bluuebunny/crossref_metadata_2025 huggingface dataset:
Did speech slow down the human face? Human facial muscles have a high proportion of slow myosin fibers compared to other primates (918.19)
Authors: Burrows Anne , Durham Emily , Smith Timothy , Parr Lisa
Published: June 2021

Much is known about the comparative morphology of the human face relative to other primates but our understanding of comparative muscle physiology is poor. To further our insight, the present study used myofibrillar adenosine triphosphatase (mATPase) histochemical staining to investigate distribution of type I (slow) and type II (fast) myosin fibers in the zygomaticus major (ZM) and orbicularis oris muscles (OOM) of humans (n=3), chimpanzees (n=1), and rhesus macaques (n=3). Average percentages were created for fiber types from 5‐10 sections per specimen and average fiber cross‐sectional area and diameter were calculated for each muscle in each species. Non‐parametric tests were used to statistically compare averages since data were not normally distributed. Results of statistical testing showed that chimpanzees had a significantly higher percentage of fast fibers in the ZM relative to both macaques and humans. However, humans had a significantly higher percentage of slow fibers in both the ZM and OOM relative to chimpanzees and macaques, in excess of three times more than both macaques and chimpanzees. In the ZM, humans had the lowest cross‐sectional fiber area and diameter in both fast and slow fibers while chimpanzees had the greatest. Combined, these results may reflect adaptive divergence between chimpanzee and human facial muscles that may be influenced by the evolution of human speech where many facial muscles attaching to the lips act as speech articulators.

Grant Funding Source: Supported by National Institutes of Health (contract grant number MH082282)
Mature dental enamel: a diverse repository of past and present information (918.2)
Authors: Gerlach Raquel , Line Sergio
Published: June 2021

Dental enamel is the hardest, densest, and most mineralized tissue in vertebrates. It achieved its very high crystallinity with evolution of enamel specific proteins for more than 500 million years. During enamel formation (still inside the jaw bones), the proteins are degraded by the enamel‐resident proteinases and eventually “mature” enamel contains less than 0.1% of proteins 96% of minerals. This pre‐fossilized rocky tissue is essential for proper nutrition and survival, even today.

The past century witnessed the characterization of 3 enamel structural proteins and 2 enamel proteinases, achieved by cumbersome work with samples of thousands of developing teeth to obtain micrograms of protein for mass spectrometry analyses and Edman degradation. Newer equipments enable today that hundreds of different peptides from all 3 enamel proteins can be obtained from enamel acid extracts, obtained with a much less invasive procedure compared to the ones used before. Mammal´s mature enamel harbors Y‐chromosome amelogenin specific peptides that allow the determination of sex, which we show at the EB2014 for the first time in humans samples. Enamel is also a good biomarker for toxic metals like lead and cadmium. Superficial enamel etch (for 2 minutes) was sufficient to obtain enamel specific peptides from shed primary teeth. Thus, dental enamel is an immense and diverse repository with relevant information for paleontology, archaeology, forensics, toxicology, medicine and developmental biology.

Grant Funding Source: Supported by FAPESP (Brazil)
The devil is in the details: identification of postcranial elements of Canis dirus and Canis lupus from Rancho La Brea using discriminant function and morphometric analyses (919.7)
Authors: Koper Lindsey
Published: June 2021

The preservation of fossils in the tar pits at Rancho La Brea makes identification of postcranial material difficult, especially separating Canis lupus form Canis dirus. Generally, distinguishing C. dirus from C. lupus is achieved by study of the dentition and cranial features; otherwise most postcranial bones are identified by size. Because the tar pits at Rancho La Brea disarticulate the remains during the fossilization process, one bony element can rarely be associated with any other. Therefore, another means of identification is needed for postcranial canid materials. I compiled a series of measurements of scapulae, humeri, and ulnae of C. dirus to compare with similar measurements of modern C. lupus. The wolf specimens in this study represent wild populations of North American subspecies that often included sex and age information. By comparing linear measurements of both taxa, I was able to determine statistically relevant differences in the forelimb bone using discriminant function analysis and morphometrics. Being able to provide composite size ranges for individual bony elements, these disarticulated canid specimens of C. dirus and C. lupus can be more accurately identified only from postcranial material. These comparisons can serve as a reference tool for accurately comparing other fossil canid specimens as well as modern species when cranial material is unavailable.
Jagged1 is essential and sufficient for osteoblast development during maxillary ossification (921.2)
Authors: Hill Cynthia , Yuasa Masato , Schoenecker Jon , Goudy Steven
Published: June 2021

Maxillary hypoplasia occurs due to insufficient maxillary intramembranous ossification, leading to poor dental occlusion, respiratory obstruction and cosmetic deformities. Conditional deletion of Jagged1 (Jag1) in cranial neural crest (CNC) cells using Wnt1‐cre (Jag1CKO) led to maxillary hypoplasia characterized by intrinsic differences in bone morphology and density using μCT evaluation. Jag1CKO maxillas had altered collagen deposition, delayed ossification, and reduced expression of early and late determinants of osteoblast development during maxillary ossification. In vitro bone cultures on Jag1CKO mouse embryonic maxillary mesenchymal (MEMM) cells demonstrated decreased mineralization that was also associated with diminished induction of osteoblast determinants. BMP receptor expression was reduced in the Jag1CKO MEMM cells suggesting that these cells were unable to respond to BMP‐induced differentiation. Jag1‐Fc rescued in vitro mineralization and osteoblast gene expression changes. These data suggest that Jag1 signaling in CNC‐derived MEMM cells is required for osteoblast development and differentiation during maxillary ossification.

