and HIV PIs. The active form of caspase‐1 will be increased.

Conclusions

Activation of the KC infllammasome is a key cellular mechanism underlying alcohol and HIV PI‐induced hepatotoxicity.
Integration of Biochemistry and Problem Solving‐Based Pedagogy Throughout the Chemistry Curriculum in Support of Learning Outcomes Articulated by the Association of American Medical Colleges (AAMC), the ASBMB and the American Chemical Society (ACS)
Authors: Jakubowski Henry V. , Graham Kate , McIntee Ed , Schaller Chris
Published: November 2023

The Chemistry Department at the College of St. Benedict/St. John's University has integrated biochemistry throughout a dynamic and modern curriculum to enhance its appeal to students, to better prepare them for modern science and an early entry into research, and to increase our number of majors. In support of these changes we have adopted modern pedagogy that use a flipped pre‐class format and in‐class group problem solving activities. These changes align with biochemistry content and the scientific reasoning and inquiry skills (SIRS) represented in the MCAT2015. They support ASBMB calls for students to be trained in an interdisciplinary fashion that allows them to explain cross‐disciplinary concepts. They also align with the American Chemical Society (ACS) encouragements to create integrated foundation courses that combine traditional sub‐disciplines of chemistry and that are organized on integrative principles such as reactivity. Student start with a single introductory course (Chemical Structure and Properties) and then take three following courses in Chemical Reactivity which integrate one semester each of organic, inorganic and biochemistry. Traditional biochemistry lab skills are integrated into separate foundation lab courses in Chromatography, Synthesis, and Measurement. Upper division students take half‐semester in‐depth courses in such contemporary fields as Chemical Biology, Signal Transduction, and Medicinal Chemistry. Chemistry majors also taking an in‐depth Integrated Lab course with a three week “biophysical module”. Descriptions of this novel and integrative curriculum, how it prepares students for the MCAT and SIRS in general, and assessment of its effects will be presented. Especially interesting are changes in our number of majors and their diversity.

Support or Funding Information

This work was supported by NSF DUE 1043566
Dietary Depletion of Cow's Milk microRNAs Impairs Fecundity in Mice
Authors: Sadri Mahrou , Xie Fang , Wood Jennifer , Zempleni Janos
Published: November 2023

Background

Traditionally, microRNAs have been considered endogenous regulators of gene expression. Recently, we have made the paradigm‐shifting discovery that humans absorb cow's milk microRNAs (encapsulated in exosomes) by endocytosis, and that body pools of circulating microRNAs decrease by >60% in mice fed milk microRNA‐depleted diets.

Hypothesis

Dietary depletion of microRNAs impairs fecundity in mice, i.e., causes phenotypes of sub‐optimal health.

Methods

C57BL/6J mice were fed modified AIN‐93G diets depleted of microRNAs [denoted ExosomeMinus (E‐); <10% microRNAs compared with control diets, E+], starting at age 3 weeks through age 15 weeks. Mice were mated in all possible permutations (E+ males × E+ females, E+ × E−, E− × E +, E− × E−; n=8 pairs per group). Variables of fecundity were analyzed and statistical significance was assessed using one‐way ANOVA and Dunnett's post‐hoc test.

Results

Sixty‐two to 75% of mating did not result in pregnancy if at least one parent was fed the E− diet, compared with only one (12.5%) failed mating in controls. The average litter size produced by E+/E+ breeders was twice the size in other groups: 7.6±1.9 pups for E+ males/E+ females, 3.6±3.2 for E+/E−, 3.5±3.0 for E−/E+, and 4.1±3.4 for E−/E−; P<0.05, n=8). The average birth weight of pups born to dams fed the E− diet (E+ males) was 1.13±0.11 g compared to 1.30±0.09 g in E+/E+ breeders (P<0.05). MicroRNA depletion also impaired survival of litters to weaning (3 weeks) if dams were continued on the previous diets: 100% survival in E+/E+, zero survival in E+/E− and E−/E+, and one surviving litter in E−/E−. The hepatic concentrations of purine metabolites were 65% higher in E‐females compared to E+ females (16.6±3.3 vs. 10.1±1.0 μmol/L xanthine; P<0.05, n=5), suggesting a potential mechanism leading to low fecundity.

Conclusions

Depletion of dietary microRNAs elicits disease phenotypes.

Support or Funding Information

Supported by NIFA/USDA 2015‐67017‐23181, NIH 1P20GM104320, Gerber Foundation, Egg Nutrition Center, USDA W‐3002 and Hatch