Biochemistry, Biophysics, and Structural Biology Commons^™

Why Should Early-Career Scientists Publish In Society Journals, Stephen K. Dolan, Lori D. Banks, Wenqi Yu

Molecular Biosciences Faculty Publications

In this editorial, written by early-career scientists, we advocate for the invaluable role of society journals in our scientific community. By choosing to support these journals as authors, peer reviewers, and as editors, we can reinforce our academic growth and benefit from their re-investment back into the scientific ecosystem. Considering the numerous clear merits of this system for future generations of microbiologists and more broadly, society, we argue that early-career researchers should publish our high-quality research in society journals to shape the future of science and scientific publishing landscape.

The Inaugural Mbio Junior Editorial Board—Lessons Learned And The Path Forward Toward Improving The Peer Review Process, Cynthia Ayefoumi Adinortey, Stephen K. Dolan, Sarah Doore, Rebeccah Lijek, Diana Priscila Pires, Wenqi Yu, Elizabeth B. Draganova, Lennart Schada Von Borzyskowski

Molecular Biosciences Faculty Publications

The inaugural Junior Editorial Board (JEB) of mBio consisted of 64 early-career researchers active from 2022 to 2023. The goal of the JEB was to train early-career researchers in the art of peer review under the guidance of experienced editors. JEB members gained hands-on experience in peer review by participating in modules detailing the publishing process through the lenses of the journal, editor, and reviewer. Ultimately, JEB members applied this new knowledge by reviewing mBio manuscripts. Here, we summarize the background, the mission, and the achievements of the first mBio JEB. We also include possible trajectories for the future editions …

Dcaf14 Regulates Cdt2 To Promote Set8-Dependent Replication Fork Protection, Neysha Tirado-Class, Caitlin Hathaway, Anthony Nelligan, Huzefa Dungrawala

Molecular Biosciences Faculty Publications

DDB1- and CUL4-associated factors (DCAFs) CDT2 and DCAF14 are substrate receptors for Cullin4–RING E3 ubiquitin ligase (CRL4) complexes. CDT2 is responsible for PCNA-coupled proteolysis of substrates CDT1, p21, and SET8 during S-phase of cell cycle. DCAF14 functions at stalled replication forks to promote genome stability, but the mechanism is unknown. We find that DCAF14 mediates replication fork protection by regulating CRL4CDT2 activity. Absence of DCAF14 causes increased proteasomal degradation of CDT2 substrates. When forks are challenged with replication stress, increased CDT2 function causes stalled fork collapse and impairs fork recovery in DCAF14-deficient conditions. We further show that stalled fork protection …

Otud5 Limits Replication Fork Instability By Organizing Chromatin Remodelers, Angelo De Vivo, Hongseon Song, Yujin Lee, Neysha Tirado-Class, Anthony Sanchez, Sandy D. Westerheide, Huzefa Dungrawala, Younghoon Kee

Molecular Biosciences Faculty Publications

Proper regulation of replication fork progression is important for genomic maintenance. Subverting the transcription-induced conflicts is crucial in preserving the integrity of replication forks. Various chromatin remodelers, such as histone chaperone and histone deacetylases are known to modulate replication stress, but how these factors are organized or collaborate are not well understood. Here we found a new role of the OTUD5 deubiquitinase in limiting replication stress. We found that OTUD5 is recruited to replication forks, and its depletion causes replication fork stress. Through its C-terminal disordered tail, OTUD5 assembles a complex containing FACT, HDAC1 and HDAC2 at replication forks. A …

The Identification Of Two M20b Family Peptidases Required For Full Virulence In Staphylococcus Aureus, Nathanial James Torres, Devon Rizzo, Maria A. Reinberg, Mary-Elizabeth Jobson, Brendan C. Totzke, Jessica K. Jackson, Wenqi Yu, Lindsey Neil Shaw