Grant Funding Source: 5K08DE17953‐5
Ephrin reverse signaling induces mouse palatal fusion without TGFβ3 signaling (921.4)
Authors: Serrano Maria , Hills Patricia , Pourmand Iksa , Varghese Allen , Svoboda Kathy , Benson M
Published: June 2021

Orofacial clefts are the most prevalent craniofacial birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium which adheres to form the midline epithelial seam (MES). MES cells then proceed through epithelial to mesenchymal transition (EMT) forming a fused palate. TGF‐β3 is essential for the disintegration of the palatal MES. Eph receptor tyrosine kinases and their ephrin ligands are responsible for many developmental processes. Binding of ephrins causes receptor activation in Eph‐bearing cells (forward signaling), and intracellular signaling inside ephrin‐bearing cells (reverse signaling). We asked if activation of ephrin reverse signaling is sufficient to fuse mouse palates if we blocked TGFβ receptors. We cultured E13.5 mouse palatal shelves in different conditions: Control IgG Fc; anti‐TGF‐β3 with or without clustered recombinant EphB2/Fc protein; TGF‐β RI kinase inhibitor (SB 431542) with or without EphB2/Fc. Palatal fusion was quantified and analyzed with a mean fusion score (MFS) in anterior, medial and posterior regions. Palates treated with anti‐TGF‐β3 or (SB 431542) did not fuse while those treated with SB 431542 and EphB2/Fc and those treated with anti‐TGF‐β3 and EphB2/Fc were partially fused. Palates from the control groups fused. These results demonstrate that ephrin reverse signaling induces fusion of the mammalian palate without TGF‐β3 signaling.

Grant Funding Source: Biomedical Sciences Department, baylor College of Dentistry, Baylor Oral Health Foundation
A noncanonical nuclear role for fibroblast growth factor signaling in skeletal development (921.6)
Authors: Salva Joanna , Neben Cynthia , Merrill Amy
Published: June 2021

Fibroblast Growth Factor (FGF) signaling plays crucial roles in osteochondral progenitor cell proliferation and differentiation as seen by the multiple skeletal dysplasias resulting from abnormal FGF signaling. We identified that the skeletal dysplasia Bent Bone Dysplasia Syndrome (BBDS) results from unique FGF Receptor 2 (FGFR2) mutations that reduce canonical membrane signaling, while also enhancing a noncanonical nuclear role for the receptor in ribosome biogenesis. To determine how canonical and noncanonical FGFR2 signaling individually contribute to skeletal development and the pathogenesis of BBDS, we blocked membrane FGFR2 signaling with a chemical inhibitor and enhanced nuclear FGFR2 signaling by employing the BBDS mutations. In a bone organ culture assay, avian mandibles grown with inhibitors of membrane FGF signaling displayed reduced bone formation and exhibited uncharacteristic hypertrophy of Meckel’s cartilage. Expression of the BBDS FGFR2 mutations in developing chicken embryos caused increased proliferation and abnormal patterning in bone and cartilage. These results suggest that membrane FGF signaling is important in osteochondral progenitor differentiation and maturation while nuclear FGF signaling regulates proliferation. Combined our research shows that canonical and noncanonical FGF signaling have distinct roles during skeletal development.

Grant Funding Source: Supported by NIH#5T32HD060549‐02 to JES, MOD #5‐FY12‐166 to AEM, NIH #5P30DE020750‐02
A new class of osteoarthritis therapeutic, extracellular matrix protection factor, alters cytokine production in chondrocytes (922.12)
Authors: Docherty Courtney , Belogorodsky Dimitry , Cho Ellen , Chmielewski Sarah , Lopez Samuel , Holmes Tiffany , D'Angelo Marina
Published: June 2021

Articular cartilage damage leads to osteoarthritis (OA), a disease characterized by altered cartilage homeostasis. Several cellular elements including growth factors and proteases are involved in this pathology. Our lab has developed a new class of therapeutic, extracellular matrix protection factors (ECPFs), that protect cartilage from the devastation associated with OA. ECPF‐1 targets the interaction between matrix metalloprotease 13 (MMP‐13) and transforming growth factor β (TGF‐β), but the cellular mechanism of ECPF‐1 chondroprotection is unknown. Inflammatory cytokines play a role in the pathology associated with OA, and are regulated, in some part, by TGF‐β biology. To test the downstream effects of blocking TGF‐β activation on cytokine production, cultured chondrocytes from embryonic avian sterna were treated for 24 hours with ECPF‐1 at 250nM, 2.5μM and 5.0μM concentrations and the production of the inflammatory cytokines, IL‐1β and TNF‐α, were monitored. Activated TGF‐β was reduced and production of IL‐1β and TNF‐α were decreased in response to ECPF‐1 treatment including an unexpected increase in these cytokines following 5.0μM addition of ECPF‐1. This biphasic effect of ECPF‐1 indicates a duality to the protective therapeutic nature and overall mechanism of action of ECPF‐1 in the protection of articular cartilage.
Mechanistic insights into PriA mediated DNA replication restart (924.2)
Authors: Thurmes Tiffany , Bhattacharyya Basudeb , Zhou Ruobo , Leroux Maxime , Sandler Steven , Ha Taekjip , Keck James
Published: June 2021

