bluuebunny/crossref_metadata_2025_split huggingface dataset:
Redundancy Between Proteasome Chaperones Nas2 and Hsm3 During Proteasome Assembly
Authors: Suppahia Anjana , Chingakham Ranjit Singh , De La Mota Peynado Alina , Roelofs Jeroen 
Published: November 2023

Eukaryotic proteasomes consist of a 20S cylindrical core particle (CP) capped with a 19S regulatory particle (RP) on one or both sides. The regulatory particle can be subdivided into base and lid complexes. The base contains a ring of six homologous AAA‐ATPase subunits; Rpt1 to Rpt6. The efficient assembly of the base requires several proteasome‐specific chaperones. Two of these, Nas2 and Hsm3, show redundancy and bind to neighboring Rpt subunits, Rpt5 and Rpt1 respectively. However, Nas2 and Hsm3 are never found on the same sub complex and it is unclear why they are redundant. Here, we show that the reported rescue of Hsm3 deletion strains by over expression of Rpt1 depends on the presence of Nas2. Interestingly, in an Hsm3ΔNas2Δ strain no increased levels of Rpt1 were detected upon overexpression of Rpt1. This suggest that, at least in part, the Nas2 and Hsm3 redundancy depends on the ability of either protein to stabilize Rpt1 in the cell. Consistent with a potential role of Nas2 in Rpt1 stabilization, we observed a direct interaction between Nas2 and Rpt1 in vitro. This binding site mapped to the N‐terminal region of Rpt1. To understand the importance of the domains in Nas2, we studied the effects of over expression of N‐terminal domain or the PDZ domain of Nas2 in hsm3Δnas2Δ strain and observed differences in their ability to rescue the temperature sensitivity of these cells. Biochemical and structural studies have suggested the binding of Nas2 and Hsm3 to proteasome sub complexes is mutually exclusive and suggests a role of Hsm3 in Nas2 expulsion. Consistent with this, we observed the presence of Nas2 on larger assembly intermediates in strains deleted for HSM3. In all, our data suggest Nas2 and Hsm3 show redundancy at the step of Rpt1 incorporation into proteasome complexes and indicate pliability in the assembly pathway.Support or Funding InformationNIH‐NIMGS (1R15GM112142‐01A1)
Elucidating the Molecular Details of Catalysis for Type III Polyketide Synthases Using Near Natural Substrate/Intermediate Analogs
Authors: Richards Logan R , Stunkard Lee , Benjamin Aaron , Manning Bethany P. , Lohman Jeremy 
Published: November 2023

Natural products are a well‐established source of drugs and other commodity chemicals. Polyketide natural products have a high degree of chemical variability and play many roles in biology, making them targets for protein engineering to generate analogs. Polyketide synthases (PKS) exist as three different types, but all require the function of a ketoacyl synthase or ketosynthase (KS). The KS catalyzed reaction is carried out in three steps, transthiolation of an acyl‐chain onto an active site cysteine yielding an acyl‐enzyme intermediate, binding and decarboxylation of a malonyl‐group to yield an enolate, and a Claisen condensation of the enolate onto the acyl‐enzyme intermediate yielding a carbon‐carbon bond. The molecular details of decarboxylation and Claisen condensation are only poorly characterized, yet these details are needed for protein engineering efforts. Type III PKSs exist as an independently functioning KS, which primarily use coenzyme A (CoA) rather than acyl carrier proteins to carry the acyl‐chains and malonyl‐groups. As type III PKS are the simplest system we chose them as our starting point to elucidate the molecular details of KS catalysis. We have expressed and purified wild‐type and mutant forms of germicidin synthase (GCS) and tetrahydroxynaphthlene synthase (THNS) from Streptomyces coelicolor, and chalcone synthase‐2 (CHS‐II) from Arabidopsis thaliana in E. coli. Mutation of the active site cysteine to glutamine results in enzymes that mimic the acyl‐enzyme intermediate state, and can decarboxylate malonyl‐CoA. Interestingly, these mutants are much less stable than the wild‐type enzymes, suggesting that conformational changes occur upon formation of the acyl‐enzyme intermediate. We propose that using near natural substrate and intermediate mimics will allow us to capture the molecular details of catalysis. As such, X‐ray crystallography is underway with these enzymes in complex our synthetic analogs.Support or Funding InformationWe would like to thank the Department of Biochemistry, Purdue University for their sponsorship of the project. We would also like to thank Jeremy Lohman for providing the space needed to conduct our project.
Preliminary Investigation by NMR of the Electron Transfer Complex Between the Vitamin D Metabolizing Cytochrome P450 24A1 and Adrenodoxin
Authors: Estrada D. Fernando , Tu Youbin 
Published: November 2023