Molecular Biosciences Faculty Publications

We have previously demonstrated that deletion of an intracellular leucine aminopeptidase results in attenuated virulence of S. aureus. Herein we explore the role of 10 other aminopeptidases in S. aureus pathogenesis. Using a human blood survival assay we identified mutations in two enzymes from the M20B family (PepT1 and PepT2) as having markedly decreased survival compared to the parent. We further reveal that pepT1, pepT2 and pepT1/2 mutant strains are impaired in their ability to resist phagocytosis by, and engender survival within, human macrophages. Using a co-infection model of murine sepsis, we demonstrate impairment of dissemination and survival …

Phip Variants Associated With Chung–Jansen Syndrome Disrupt Replication Fork Stability And Genome Integrity, Neysha Tirado-Class, Caitlin Hathaway, Wendy K. Chung, Huzefa Dungrawala

Molecular Biosciences Faculty Publications

Chung–Jansen syndrome (CJS) is a rare, autosomal dominant disorder characterized by developmental delay, intellectual disability/cognitive impairment, behavioral challenges, obesity, and dysmorphic features. CJS is associated with heterozygous variants in PHIP (Pleckstrin-Homology Interacting Protein), a gene that encodes one of several substrate receptors for Cullin4-RING (CRL4) E3 ubiquitin ligase complex. Full-length PHIP, also called DCAF14, was recently identified to function as a replication stress response protein. Herein, we report the identification of two PHIP missense variants identified by exome sequencing in unrelated individuals with CJS. The variants p.D488V and p.E963G occur in different functional elements of DCAF14-WD40 repeat domain and pleckstrin …

Sequence Properties Of An Intramolecular Interaction That Inhibits P53 Dna Binding, Emily Gregory, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

An intramolecular interaction between the p53 transactivation and DNA binding domains inhibits DNA binding. To study this autoinhibition, we used a fragment of p53, referred to as ND WT, containing the N-terminal transactivation domains (TAD1 and TAD2), a proline rich region (PRR), and the DNA binding domain (DBD). We mutated acidic, nonpolar, and aromatic amino acids in TAD2 to disrupt the interaction with DBD and measured the effects on DNA binding affinity at different ionic strengths using fluorescence anisotropy. We observed a large increase in DNA binding affinity for the mutants consistent with reduced autoinhibition. The ΔΔG between DBD and …

Unraveling The Role Of Novel G5 Peptidase Family Proteins In Virulence And Cell Envelope Biogenesis Of Staphylococcus Aureus, Stephanie M. Marroquin

USF Tampa Graduate Theses and Dissertations

Virulence factors and the bacterial cell envelope are two important components in S. aureus pathogenesis and survival. More importantly, understanding the regulation of these cellular processes is crucial to further understanding and combating this successful pathogen. To date, numerous factors have been identified as playing a role in the regulation of Agr activity in S. aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigate the product of SAUSA300_1984 (termed MroQ), a transmembrane G5 peptidase family protein, as a novel effector of this system. Using a USA300 mroQ mutant we observed a drastic reduction in proteolysis, hemolysis, and …

Investigation Of An Alternative Protocol For The Production Of Sars-Cov-2 Antigenic Proteins, Nichole Ninaltowski

USF Tampa Graduate Theses and Dissertations

With the COVID-19 pandemic showing no signs of slowing down, large-scale antigenic protein production is still needed for surveillance using serologic assays. From screening to vaccines to biotherapeutics, being able to produce the proteins for these assays is essential; however, the current gold standard method for producing SARS-CoV-2 spike proteins is prohibitively expensive for most research groups.