DNA replication restart is an essential genome maintenance process by which replication complexes (replisomes) are reloaded onto abandoned DNA replication forks. In Escherichia coli and related bacteria this process is orchestrated by the PriA DNA helicase, which binds to replication forks in a structure‐specific manner. PriA also binds to single‐stranded DNA‐binding protein (SSB), a protein that coats the lagging‐strand template at DNA replication forks. Complex formation with SSB stimulates PriA DNA unwinding DNA and, as we show here, this interaction is required for PriA remodeling of SSB‐DNA nucleoprotein complexes. Proteins that interact with SSB typically do so by binding directly to the evolutionarily conserved amphipathic C‐terminal tail of SSB (SSB‐CT). Although previous studies indicated that PriA also binds to SSB via the SSB‐CT, a lack of structural information on PriA has left the SSB binding site on PriA unknown. We determined the 4 Å resolution structure of PriA bound to the SSB‐CT. The electrostatic characteristics of the PriA SSB‐Ct binding site resemble those of other known SSB‐interacting proteins. Single‐molecule FRET studies show that a PriA variant that has lost the ability to bind the SSB‐CT fails to modulate SSB‐DNA complexes. Our studies point to the importance of the PriA/SSB interaction in allowing PriA to reload replisomes at damaged replication forks where SSB is bound to the lagging‐strand template.
Biophysical analysis of the human POT1‐TPP1 complex reveals functional protein‐protein and protein‐DNA interactions involved in telomere maintenance (926.1)
Authors: Taylor Derek , Rajavel Malligarjunan , Baus Diane , Orban Tivadar , Palczewski Krzysztof
Published: June 2021

Telomeres are nucleoprotein complexes that reside at the ends of linear chromosomes. Six specialized proteins interact specifically with telomere DNA and one another to form a protective moiety called shelterin. Telomere repeat‐binding factors (TRF) 1 and 2 bind to the double‐stranded telomere DNA sequence and the Protection of telomeres 1 (POT1) protein binds to the single‐stranded DNA overhang. RAP1, TIN2, and TPP1 interact with TRF1, TRF2, and POT1 to form shelterin. TPP1, specifically, interacts with POT1 to increase its affinity and selectivity for telomere DNA. In addition, TPP1 recruits telomerase to the telomere, where it can synthesize telomere DNA. X‐ray crystallography has revealed the atomic structures of individual domains of POT1 and TPP1, but the molecular interactions between the two proteins with and without DNA remains obscure. Using amide hydrogen‐deuterium exchange and biochemical analysis, we have determined the regions of TPP1 that are responsible for POT1‐interactions and those that are altered upon DNA binding. Combining these experimental data with homology modeling allowed us to construct a model of the POT1‐TPP1‐DNA complex and interpret its biological function in DNA recognition and in regulating telomerase activity.

Grant Funding Source: Supported by American Heart Association 11SDB5580010 and American Cancer Society RSG‐13‐211‐01‐DM
Biological activity of a set of new copper(II) complexes: toward understanding structure‐reactivity correlations (928.2)
Authors: Aitken Chelsea , Tanski Joseph , Fox Kristin , Tyler Laurie
Published: June 2021

Cisplatin, carboplatin and oxaliplatin are all platinum‐containing complexes that have been approved for use as chemotherapy drugs. The success of these complexes as well as their downfalls have sparked interest into alternative transition metal based compounds as potential chemotherapeutic agents. The use of platinum based complexes in cancer treatment has faced complications including cytotoxicity and drug resistance. The use of copper(II) as an alternative metal center is of specific interest because of copper’s bioavailability and elevated concentrations within tumor cells. We have synthesized and characterized several copper complexes that utilize a nitrogen based ligand system including some that contain a thiazole moiety. The biological activity and potential cytotoxicity of these complexes was measured on the basis of nuclease activity, DNA binding, and interaction with BSA. These characteristics were determined by quantitative analysis of studies using ethidium bromide, gel electrophoresis, and UV‐Vis spectroscopy. We present comparisons between the activity of these newly synthesized compounds and those previously studied to aid in developing a correlation between ligand structure and complex activity.

Grant Funding Source: Supported by Union College
Rethinking freshman chemistry (93.2)
Authors: Drennan Catherine , Olivier‐Mason Anique , Taylor Elizabeth , Mitchell Rudolph
Published: June 2021

Too many students see freshman chemistry as just a hoop to jump through_a field disconnected from the exciting research today in medicine, energy, and the environment. Studies show that when students fail to see either the relevance of their course content or scientists who look like them, they are much less likely to persist in science‐related majors. At MIT, we are working to make small changes to 1) motivate all students, particularly female and underrepresented minority students, to learn chemistry and tackle important scientific problems in their future careers, and 2) expose all students to examples of high performing female and minority scientists. To impact students at and beyond MIT, my team produced, implemented, and assessed a series of two‐minute videos that feature research scientists, each discussing a general‐chemistry concept in the context of their own cutting‐edge work in medicine, cancer research, or the environment (http://chemvideos.mit.edu). Through an online learning‐experience survey (N=328) and in‐person interviews (N=15), we found that the students made statistically significant gains in their motivation to learn chemistry and their awareness of diversity among bench chemists. By re‐thinking freshman chemistry to highlight the WHY and the WHO, we can increase the number of students motivated to learn the WHAT of chemistry and help retain the scientists of tomorrow.
Proteasomal inhibition effects Notch signaling (937.2)
Authors: Chambers Elaina , Hsu Aaron , White Brandon
Published: June 2021

