bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Mammalian Cell Engineering
Authors: Khodthong Chuenchanok , Snow Josh , Juckem Laura 
Published: November 2023

The CRISPR/Cas9 genome‐editing platform is a versatile and powerful technology to efficiently create genetically engineered living cells and organisms. This system requires a complex of Cas9 endonuclease protein with a gene‐targeting guide RNA (gRNA) to introduce double‐strand DNA breaks (DSBs) at specific locations in the genome. DSBs are then repaired by the error‐prone Non‐Homologous End Joining (NHEJ) pathway, resulting in insertions and/or deletions (indels) which disrupt the targeted locus.The success of CRISPR genome editing experiments is limited by the intracellular delivery and expression of Cas9 protein and gRNA. Many methods for achieving CRISPR mediated cleavage have been identified and the choice of DNA, RNA or ribonucleoprotein (RNP) format is dictated by experimental goal and cell type. Transfection of each type of molecule requires specific considerations for efficient functional delivery. We performed transfections using different combinations of molecules including: plasmid DNA, messenger RNA, Cas9 protein and gRNA to maximize targeting of the Cyclophilin B (PPIB) gene in HEK 293T/17, U2OS, and other mammalian cell types. Our results extend the utility of the CRISPR/Cas9 system by identifying optimal transfection conditions for intracellular delivery of Cas9 and gRNA in different formats.
Design and construct a type of fluorescently labeled circular DNA molecules to study DNA topology and topoisomerases by fluorescence resonance energy transfer
Authors: Leng Fenfei , Gu Maxwell 
Published: November 2023

DNA topology, an essential property of the DNA double helix, is under tight homeostatic control in vivo. Studies of DNA topology and topoisomerases are critical for understanding the mechanism underlying several infectious and genetic diseases and also for designing new anti‐cancer and anti‐microbial drugs targeting DNA topoisomerases. Typically agarose gel electrophoresis is used to study DNA supercoiling and topoisomerases. However, gel electrophoresis is time‐consuming and labor‐intensive. It is desirable to develop other assays, such as fluorescence‐based assays, for such studies. In this article, we developed a new method to construct a type of fluorescence‐labeled circular DNA molecules with high yields to study DNA topology and topoisomerases by using fluorescence resonance energy transfer (FRET). Specifically, we successfully produced relaxed and supercoiled pAB1_FL905 that carries fluorescein and dabcyl labels in the 42 bp AT sequence. Our results showed that pAB1_FL905 is a powerful tool to study DNA topology and topoisomerases by FRET. pAB1_FL905 can also be developed into rapid and efficient high‐throughput screening assays to identify inhibitors from the millions of compounds found in small molecule libraries that may target DNA topoisomerases, such as bacterial DNA gyrase and human DNA topoisomerase I and II.Support or Funding Information1R15GM109254‐01A1 from the National Institutes of Health
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 InformationThis 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 InformationThis 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 InformationFunding 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 InformationHispanic‐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 InformationThis material is based upon work supported by the National Science Foundation under Grant No. 1515229.
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 InformationNIH – 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 InformationThe 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 InformationOffice of Science, Basic Energy Sciences