Alternative methods of transfecting mammalian cells to produce recombinant proteins that are relatively inexpensive have been used for years. Unlike the expensive, commercially available lipid-based methods, other established methods such as polyethyleneimine (PEI), are considerably easier, and cheaper to meet the needs of …

Transcriptomic And Functional Investigation Of Bacterial Biofilm Formation, Brooke R. Nemec

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus and Acinetobacter baumannii are two highly successful human pathogens, which have adopted very different, but effective survival strategies. The success of S. aureus is attributed to the tight regulation of an arsenal of virulence factors. Conversely, A. baumannii lacks what would be considered traditional virulence factors and, instead, has developed a high tolerance for environmental stress, which allows it to persist in unforgiving environments, including nosocomial settings and the human body. One common characteristic of these two organisms is their proclivity for biofilm formation. Herein, we discuss the diverse mechanisms governing biofilm formation for A. baumannii and S. …

Dcaf14 Promotes Stalled Fork Stability To Maintain Genome Integrity, Arik Townsend, Gabriella Lora, Justin Engel, Neysha Tirado-Class, Huzefa Dungrawala

Molecular Biosciences Faculty Publications

No abstract provided.

Tracking The Subcellular Localization Of Surface Proteins In Staphylococcus Aureus By Immunofluorescence Microscopy, Salvatore J. Scaffidi, Mac A. Shebes, Wenqi Yu

Molecular Biosciences Faculty Publications

Surface proteins of Staphylococcus aureus and other Gram-positive bacteria play essential roles in bacterial colonization and host-microbe interactions. Surface protein precursors containing a YSIRK/GXXS signal peptide are translocated across the septal membrane at mid-cell, anchored to the cell wall peptidoglycan at the cross-wall compartment, and presented on the new hemispheres of the daughter cells following cell division. After several generations of cell division, these surface proteins will eventually cover the entire cell surface. To understand how these proteins travel from the bacterial cytoplasm to the cell surface, we describe a series of immunofluorescence microscopy protocols designed to detect the stepwise …

Posttranslational Modification And Protein Disorder Regulate Protein-Protein Interactions And Dna Binding Specificity Of P53, Robin Levy

USF Tampa Graduate Theses and Dissertations

p53 is an intrinsically disordered transcription factor that suppresses tumor development by arresting the cell cycle and promoting DNA repair. p53 deletions or mutations can lead to cancer due to the inability of cells to respond to stress. The protein levels and post-translational modification state of p53 changes in response to cellular stress like DNA damage. Previous studies have shown that p53 can undergo coupled folding and binding with the E3 ubiquitin ligase, Mdm2, and the histone deacetylase, p300. In normal cells, p53 is kept at a low level by Mdm2, which marks it with ubiquitin, targeting p53 for proteasome …

Surface Runoff Alters Cave Microbial Community Structure And Function, Madison Davis, Maria A. Messina, Giuseppe Nicolosi, Salvatore Petralia, Melvin D. Baker, Christiana K. S. Mayne, Chelsea M. Dinon, Christina J. Moss, Bogdan P. Onac, James R. Garey

Molecular Biosciences Faculty Publications

Caves formed by sulfuric acid dissolution have been identified worldwide. These caves can host diverse microbial communities that are responsible for speleogenesis and speleothem formation. It is not well understood how microbial communities change in response to surface water entering caves. Illumina 16S rRNA sequencing and bioinformatic tools were used to determine the impact of surface water on the microbial community diversity and function within a spring pool found deep in the Monte Conca Cave system in Sicily, Italy. Sulfur oxidizers comprised more than 90% of the microbial community during the dry season and were replaced by potential anthropogenic contaminants …

To Mid-Cell And Beyond: Characterizing The Roles Of Gpsb And Ypsa In Cell Division Regulation In Gram-Positive Bacteria, Robert S. Brzozowski

USF Tampa Graduate Theses and Dissertations

The bacterial cell division protein FtsZ is a tubulin homolog that forms a ring-like structure at the site of cell division in most bacterial species. There it acts as a scaffold, aiding in the recruitment of other divisome proteins to the site of cell division. Furthermore, studies focusing on the role of FtsZ treadmilling and septal peptidoglycan synthesis implicates that FtsZ plays a direct role in the ultimate closure of the division septum. Thus, many studies in the field of bacterial cell division have focused on FtsZ in terms of its spatial and temporal regulation as well as its ability …