T‐cell acute lymphoblastic leukemia (T‐ALL) is an aggressive cancer with poor clinical outcome and aberrant Notch signaling has been shown to play a central role in disease pathogenesis. Therefore, it is crucial to elucidate the mechanism underlying Notch deregulation. Upon release from the membrane, the Notch Intracellular Domain (NICD) translocates to the nucleus where is associates with CSL, displacing co‐repressors and recruiting co‐activators (such as Mastermind and CBP/p300), thus resulting in the transcription of target genes (Hes1 and Myc). Our earlier work demonstrated that the NICD could be turned over through a phosphorylation, and subsequent ubiquitination mechanism of degradation. However, it remains unclear if turnover occurs on chromatin or in the nucleoplasm. It is also known that Notch translocation can be pharmacologically inhibited by the drug DAPT. The current work examines the interplay at hand between the inhibition of Notch translocation and the ubiquitin‐mediated proteasomal degradation of Notch. Current data suggest that proteasomal inhibition enhances or “rescues” the transcription of Notch target genes which have been inhibited by DAPT, thus implying that Notch has the ability to “recycle” itself, despite the ubiquitin signal, and that Notch continues participating in gene transcription. These data clearly indicate that Notch signaling is more complex than previously believed, and elucidating the mechanisms underlying its deregulation will greatly benefit the biomedical community as it will effectively enable us to target the Notch pathway in cancer treatment.

Grant Funding Source: Support of research was through NSF‐RUI Grant#1052039 to JBW.
The hunt for a PAX3‐FOXO1 motif (939.4)
Authors: Johanson Kelly , Collins Chantelle , Stewart Desmond , Cherlin Tess
Published: June 2021

Alveolar Rhabdomyosarcoma (ARMS) patients carrying a chromosomal translocation of PAX3 and FOXO1 have the highest mortality rates, indicating the importance of understanding how this transcription factor influences gene expression. Many studies have been performed to identify PAX3‐FOXO1 target genes and while some overlapping gene sets have been identified, there exists great variation in the genes identified. The search for these target genes is hampered by a lack of understanding of the full DNA‐binding capabilities of this fusion protein. We have examined the truncated FOXO1 DNA‐binding domain in PAX3‐FOXO1 and found that it retains activity, but does not have the same sequence specifications for binding as full length FOXO1. A search of 1463 PAX3‐FOXO1 putative binding sites revealed that 37% contained both a PAX3 paired domain and FOXO1 recognition sequence within close proximity. The sequences containing the dual motif mapped to 160 genes with the most interesting of these known to be regulated by PAX3‐FOXO1 such as MET or upregulated in response to PAX3‐FOXO1 expression such as ABAT and PBK. Here we present additional data exploring the sequence variations on the PAX3 and FOXO1 recognition sequences found within Pax3‐FOXO1 putative binding sites as well as in vitro confirmation of the interaction.

Grant Funding Source: Supported by NIH SC2GM095430
A mitochondrial etiology of metabolic and degenerative diseases, cancer and aging (94.1)
Authors: Wallace Douglas
Published: June 2021

Mitochondrial defects have been implicated in a broad spectrum of metabolic and degenerative diseases and can result from mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) genes, changes in the epigenome, and environmental perturbations. Clinically relevant mtDNA variation can result from ancient polymorphisms, recent deleterious mutations, and somatic mutations. Proof that mtDNA mutations can cause disease has been obtained by introducing mtDNA mutations into the mouse via embryonic stem cells (mESCs). Introduction of a COI (nt T6589C, V421A) mutation resulted in myopathy and cardiomyopathy, an ND6 nt G13997A (P25L) mutation in optic atrophy and a parkinsonian phenotype, and mixing two normal but different mtDNAs resulted in neuropsychiatric dysorder and learning problems. Mixtures of mutant and normal mtDNAs (heteroplasmy) can generate very different phenotypes. The tRNALeu(UUR) nt 3243A>G mutation is associated with autism or diabetes at 0‐37% 3243G mutant, neuromuscular degenerative disease at 50‐90% mutant, and perinatal lethal diseases at ~100% mutant. Cell cybrids harboring different percentages of the 3243G mutant have revealed that the different phenotypes correspond with marked changes in the nDNA gene expression profile and hence in the epigenome and signal transduction systems.
New paradigms for regulation of human mitochondrial transcription (94.2)
Authors: Cameron Craig
Published: June 2021

Transcription of human mitochondrial DNA (mtDNA) is directed by three promoters: light‐strand promoter (LSP); heavy‐strand promoter 1 (HSP1) and HSP2. Initiation from all promoters requires the mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factor B2 (TFB2M). Initiation from all promoters is now known to be differentially regulated by the concentration of mitochondrial transcription factor A (TFAM). Full activation of HSP1 requires sequences upstream of the sequences thought to be necessary and sufficient for HSP1 utilization. These “upstream sequences” are highly polymorphic in the human population and are also sites of somatic mutations linked to disease. The contribution of these somatic mutations to the disease phenotypes is not know. The primary obstacle to assessing the causal or consequential relationship between somatic mutations in mtDNA and disease is the retrospective nature of the analysis. In collaboration with the Ridky lab (Nat Med. 2010 Dec; 16(12):1450‐5), we have performed a prospective analysis using squamous cell carcinomas established ex vivo. We observe mutations reminiscent of those observed retrospectively. The implications of this system and our findings on the consequence of these mutations for misregulated mitochondrial transcription in cancer will be discussed.
Vitamin D hormone regulates the expression of hedgehog coreceptor genes Boc and Cdon in osteocytes (946.4)
Authors: Osinski Victoria , St. John Hillary , Pike J
Published: June 2021