BackgroundDeactivation of the bio‐activated form of vitamin D (1,25‐dihydroxy vitamin D3) represents a key metabolic step that helps determine the bioavailability of the active hormone. Deactivation is accomplished by hydroxylation of carbon 23 or carbon 24 of the vitamin D side chain as mediated by the mitochondrial cytochrome P450 enzyme CYP24A1. CYP24A1 belongs to a sub‐set of P450 enzymes that function in the mitochondrial membrane, where interactions with the soluble [2FE‐2S] cluster protein adrenodoxin (Adx) triggers the electron transfer required for P450 catalysis. Adx also serves as the principle redox partner for all other mitochondrial P450 enzymes and their respective reactions, including the activation of vitamin D by CYP27B1. In light of the importance of this electron transfer step in activation and deactivation of vitamin D, there remain basic questions regarding the formation of the Adx‐P450 enzyme complex. Among these are 1) the interface of the protein complex has yet to be mapped out for a vitamin D metabolizing P450, and 2) it is unknown whether Adx binding influences other regions of CYP24A1 that relate to substrate recognition and access.ObjectiveIn this preliminary study, we have set out to characterize the Adx‐CYP24A1 complex via a combination of spectrophotometric and nuclear magnetic resonance techniques.ResultsOur spectral CYP24A1‐ligand binding data indicate that the affinity between rat CYP24A1 and the vitamin D analog alfacalcidol is strengthened by approximately two‐fold in the presence of adrenodoxin, thereby suggesting a long‐range effect between the putative Adx binding surface of CYP24A1 and the canonical P450 substrate recognition and binding sites. Additionally, we have also optimized the Adx‐CYP24A1 complex for analysis by solution NMR, in which titration with CYP24A1 induces differential line broadening of the spectrum of 15N labeled Adx, with enhanced line broadening occurring in the short α‐helix adjacent to the [2FE‐2S] cluster.ConclusionsThe interaction of Adx with CYP24A1 induces a change in ligand binding, leading to greater affinity for alfacalcidol. Additionally, line broadening of residues in the short α‐helix adjacent to the [2FE‐2S] cluster is suggestive of participation of this region in formation of the complex with CYP24A1. These preliminary studies lay the groundwork for a comprehensive study of the Adx‐CYP24A1 complex under a variety of conditions, including a comparison between the oxidized and reduced states of Adx, and thereby providing insight toward this important step in vitamin D metabolism.Support or Funding InformationThis work was funded in combination by R00GM112862 and SUNY Buffalo startup funds.
Lybatide: Naturally‐Occurring Disulfide‐Stapled Helical Peptides from Lycium barbarum
Authors: Tan Wei Liang , Wong Ka H. , Tam James P. 
Published: November 2023

Small molecules and proteins represent the two major groups of compounds that are currently approved for human use. In between the molecular size of these two groups are the constrained peptides, a class of compounds that integrate their drug‐like advantages. Constrained peptides share the advantage of proteins for high on‐target specificity and low off‐target adverse side effects. They also have the robustness of small molecules to tolerate thermal, chemical and enzymatic degradation. Currently, naturally‐occurring constrained peptides in plants is an underexplored chemical space for drug discovery. Here we report the discovery and characterization of Lybatides, a constrained peptides with unusual cysteine motif and disulfide connectivity from the root bark of Lycium babarum, the plant that produces the popular functional food and herb, wolfberries. Importantly, an X‐ray crystallographic study revealed the presence of a helix as a dominant feature in the lybatide structure, which is stabilized by four cystine bonds. The presence of a single helix without stabilization by another helix or β‐strand is most unusual and has not been reported. As such, the lybatide structure resembles a naturally‐occurring stapled helical peptide. Lybatides are non‐cytotoxic, up to 100 μM, to the cell lines tested and are stable against thermal, acidic and proteolytic denaturation for up to 2 hours. The presence of the helix in the lybatide scaffold should allow the grafting of helical bioactive peptides for developing peptidyl therapeutics. Overall, this study provides a new scaffold and expands the number of existing constrained peptide scaffolds for drug design.Support or Funding InformationThis research was supported by a Competitive Research Grant from the National Research Foundation in Singapore (NRF‐CRP8‐2011‐05) and an NTU iFood Research Grant (M4081467.080).
Profiling and Authentication of Herbal Products
Authors: Huang Jiayi , Wong Ka Ho , Tam James P 
Published: November 2023