Draft Genome Sequence Of Verrucosispora Sp. Strain Cwr15, Isolated From A Gulf Of Mexico Sponge, Sarah J. Kennedy, Eleonora Cella, Mohammad Jubair, Taj Azarian, Bill J. Baker, Lindsey N. Shaw

Molecular Biosciences Faculty Publications

Here, we present the draft genome sequence of Verrucosispora sp. strain CWR15, a bacterial symbiont of a Gulf of Mexico sponge. The genome consists of 35 contigs encoding 5,840 genes. The genome is the basis for future study and presents an underexplored taxonomy and biosynthetic potential.

Mir146a-Mediated Targeting Of Fancm During Inflammation Compromises Genome Integrity, Devakumar Sundaravinayagam, Hye Rim Kim, Tingting Wu, Hyun Hee Kim, Semo Jun, Jeong-Heon Cha, Younghoon Kee, Ho Jin You, Jung-Hee Lee

Molecular Biosciences Faculty Publications

Inflammation is a potent inducer of tumorigenesis. Increased DNA damage or loss of genome integrity is thought to be one of the mechanisms linking inflammation and cancer development. It has been suggested that NF-κB-induced microRNA-146 (miR146a) may be a mediator of the inflammatory response. Based on our initial observation that miR146a overexpression strongly increases DNA damage, we investigated its potential role as a modulator of DNA repair. Here, we demonstrate that FANCM, a component in the Fanconi Anemia pathway, is a novel target of miR146a. miR146a suppressed FANCM expression by directly binding to the 3’ untranslated region of the gene. …

Cornus Officinalis Promotes Igfbp2 And Autophagy In Human 1.1b4 Pancreatic Cell Line As Revealed By Employing A Global Proteomic Approach Via Mass Spectrometry, Arielle Elizabeth Tawfik, Jennifer Guergues, Stanley M. Stevens Jr, Brant Roger Burkhardt

Molecular Biosciences Faculty Publications

Type 1 diabetes (T1D) results in the loss of pancreatic beta cells and subsequent loss of insulin production. Exogenous insulin is the only effective treatment, but there is still no cure or interventional therapy available to inhibit progression of T1D. Successful T1D interventional therapy must protect pancreatic beta cells from autoimmunity while enhancing beta cell survival and function. Our data suggests Cornus officinalis (CO) may be a candidate for interventional therapy to protect pancreatic beta cells from autoimmune attack and increase their function. CO has been used in traditional Chinese medicine (TCM) for over 2,000 years and has shown characteristics …

The Role Of Secreted Proteases In Regulating Disease Progression In Staphylococcus Aureus, Brittney D. Gimza

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus is a highly successful pathogen capable of producing a wealth of virulence factors in the human host. Of note, ten extracellular proteases are produced alongside these virulence factors and play a multifaceted role during infection. They not only cleave host proteins to promote bacterial invasion, immune evasion and survival, but also control disease progression by modulating the stability of self-derived pathogenic determinants. The importance of the secreted proteases modulating virulence factor stability is evidenced by our groups previous finding that a protease-null strain has a substantially increased infectious capacity in a murine model of sepsis; resulting from the …

Targeting Bacterial Resistance And Cancer Metastasis: A Structure Based Approach, Kyle Galen Kroeck

USF Tampa Graduate Theses and Dissertations

Current research in pharmaceutical development commonly utilizes a profusion of methods in molecular modeling in order to probe intricate biological problems. Many original and promising compounds have been identified and developed by integrating experimental and computational methods. Structural biology utilizes many different research techniques including x-ray crystallography, NMR, and electron microscopy in order to develop molecular models of macromolecules that are of biological interest. Such techniques can be used in conjunction with molecular docking, which utilizes those molecular models in order to target macromolecules of therapeutic interest by computationally analyzing the conformations adopted by ligands upon interaction with a desired …