Osteocytes are terminally differentiated cells descended from mesenchymal stem cells that are embedded in bone. Osteocytes are known to play an essential role in maintaining bone health, acting as mechanical sensors in bone: translating mechanical load into biochemical signals. Osteocytes have diverse regulatory functions that extend beyond bone, such as lipid regulation and immune system development. These cells are regulated by a number of hormones and cofactors, including the active form of vitamin D, 1,25 dihydroxyvitamin D3 (1,25D3). 1,25D3 binds and activates the vitamin D receptor (VDR) and this complex serves as a transcriptional regulator of various genes. Previously, chromatin immuno‐precipitation coupled to high throughput sequencing (ChIP‐seq) revealed that VDR binds to regions upstream and downstream of the genes coding for Cell adhesion molecule‐related/down‐regulate by oncogenes protein (Cdon) and Brother of Cdon (Boc). These two genes encode cell surface receptor proteins in the Hedgehog signaling pathway, that play an important role in animal tissue development. To better understand 1,25D3’s role in regulating Boc and Cdon, MC3T3‐E1 cells were transfected with luciferase reporter plasmids containing regions of VDRE occupancy identified by ChIP‐seq. These results revealed that these regions of Boc and Cdon mediate up‐regulation by 1,25D3. Specific vitamin D response elements (VDREs) within the enhancer regions were then identified by in silico analyses and selectively mutated. Analysis of these VDRE mutated luciferase reporters revealed an elimination of 1,25D3 activity. In summary, we have shown 1,25D3’s novel role in the up‐regulation of Boc and Cdon and identified the specific VDREs that are responsible for the 1,25D3 response of these genes in osteocytes. Further studies are underway to define the impact of 1,25D3 on Hedgehog signaling in osteocytes. These results contribute to an understanding of 1,25D3’s role in osteocyte and bone health and development.

Grant Funding Source: Supported by NIH DK072281
Experimental and computational analysis of peroxisome protein targeting (948.10)
Authors: Berg Jeremy
Published: June 2021

The targeting of proteins from the cytoplasm to the peroxisomal matrix is determined largely by targeting signals with the sequences of the proteins to be targeted. The Peroxisomal Targeting Signal‐1 (PTS1) is particularly important and comprises sequences at the carboxyl terminus of the protein that approximate X‐X‐X‐Ser‐Lys‐Leu‐COO‐. PTS1s are recognized by the protein Pex5p which binds them and carries the targeted protein to the peroxisome membrane at which point the Pex5p‐protein complexes translocate to the peroxisomal matrix. The structures of Pex5p‐PTS1 peptide and protein complexes as well as apo‐Pex5p have been determined. An experimental human proteome‐wide survey of Pex5p‐PTS1 peptides using a fluorescence anisotropy‐based assay revealed dissociation constants for bona fide peroxisomal matrix proteins ranging from 1.6 nm to > 1 micromolar. Further analysis revealed correlation between protein tissue‐specific expression levels and these dissociation constants, suggesting co‐evolution of PTS1‐Pex5p affinities and expression levels. These observations also suggest testable differences in tissue‐specific peroxisome composition and function. The Pex5p‐PTS1 system also presents a well‐characterized system for the exploration of free energy perturbation methods for calculating binding free energies from structural data. It has been possible to calculate relative free energies with reasonable agreement with experimental results for a series of peptides with variations within the PTS1 sequence as well as some mutated forms of Pex5p.

Grant Funding Source: Supported in part by NIGMS and NIDDK
Alternative autophagy alleviates A1AT‐Z accrual (949.2)
Authors: Wen John , Dankle Geoff , Glenn Kevin
Published: June 2021

Alpha‐1 antitrypsin deficiency is the leading cause of childhood liver failure, one of the most common lethal genetic diseases, and is present in 1 in 2,500 people of European descent. The disease‐causing mutant A1AT‐Z fails to fold correctly and accumulates in the endoplasmic reticulum of the liver, resulting in hepatic fibrosis and hepatocellular carcinoma. A1AT‐Z sequestration in hepatocytes leads to reduced secretion, causing centrolobular emphysema in adults. The purpose of this work was to study the degradation of A1AT‐Z in hepatic tissue and cultured cell lines. We identified the ubiquitin ligase FBG1, which has been previously shown to degrade proteins by both the ubiquitin proteasome pathway and autophagy, as being key to A1AT‐Z degradation. Using chemical and genetic approaches we show that FBG1 degrades A1AT‐Z via both the ubiquitin proteasome system and autophagy. Overexpression of FBG1 decreases the half‐life of A1AT‐Z. Knocking down FBG1 in a hepatic cell line results in an increase in ATAT. Heterozygous A1AT‐Z mutant mice have more Periodic Acid Schiff Base positive inclusions in an FBG1 heterozygous knockdown background compared to mouse lines containing the full amount of FBG1. Finally, we show that FBG1 degrades A1AT‐Z through an ATG5‐independent arm of autophagy, suggesting that alternative autophagy may be important in treating A1AT‐Z deficiency.