Traditional Chinese medicine (TCM) is gaining popularity as an alternative medicine. A central element of TCM is the use of herbal medicine, which could be benefited by a novel authentication method. Astragalus membranaceus, belongs to the Fabaceae family, is a flowering plant native to China. Its root, commonly known as Huang Qi, is a traditional Chinese medicine used for treating diabetes, promoting Qi and boosting immune system. However, there is another medicinal herb, Hedysarum polybotrys, which also belongs to Fabaceae family, sharing the similar Chinese name (Hong Qi) and morphological characteristics with A. membranaceus, making it difficult to differentiate these two species. Here, we report the use of hyper‐constrained, heat‐stable and enzyme‐resistant peptides as a biologic fingerprint for the identification and authentication of herbal medicines. In particular, we use proteomics fingerprints to differentiate 20 samples from each species profiled by MALDI‐TOF MS analysis. Our results showed that A. membranaceus and H. polybotrys are divided into two clusters in a scores plot, suggesting that these two species had distinct proteomic profiles and can be successfully distinguished from each other. Together, the ability to authenticate provides confidence for customers to use herbal medicine and in TCM treatments.Support or Funding InformationAcknowledgementsThis research was supported by a Competitive Grant from the National Research Foundation in Singapore (NRF‐CRP8‐2011‐05) and an NTU iFood Research Grant (M4081467.080)
Bioorthogonal Protein Conjugation: Application for the Development of a Highly Sensitive Bioluminescent Immunoassay for the Detection of Interferon‐γ
Authors: Moutsiopoulou Angeliki , Hunt Eric , Broyles David , Pereira Christie Ataides , Woodward Kristen , Dikici Emre , Kaifer Angel , Daunert Sylvia , Deo Sapna 
Published: November 2023

Interferon‐γ (IFN‐γ) is a 144‐amino acid long N‐terminal methionylated recombinant protein and is a potent multifunctional cytokine which is secreted primarily by activated NK cells and T cells. Originally came to attention due to its anti‐viral activities, IFN‐γ can also act as an anti‐proliferative, and immunoregulator, as well as a proinflammatory compound. A continuous production of the IFN‐γ along with other inflammatory cytokines is triggered in order to activate the immune system when the virus such as the HIV infects the immune system. Therefore, IFN‐γ is an important biomarker for monitoring infections.The hypothesis for this study is that a bioluminescence‐based immunoassay would provide for highly sensitive detection of IFN‐γ due to high signal‐to‐noise ratio obtained with bioluminescence signal. For this purpose, monoclonal anti‐IFN‐γ antibody was conjugated to a mutant of bioluminescent protein, Gaussia luciferase (GLuc) containing a tyrosine at the N‐terminus. The bioconjugation was achieved through orthogonal chemical conjugation at the N‐terminal tyrosine. It has been demonstrated that the Formylbenzene Diazonium Hexafluorophosphate (FBDP) reagent provides bioorthogonal aldehyde functionality and reacts specifically with the ortho position of the phenol group of the tyrosine residue. The conjugation was efficient and retained the functionalities of both the antibody and the luciferase.A sandwich‐type immunoassay was developed assay and validated for its accuracy and precision. A detection limit of 10−12 M was achieved for the detection of IFN‐γ. We also demonstrated the applicability of the assay for the detection of IFN‐γ in biological fluids such as serum samples.Support or Funding InformationNIGMS funding (R01GM047915)
Association Between Women with Endometriosis and Human Leukocyte Antigen‐C Genotyping
Authors: Chou Ya‐Ching , Tzeng Chii‐Ruey 
Published: November 2023

Study objectiveEndometriosis is a frequent benign gynecological disorder that affects 6%–10% of fertile women and causes severe pelvic pain, suffering, infertility, and hysterectomy. The etiology of endometriosis remains unclear and several evidence suggest cell‐mediated and humoral immunity are involved in its pathogenesis. HLA‐C antigen expression is low and only a minority of immunodominant cytotoxic CD8+ T cells response are restrict by HLA‐C. Moreover, HLA‐C has been regarded to act as a ligand for killer immunoglobulin receptors expressed in natural killer cells and inhibit NK cells. Previous studies have showed that HLA‐C affects viral infection and has been implicated in several human diseases. The aim of this study is to investigate the human leukocyte antigen‐C genotype in women with endometriosis.MethodA total of 52 endometriosis and 47 non‐endometriosis women were diagnosed by laparoscopy and typed for the HLA‐C antigen. The total DNA were extracted from blood and analyzed HLA‐C alleles through sequence based typing methodology.ResultsThe prevalence of HLA‐C*0304 in endometriosis group was 18.3% (19/104 alleles), compared with 6.4% in non‐endometriosis group (6/94 alleles). Thus, the prevalence of HLA‐C*0304 in endometriosis women was significantly higher than non‐endometriosis [OR 3.278, 95% CI=1.249 to 8.607, p= 0.0119 (χ2 analysis)].ConclusionThe result may be useful to implicate the role of HLA‐C antigens and NK cells in the pathogenesis of endometriosis.
Effect of Outer Hair Cell‐Specific STAT3 Deletion in the Noise‐induced Cellular Stress Response
Authors: Dziennis Suzan , Wilson Teresa L , Foster Sarah L , Nuttall Alfred L 
Published: November 2023