A Small Rna And Dna Binding Protein Contribute To Biofilm Development In Bartonella Henselae, Udoka Okaro

USF Tampa Graduate Theses and Dissertations

A biofilm, which is associated with 80% of chronic infections in humans, is formed when bacteria aggregate, attach to a substrate and secrete a matrix protecting the bacteria from host cell defenses and antibiotics. Bartonella henselae (B. henselae) is the causative agent of cat scratch disease, persistent bacteremia, and one of the most frequently reported causes of blood-culture negative endocarditis (BCNE) in patients. The ability of B. henselae to adhere to the heart valve, form a biofilm and vegetation to cause endocarditis increases the morbidity and mortality rate in infected patients. The presence of a trimeric autotransporter adhesin (TAA) called …

Functional Inhibition Of Acid Sphingomyelinase Disrupts Infection By Intracellular Bacterial Pathogens, Chelsea L. Cockburn, Ryan S. Green, Sheela R. Damle, Rebecca K. Martin, Naomi N. Ghahrai, Punsiri M. Colonne, Marissa S. Fullerton, Daniel H. Conrad, Charles E. Chalfant, Daniel E. Voth, Elizabeth A. Rucks, Stacey D. Gilk, Jason A. Carlyon

Molecular Biosciences Faculty Publications

Intracellular bacteria that live in host cell–derived vacuoles are significant causes of human disease. Parasitism of low-density lipoprotein (LDL) cholesterol is essential for many vacuole-adapted bacteria. Acid sphingomyelinase (ASM) influences LDL cholesterol egress from the lysosome. Using functional inhibitors of ASM (FIASMAs), we show that ASM activity is key for infection cycles of vacuole-adapted bacteria that target cholesterol trafficking—Anaplasma phagocytophilum, Coxiella burnetii, Chlamydia trachomatis, and Chlamydia pneumoniae. Vacuole maturation, replication, and infectious progeny generation by A. phagocytophilum, which exclusively hijacks LDL cholesterol, are halted and C. burnetii, for which lysosomal cholesterol accumulation is bactericidal, …

The Otud5–Ubr5 Complex Regulates Fact-Mediated Transcription At Damaged Chromatin, Angelo De Vivo, University Of South Florida, Jose Yegres, Jeonghyeon Kim, Sylvia Emly, Younghoon Kee

Molecular Biosciences Faculty Publications

Timely stalling and resumption of RNA polymerases at damaged chromatin are actively regulated processes. Prior work showed an importance of FACT histone chaperone in such process. Here we provide a new role of OTUD5 deubiquitinase in the FACT-dependent process. Through a DUB RNAi screen, we found OTUD5 as a specific stabilizer of the UBR5 E3 ligase. OTUD5 localizes to DNA double strand breaks (DSBs), interacts with UBR5 and represses the RNA Pol II elongation and RNA synthesis. OTUD5 co-localizes and interacts with the FACT component SPT16 and antagonizes the histone H2A deposition at DSB lesions. OTUD5 interacts with UBR5 and …

Structural Basis Of Phosphatidylcholine Recognition By The C2–Domain Of Cytosolic Phospholipase A,2Α, Yoshinori Hirano, Yong-Guang Gao, Daniel J. Stephenson, Ngoc T. Vu, Lucy Malinina, Dhirendra K. Simanshu, Charles E. Chalfant, Dinshaw J. Patel, Rhoderick E. Brown

Molecular Biosciences Faculty Publications

Ca²⁺-stimulated translocation of cytosolic phospholipase A2α (cPLA2α) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLA2α preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLA2α C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and Ca2+ ions. Two Ca2+ are complexed at previously reported locations in the lipid-free C2-domain. One of these Ca2+ions, along with a third Ca2+, bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid …

Cornus Officinalis Significantly Improves Oxidative Capacity And Promotes The Calcium-Dependent Transcription Factor, Nfatc2, In Human 1.1b4 Pancreatic Cell Line, Arielle Sharp, Alexis Coiner, Brant Burkhardt