Grant Funding Source: VA‐Career Development Award
Regulation of Ras localization and cell transformation by evolutionarily conserved palmitoyltransferases (950.5)
Authors: Young Evelin , Zheng Ze‐Yi , Wilkins Angela , Jeong Hee‐Tae , Min Li , Lichtarge Olivier , Chang Eric
Published: June 2021

Ras GTPases act on the plasma membrane (PM) to mediate signaling that can lead to tumorigenesis. Ras trafficking to the PM is influenced by a series of post‐translational modifications of the Ras C‐terminus, however the identity and necessity of the regulatory proteins are not clear. In an effort to find proteins that are necessary for Ras trafficking to the PM, we conducted an unbiased genomic mutagenesis screen in fission yeast where we sought mutants with diminished PM Ras. Only 5 mutants were found, all defective in the same gene, sp‐erf2, creating sp‐Erf2p, with varying activities. Sp‐Erf2p is a DHHC protein acyl transferase (DHHC‐PAT) functioning at the Golgi for efficient Ras palmitoylation. Sp‐Erf2p is one of five fission yeast DHHC‐PATs and it is closely related to human zDHHC9, previously shown as a Ras PAT in vitro. We show that zDHHC9 efficiently rescued sp‐erf2 null phenotypes and, when repressed in mammalian cells, N/H‐Ras became diminished at the PM and mislocalized into the cytoplasm, and Ras‐induced transformation is reduced. These data illustrate that in fission yeast, sp‐Erf2p is the most critical component in selectively palmitoylating Ras at the Golgi for efficient PM localization, and that in mammals, zDHHC9 is now firmly established as a Ras DHHC‐PAT.
Structural and biophysical analysis of UCH37: ubiquitin recognition and binding to ADRM1 (952.3)
Authors: Morrow Marie , Kim Myung‐Il , Paul Lake , Ronau Judith , Das Chittaranjan
Published: June 2021

Ubiquitin C‐terminal hydrolase 37 (UCH37) is a deubiquitinating enzyme capable of dismantling Lys48‐linked polyubiquitin chains when associated with the 26S proteasome. Upon binding to the proteasomal subunit ADRM1, UCH37 is activated for cleavage of the distal monomer in a chain, an exo‐specific activity. Prior to binding with ADRM1, it is thought that UCH37 is inhibited by its own C‐terminal UCHL5‐like domain (ULD). Our interests lie in UCH37’s mechanism of activation by ADRM1 as well as its exo‐specificity. Previously we have reported the crystal structure of T. spiralis UCH37 bound to ubiquitin vinyl methyl ester, which revealed that the ULD domain interacts with ubiquitin’s Lys48, an event we believe accounts for its exo‐specificity. In order to further examine specificity and activation, we have generated a series of ULD mutants of UCH37. To probe exo‐specificity, we are purifying the 19S regulatory particle of the proteasome from rabbit skeletal muscle, will reconstitute each ULD mutant within the 19S, and then test their cleavage with an asymmetric triubiquitin substrate. When mutated, ULD residues involved in ubiquitin recognition may lose exo‐specificity against the asymmetric substrate. Additionally, we have analyzed UCH37 and ADRM1’s biophysical interactions through isothermal titration calorimetry, finding that the two bind with 1:1 stoichiometry and a dissociation constant of 22 nM. Due to this tight binding and our structural data, we hypothesize that ADRM1’s activation of UCH37 may not be due to an enhancement of UCH37’s turnover rate, but rather due to an increase in ubiquitin’s koff rate; that is, the ULD merely slows product dissociation. We hope to characterize this phenomenon through surface plasmon resonance studies of ubiquitin binding to ULD mutants in the presence of ADRM1. These findings should not only provide a better understanding of UCH37’s activation and exo‐specificity, but will further probe the role of deubiquitination at the proteasome.

Grant Funding Source: Supported by National Institutes of Health Grant 1R01RR026273 (C.D.)
ARRDC5 cooperates with ACAP4 to regulate ARF6 activity at the cell membrane (964.1)
Authors: Zhou Jiajia , Liu Hongsheng , Zhao Xuannv , Wang Dongmei , Wang Fengsong , Liu Fusheng , Yao Xuebiao
Published: June 2021

The ARF6 GTPase is essential for regulating multiple cellular processes, such as, secretion, endocytosis, membrane trafficking, cell migration. ACAP4 is an ARF6 GTPase‐activating protein is critical EGF‐elicited cell migration (Mol. Cell Proteomics. 5, 1437‐49). However, how ACAP4 regulates proper levers of active ARF6 is not well understood. Here we show that, arrestin domain‐containing 5 (ARRDC5), a novel protein of α‐ arrestin family, competes with ARF6 for binding ACAP4. ARRDC5 can inhibit ACAP4 mediated inactivation of ARF6 in a dose‐dependent manner. Knock‐down of ARRDC5 decreases cellular ARF6‐GTP, whereas ARRDC5 overexpression increases cellular ARF6‐GTP. Surprisingly, EGF stimulation of cells causes phosphorylation of ARRDC5 at Ser247 by AKT1, which is essential for cell motility. Significantly, our biochemical characterization demonstrated that the phosphorylation of Ser247 strengthens ACAP4‐ARRDC5 association. The co‐localization of ACAP4 with ARRDC5 occurred in ruffling membranes formed upon EGF stimulation. In addition, expression of the phospho‐mimicking mutant of ARRDC5 promotes ARF6 activation and cell migration, knock‐down of AKT1 inhibits ARF6 activation and cell migration. Our date thus uncover ARRDC5 as a modifier of cellular ARF6‐GTP through regulation of ACAP4. AKT1‐mediated ARRDC5 phosphorylation facilitates cell migration by sequestering ACAP4 at the cell membrane to stabilize active ARF6 in response to EGF stimulation.