BackgroundSignal transducer and activator of transcription 3 (STAT3) is part of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. STAT3 is activated by phosphorylation and translocates to the nucleus in response to inflammatory cytokines to induce a transcriptional response. Non‐canonical actions of STAT3 are becoming elucidated such as those effected in the cytoplasm and mitochondria. Loud sound exposure induces several stress responses including increased reactive oxygen species (ROS) which contribute to noise‐induced cochlear tissue damage. We previously reported the general JAK2/STAT3 inhibitor JSI‐124 reduced levels of ROS after noise exposure suggesting STAT3 contributes to noise‐induced cellular damage.ObjectiveHere, we generated outer hair cell (OHC)‐specific STAT3 knock‐out mice to better understand the function of STAT3 in OHCs in response to cellular stress pathways induced after loud sound exposure.Methods and ResultsFloxed STAT3 mice were crossed with prestin‐CreERT2 mice for several generations to produce STAT3fl/fl × prestin‐CreERT2+/− (STAT3fl/fl) mice. Tamoxifen treatment resulted in STAT3 deletion specifically in OHCs (STAT3OHCΔ/Δ). The OHCs of 8 week old STAT3OHCΔ/Δ mice appeared morphologically normal and a full complement of OHCs was present. Ex vivo cochlea from STAT3fl/fl and STAT3OHCΔ/Δ mice were treated with IL‐6, antimycin A or corresponding vehicle followed by addition of the ROS detector CellROX. Nuclei were stained with Hoechst. IL‐6 or antimycin A treatment resulted in contrasting effects on nuclear morphology and ROS based on the loss of OHC‐specific STAT3, demonstrating the diverse contribution of STAT3 in the noise‐induced cellular stress response.ConclusionsSTAT3 either prevents or contributes to detrimental cellular stress pathways in OHCs. This may be due to multiple and diverse activities of STAT3 including both genomic actions as a transcription factor and non‐genomic actions such as interaction with the mitochondrial electron transport chain.Support or Funding InformationSupported by grants 5R01DC000105 (ALN) and P30DC005983.
CD95‐Ligand from Myeloid Cells Contributes to Abdominal Aortic Aneurysm Formation
Authors: Wiong Wanfen , Meisinger Trevor , Fitzgerald Matthew , Batra Rishi , Baxter B. Timothy 
Published: November 2023

Abdominal aortic aneurysm (AAA) is one of a number of diseases associated with a prominent inflammatory cell infiltration, matrix protein degradation, and smooth muscle cell apoptosis. This chronic infiltrate is predominantly composed of macrophages and T lymphocytes. Active T cells produce a variety of cytokines, including CD95‐ligand (CD95L). Elevated expression of CD95L in the aortic tissue was observed in patients with AAA, suggesting that CD95L may play a role in the pathogenic mechanism of AAA. The purpose of this study was to investigate the role of CD95L in AAA formation. By studying a murine model of aneurysm, we found that both mRNA and protein levels of CD95L were increased in aneurysm tissue compared with normal aortic tissue. We further tested the response of mice lacking expression of CD95L to aneurysm formation. These mice were resistant to aneurysm formation. Our results show that CD95L deficiency attenuates macrophage and T cell infiltration into the aortic tissue. To study the role of CD95L in the myelogeous cells in AAA formation, we created chimeric mice and showed that myelogeous cell‐derived CD95L plays a dominant role in disease progression. These studies demonstrated that inflammatory cell infiltration during AAA formation is dependent on myelogeous cell‐derived CD95L.Support or Funding InformationR01HL130623 (To W. X) and R01HL062400 (To B.T.B)
C‐Terminal 15kDa Fragment from β‐Actin Causes Apoptotic Cell Death
Authors: Ito Nanako , Tone Shigenobu , Tanaka Masato 
Published: November 2023