Molecular Biosciences Faculty Publications

Type 1 diabetes (T1D) is an autoimmune disease resulting in the destruction of pancreatic β cells (β-cells) and subsequent loss of insulin production. The only treatment for T1D is using exogenous insulin coupled with continual glucose monitoring following significant autoimmune destruction of β-cells. Novel interventional therapies are needed that can preserve and protect existing pancreatic β cells in individuals with early identified T1D autoimmunity. Our initial in-vitro evidence indicates Cornus officinalis (CO) may be able to serve in this function. What sets ethnopharmacology apart from conventional medicine is the simultaneous targeting of multiple mechanisms using a single herb due to …

Functional Analysis Of The Replication Fork Proteome Identifies Bet Proteins As Pcna Regulators, Sarah R. Wessel, Kareem N. Mohni, Jessica W. Luzwick, Huzefa Dungrawala, David Cortez

Molecular Biosciences Faculty Publications

Identifying proteins that function at replication forks is essential to understanding DNA replication, chromatin assembly, and replication-coupled DNA repair mechanisms. Combining quantitative mass spectrometry in multiple cell types with stringent statistical cutoffs, we generated a high-confidence catalog of 593 proteins that are enriched at replication forks and nascent chromatin. Loss-of-function genetic analyses indicate that 85% yield phenotypes that are consistent with activities in DNA and chromatin replication or already have described functions in these processes. We illustrate the value of this resource by identifying activities of the BET family proteins BRD2, BRD3, and BRD4 in controlling DNA replication. These proteins …

Conserved Glycines Control Disorder And Function In The Cold-Regulated Protein, Cor15a, Oluwakemi T. Sowemimo, Patrick Knox-Brown, Wade M. Borcherds, Tobias Rindfleisch, Anja Thalhammer, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly α-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil–helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A …

P53 Phosphomimetics Preserve Transient Secondary Structure But Reduce Binding To Mdm2 And Mdmx, Robin Levy, Emily Gregory, Wade Borcherds, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient …

Uncoupling The Folding And Binding Of An Intrinsically Disordered Protein, Anusha Poosapati, Emily Gregory, Wade M. Borcherds, Lucia B. Chemes, Gary Daughdrill

Molecular Biosciences Faculty Publications

The relationship between helical stability and binding affinity was examined for the intrinsically disordered transactivation domain of the myeloblastosis oncoprotein, c-Myb, and its ordered binding partner, KIX. A series of c-Myb mutants was designed to either increase or decrease helical stability without changing the binding interface with KIX. This included a complimentary series of A, G, P, and V mutants at three non-interacting sites. We were able to use the glycine mutants as a reference state and show a strong correlation between binding affinity and helical stability. The intrinsic helicity of c-Myb is 21%, and helicity values of the …

Anemarrhena Asphodeloides Bunge And Its Constituent Timosaponin‐Aiii Induce Cell Cycle Arrest And Apoptosis In Pancreatic Cancer Cells, Catherine B. Marelia, Arielle Sharp, Tiffany A. Shemwell, Y. C. Zhang, Brant R. Burkhardt

Molecular Biosciences Faculty Publications

Pancreatic cancer is one of the most recalcitrant and lethal of all cancers. We examined the effects of Anemarrhena asphodeloides (AA) and timosaponin‐AIII (TAIII), a steroidal saponin present in AA, on pancreatic cancer cell proliferation and aimed to elucidate their potential apoptotic mechanisms of action. Viability assays and cell cycle analysis revealed that both AA and TAIII significantly inhibited pancreatic cancer cell proliferation and cell cycle progression compared to treatment with gemcitabine, the standard chemotherapeutic agent for advanced pancreatic cancer. We identified a dose‐dependent increase in caspase‐dependent apoptosis and activation of pro‐apoptotic PI3K/Akt pathway proteins, with a subsequent downregulation of …