Grant Funding Source: DK56292
Mutant Taq polymerase recognition of 2’ modified nucleotides (967.1)
Authors: Chia Hannah , Ogonowsky Alexie , Hadley Emma , Leconte Aaron
Published: June 2021

Mutant Taq DNA Polymerases with the ability to recognize modified nucleotides at the 2’ sugar position have been isolated through directed evolution; these nucleotide modifications increase DNA’s utility by preventing degradation in biological systems. However, these previously identified mutants are only capable of extending DNA templates by two to six base pairs with modified nucleotides. This project focuses on characterizing the increased promiscuity of these enzymes and their ability to recognize a greater substrate range beyond the nucleotides used for their selection. Three previously evolved mutants are able to recognize a surprisingly wide range of 2’ modified nucleotides (dNTP, O‐Me, F, NH3, N3, OH) with varying efficiencies, providing insight into possible new enzyme‐substrate pairings. We describe initial kinetic characterization and further engineering efforts, which may lead to valuable new mutant enzyme‐modified substrate pairs capable of full length synthesis.
Progress toward a novel pro‐drug approach for temporal manipulation of protein kinase A with a commercially available ruthenium catalyst (968.1)
Authors: Hunt Bethany , Dahlen Shelby , Streu Craig , Mertz Pamela
Published: June 2021

Transition metal catalysts have been demonstrated in principle for temporal and spatial control of reactions, but this technology has yet to be broadly applied as a tool for biological study and cellular manipulation. Here we demonstrate the use of a ruthenium complex catalyst as a novel approach for spatial and temporal release of a pro‐drug inside cells, namely the transition of caged H89 to active H89, a Protein Kinase A (PKA) inhibitor. We synthesized caged H89 by protecting H89 as an allyl carbamate. We designed the system to intracellularly generate an active H89 inhibitor from caged H89 following addition of the ruthenium metal catalyst with the goal of inhibiting the catalytic activity of PKA and therefore reducing the phosphorylation of perilipin 1. The ruthenium catalyst activity was monitored by assessing the phosphorylation of perilipin 1, which is phosphorylated by PKA on 6 serine residues and not phosphorylated by other kinases. Experiments were performed on cell lines expressing endogenous perlipin 1, differentiated 3T3L1 cells and Y1 adrenal cells. Our results show that in its active state, H89 inhibited phosphorylation activity of PKA on substrates such as perilipin whereas the caged form of H89 lacked inhibitory activity. Experiments are planned for use of the ruthenium catalyst after the H89 and caged H89 incubation steps to determine the ability of the catalyst to activate caged H89 intracellularly.

Grant Funding Source: Supported by The Royal Society of Chemistry
Transcriptional rewiring and spatiotemporal expression of evolutionarily conserved redox active molecules during bacterial community morphogenesis (973.4)
Authors: Gokhale Rajesh , Verma Priyanka , Anand Amitesh Anand , Singh Anil
Published: June 2021

Biofilm formation by microbes is an adaptive feature common to several microorganisms, wherein community of cells is known to reconstruct a multicellular lifestyle. Such architecture results in the formation of distinct metabolic zones that are demarcated by oxygen gradients and heterogeneous cell population adapted to its microenvironment. While the quest for metabolites that would make these oxygen‐deficient zones habitable has been recognized, phenazines from Pseudomonas aeruginosa are the only characterized intracellular alternate electron acceptors to oxygen. In this study we report identification of an evolutionarily conserved biosynthetic cluster that produces redox‐active molecules ‐ bifilmoquinones and bifilmopyrones, specifically during biofilm development. Remarkably, about half of the Mycobacterium smegmatis transcriptome rewires to produce this multicellular ensemble and the hypoxia‐induced altered intracellular redox state appears to be the crucial trigger dictating this process of morphogenesis. Our analysis suggests the spatiotemporal expression of the biosynthetic cluster a way to acquire an alternate mode of respiration to survive in biofilm core. We further corroborate expression of these redox‐active molecules in the floating biofilms of Bacillus subtilis. Recognition of this novel adaptive redox‐balancing mechanism across several bacterial systems resolves the long‐standing issue of bacterial adaptability to oxygen‐depleted niches.
Dynamic elucidation of drug transport pathways in human P‐glycoprotein (974.5)
Authors: McCormick James , Vogel Pia , Wise John
Published: June 2021

Chemotherapy resistant, recurrent cancers most commonly express members of the ABCB1 class of plasma membrane transporters, specifically MDR1 P‐glycoprotein transporter (P‐gp). At high levels of expression, the P‐gp transporter lowers the intracellular concentrations of chemotherapeutics to below effective therapeutic levels. Because P‐gp can transport a broad range of substrates, such expression leads to general multidrug resistance in the population of cancerous cells. Dynamic P‐gp models which transition through putative catalytic cycles have been previously created by us using targeted molecular dynamics simulations (TMD) with four different crystallographic conformations of related proteins. In the present study, these simulations have been expanded to include transport substrates docked into different initial sites within the drug binding domain of P‐gp. Even though drug movement in these simulations was not targeted by TMD, transport of chemotherapeutics (daunorubicin and doxorubicin) and a P‐gp transport substrate (verapamil) through the membrane by human P‐gp was observed during the putative catalytic cycles. Initial results suggest that different starting positions of transport substrate in the “open to the cytoplasm” conformation resolve into a common transport pathway through the membrane as P‐gp closes to the cytoplasm and opens to the extracellular space.