Truncated 15kDa‐β‐actin fragment was originally detected in apoptotic cancer cells treated with etoposide and its over‐expression of the fragment induced apoptotic morphological changes (Mashima, T. et al. Oncogene 18, 2423–2430, 1999). Our final goal is to reveal how the 15kDa‐β‐actin fragment induces the apoptotic morphological changes. To distinguish the transfected cells from non‐transfected ones, we have developed a new expression vector in which two coding sequences are able to be simultaneously expressed in a mammalian cell. This vector enabled to express a fluorescent protein with the 15kDa‐β‐actin in an identical cell. Using these co‐expression plasmids, we have investigated the effects of the 15kDa‐β‐actin on the apoptotic processes of HEK293, COS‐7 HeLa S3, and Jurkat cells.The over‐expression of 15kDa‐β‐actin fragment was seemed to cause the loss of adhesive properties of COS‐7 cells onto culture dish, since the GFP‐labeled cells were observed to detach from dishes by losing the adhesion ability. The numbers of detached cells from HEK293, COS‐7 and HeLa S3 transfected with the 15kDa‐β‐actin cDNA were determined by counting chamber. The numbers of detached cells were significantly increased by transfection of 15kDa‐β‐actin cDNA, but not by any mock transfections. To examine the relationship between the detachment and cell death, the detached cells were stained with Hoechst33342 and NucView488 caspase‐3 vital staining. The almost detached cells showed caspase‐3 activation and nuclear condensation in HEK293, COS‐7 and HeLa S3 cells. The cell death is not seemed to result from anoikis, since the transfection of the 15kDa‐β‐actin cDNA gave the similar effects on Jurkat cells. We also confirmed that the DNA fragmentations in HEK293, COS‐7, HeLa S3 and Jurkat cells by TUNEL assay.Cytoskeletal β‐actin has three targets of caspase‐3 motif but β‐actin has been so far considered to be a poor target of caspase‐3. We showed that the 15kDa‐β‐actin C‐terminal fragment actually activated caspase‐3. These results suggest that the cleaved molecule of cytoskeletal β‐actin is not an innocent bystander but an apoptogeneic inducer. Furthermore, we found that an actin binding protein, profilin suppressed the cell death by either 15kDa‐β‐actin or etoposide, VP‐16. The cleavage of cytoskeletal β‐actin would play a key role in the process of apoptotic cell death.
Paspalum scrobiculatum polyphenol rich extracts inhibit human cervical cancer cell proliferation and induce apoptosis in vitro
Authors: Ramasamy Sakthivel , Anupam K , Ratnavathi C.V , Karunagaran D , Chandra T.S 
Published: November 2023

Natural products and their derivatives have historically been a major source of new pharmaceuticals. Many dietary derivatives act as an effective anticancer agents such as curcumin, resveratrol and tea polyphenols which selectively inhibits the growth of malignant cells by inducing apoptosis. Cereals are valuable source of bioactive compounds which are found to be very effective in preventing and treating several diseases. Paspalum scrobiculatum is a type of small seeded grain (Kodo Millet) mainly consumed by India and also in Indonesia, Philippines, Thailand, Vietnam and in Africa. It has found its use in traditional medicines against inflammatory diseases, diabetes and several other diseases. In the present study, polyphenol‐rich extract (PEKM) of kodo millet was evaluated for its ability to inhibit the growth of human cervical cancer line (HeLa), at concentrations ranging from 100–500μg/ml. With increasing concentration of PEKM, a decrease in cell proliferation was observed in HeLa cell lines. The concentrations needed for 50% growth inhibition (IC50) were found to be 212μg/ml and 178μg/ml for Kodo Millet Market (KMM) and Kodo Millet Vamban (KMV), respectively. The growth inhibition by PEKM was accompanied by membrane blebbing, appearance of apoptotic bodies, loss of mitochondrial membrane potential, increased production of reactive oxygen species and DNA damage, typically seen during apoptosis. In addition, it activated caspase‐9, decreased the expression of Bcl‐2 and induced poly ADP‐ribosome polymerase (PARP) cleavage. The phytochemical profiling of PEKM by HPLC revealed the presence of phenolic acids like quercetin and cinnamic acid in it. These results suggest that PEKM possesses the ability to inhibit cell growth in a dose dependent manner and could stimulate apoptosis in cancer cells.Support or Funding InformationIndian Council of Agricultural Research (ICAR) and Indian Institute of Millets Research (IIMR), Govt. of India, New Delhi ‐110001