Grant Funding Source: NIH 1R15GM094771 ‐ 01A1, Communities Foundations of TX
The mycobacterial pupylation pathway (98.2)
Authors: Weber‐Ban Eilika , Barandun Jonas , Delley Cyrille
Published: June 2021

Mycobacteria are prominent members of the actinobacterial phylum owing to the notoriety of the pathogen Mycobacterium tuberculosis (Mtb). The mycobacterial habitat is subject to rapid changes in nutritional sources, requiring from the bacteria a multitude of adaptive mechanisms including protein degradation. Three energy‐dependent protease systems exist in Mtb, the membrane‐associated FtsH, the ClpP protease and a bacterial proteasome assembly restricted to actinobacteria. Substrates are targeted to the proteasome by a post‐translational modification akin to ubiquitination, termed pupylation (1). Pupylation involves the covalent attachment of Pup (prokaryotic ubiquitin‐like protein) to lysine residues of the target and requires the sequential action of two enzymes, the deamidase Dop (deamidase of Pup) and the Pup ligase PafA (2). First, Dop renders Pup ligation‐competent by conversion of its C‐terminal glutamine to glutamate. Then PafA catalyzes the covalent attachment of Pup’s C‐terminal glutamate to the target lysine. We study the Pup‐proteasome pathway by biochemical and structural techniques with particular focus on how the intrinsically disordered Pup is recognized by the pupylation enzymes and how target proteins are selected for modification.

In Mtb, the Pup‐proteasome system contributes to pathogenicity by supporting the persistence within macrophages. The components of pupylation are thus attractive targets for drug development. Understanding their recognition determinants and mechanism of action provides the necessary molecular framework.

(1) Pearce (2008) Science 322: 1104.

(2) Striebel (2009) NSMB 16: 647.
Comprehensive cancer cell proteomics: a global counterpoint to genomic techniques (981.7)
Authors: Bakalarski Corey , Lianoglou Steve , Neve Richard , Yu Mamie , Selvaraj Suresh , Possematto Anthony , Beausoleil Sean , Yue Peng , Dornan David , Bourgon Richard , Forrest William , Kirkpatrick Donald
Published: June 2021

Next‐generation genomic data has produced profound insights into the genetic mechanisms of human disease. However, little is known about how rigidly these genetic features translate to protein abundance and activity. Here, we leveraged tandem mass tagging (TMT 6‐plex) proteomics to build a quantitative map of baseline protein abundance across hundreds of cell lines important to human disease.

Whole cell lysates from 189 cell lines were digested, chemically labeled and analyzed with an LTQ‐Orbitrap Velos mass spectrometer using an HCD‐MS3 method. In all, >13 million quantitative measurements were collected from 2.2 million peptide‐spectral matches. Quantitative data were normalized using a linear mixed effects model to yield relative protein abundances for >8,500 proteins and >100,000 unique peptides.

Comparisons to RNASeq revealed extensive correlation between transcript expression and protein abundance both overall and in genes with known amplification events (ERBB2, EGFR, MET). Poorly correlated expression/abundance levels for a subset of proteins revealed instances of alternate events such as mutational state (TP53) or copy number variation driving protein abundance. Ongoing work seeks to correlate these results with sensitivity/resistance to therapeutic agents.
The human kinase‐substrate phosphorylation network rewired by genetic polymorphisms is heavily associated with cancers (984.3)
Authors: Xue Yu , Wang Yongbo , Liu Zexian
Published: June 2021

Large‐scale sequencing has characterized an enormous number of genetic variations (GVs), and the functional analysis of GVs is fundamental for understanding the difference of disease susceptibility and therapeutic response among populations. Combined with a sequence‐based predictor of GPS 2.1 (http://gps.biocuckoo.org), known phosphorylation and protein‐protein interaction information, we computationally detected 4,490 potential phosSNPs (Phosphorylation‐related non‐synonymous single nucleotide polymorphisms) including 275 known disease‐associated SNPs that dramatically rewire the human phosSNP‐associated kinase‐substrate network. Also, cancer genes and drug targets were significantly over‐represented in the network, whereas proteins in the network are heavily implicated in a number of signaling and cancer pathways. Moreover, we observed that known inherited disease‐ or cancer‐associated mutations were significantly over‐represented in predicted phosSNPs, with a two‐fold enrichment. In addition, we re‐constructed four population‐specific kinase‐substrate networks and revealed several inherited disease or cancer genes such as IRS1, RAF1 and EGFR were differentially regulated by phosSNPs. Thus, phosSNPs may influence the disease and cancer susceptibility by differentially rewiring the phosphorylation networks among different populations. Taken together, our results can be helpful for further experimental analysis of the molecular mechanisms of known disease‐associated phosSNPs, and provided new leads for personalized medicine. The PhosSNP 2.0 database was freely available for academic research at http://phossnp.biocuckoo.org.

Grant Funding Source: Supported by Natural Science Foundation of China (31171263, 81272578)