bluuebunny/crossref_metadata_2025 huggingface dataset:
Incision‐independent Repair of a Psoralen‐interstrand Crosslink
Authors: Zhang Jieqiong , Budzowska Magda , Walter Johannes
Published: June 2021

DNA interstrand crosslinks (ICLs) are extremely cytotoxic because they prevent strand separation and thus interfere with DNA replication and transcription. ICLs are caused by endogenous metabolites, as well as chemotherapeutics such as cisplatin, nitrogen mustards, and psoralen. A major ICL repair pathway in metazoans is coupled with DNA replication and requires the Fanconi anemia pathway. Using Xenopus egg extracts, our lab previously described the mechanism of replication‐coupled cisplatin‐ICL repair. In this process, two DNA replication forks first converge at the ICL. Following CMG helicase dissociation, the ubiquitylated FANCI‐FANCD2 complex stimulates dual incisions on both sides of the ICL to unhook the lesion, creating a double‐strand break (DSB). Translesion DNA synthesis bypasses the lesion on one sister and the DSB in the other sister is repaired by inter‐sister recombination. Here, we investigated the repair of a psoralen‐ICL formed between two thymines. Strikingly, repair of this ICL does not require the FANCI‐FANCD2 complex and does not involve incision of the parental DNA surrounding the ICL. Instead, the ICL appears to be unhooked via cleavage of the glycosidic bond of one of the adducted thymines by an unknown glycosylase. This generates an abasic site on one parental strand while leaving a psoralen adduct on the other. No DSB is generated. Next, translesion DNA synthesis bypasses the lesions on both daughter molecules. As seen for cisplatin ICLs, the repair of psoralen ICLs requires replication fork convergence. Our data identify a novel pathway that uses DNA glycosylase activity to resolve an ICL in the absence of DSB formation. We are currently investigating which DNA glycosylase is involved in this reaction.
Crystal Structure of the Plasmodium falciparum Apicoplast DNA Polymerase
Authors: Milton Morgan , Honzatko Richard , Nelson Scott
Published: June 2021

Almost half of the world's population is at risk of malaria. The parasite Plasmodium falciparum from the phylum Apicomplexa is the leading cause of malaria infection in humans. Members of Apicomplexa contain a unique and essential organelle called the apicoplast. The apicoplast maintains its own 35 kb genome, which must undergo replication and repair. An A‐family polymerase, apPOL, has been identified as the apicoplast replicative DNA polymerase. Members of A‐family are divided into five subgroups, of which only members of the clade containing ‘typical’ A‐family DNA polymerases such as E. coli Pol I (from which the A‐family is named) and Taq polymerase have been well characterized. Currently, no structural information is available for apPOL, and its closest homolog, the Klenow fragment of E. coli Pol I, shares only 28% sequence identity. Here we report the crystal structure of P. falciparum apPOL, the first atypical A‐family polymerase to be structurally characterized. There are significant structural differences between apPOL and Klenow, primarily in the addition of 46 residues at the N‐terminus of the exonuclease domain. This region may play a role in the recognition of the DNA substrate and possibly influence the activity of the exonuclease active site. Since apPOL has no direct orthologs to mammalian polymerases, P. falciparum apPOL is an attractive anti‐malarial drug target that provides structural and biochemical insight into a poor characterized subgroup of A‐family polymerases.
Swi1Timeless Prevents Repeat Instability at Fission Yeast Telomeres
Authors: Noguchi Eishi , Gadaleta Mariana
Published: June 2021

Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. Replication stress, such as fork stalling and collapse, can arise at natural impediments present throughout the genome if not properly regulated. These impediments include telomeres, which contain repetitive DNA sequences and abundant DNA‐binding proteins. Due to such natures, telomeres are considered to be difficult to replicate. However, the mechanism of telomere replication remains elusive. In this report, we describe a genome‐wide study that identifies the Swi1Timeless protein as a critical factor to prevent repeat instability and recombination at telomeres. Loss of Swi1 causes telomere and subtelomere shortening in a telomerase‐independent manner. Our genetic studies suggest that heterochromatin and telomere‐binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Δ mutant cells, leading to the loss of repeat DNAs. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase, silencing defects, and activation of the alternative lengthening of telomeres pathway (ALT) in telomerase‐negative cells. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also contribute to the understanding the mechanism of ALT in human cancers.
Structural basis for mitochondrial DNA replication
Authors: Yin Whitney , Szymanski Michal , Shumate Christie
Published: June 2021

Human DNA polymerase gamma (Pol γ) is the sole enzyme with DNA synthesis activity in mitochondria. Pol γ is particularly susceptible to inhibition by dideoxynucleoside‐based inhibitors designed to fight viral infection. We report crystal structures of replicating Pol γ ‐DNA complex bound to either a substrate or Zalcitabine, a inhibitor designed for HIV reverse transcriptase. The structures reveal that Zalcitabine binds to Pol g active site almost identically to the normal substrate dCTP, providing a structural basis for Pol γ ‐mediated drug toxicity. Compared to apo Pol γ, the ternary complex undergoes intra‐ and inter‐subunit conformational changes upon binding to primer/template DNA. The accessory subunit Pol γB has no intrinsic enzymatic activity and does not contact the primer/template DNA directly; it serves as an allosteric regulator for holoenzyme activities. These structures suggest a mechanism for the processivity of the holoenzyme, and provide a model for understanding the deleterious effects of Pol γ mutations implicated in human diseases.
A Single‐Molecule Reconstitution of Translesion Synthesis and Competition Between DNA Polymerases
Authors: Kath James , Jergic Slobodan , Heltzel Justin , Jacob Deena , Dixon Nicholas , Sutton Mark , Walker Graham , Loparo Joseph
Published: June 2021

Translesion synthesis (TLS) alleviates replication stalling at DNA lesions. Ring‐shaped processivity clamps play a critical but ill‐defined role in mediating exchange between replicative and TLS DNA polymerases at sites of DNA damage. We have developed a single‐molecule approach to reconstitute TLS and exchange of the Escherichia coli replicative polymerase, Pol III, with the TLS polymerases Pol II and Pol IV. We observe clamp‐mediated exchange of Pol III with both TLS polymerases, and that distinct sets of interactions of each polymerase with the clamp govern the mode of exchange. These results suggest polymerase selection is not simply governed by a mass action competition, but also by positioning of polymerases on clamps at replication intermediates, suggesting a hierarchy for access to DNA lesions.
Testosterone and sex: the role of hormones in sexual dimorphism
Authors: Partsch Jonathan , Rodriguez Daniella , Van Dongen Gregory , Ketterson Ellen , Peterson Mark
Published: June 2021

Males and females have generally similar phenotypic responses to sex hormones. In both sexes, the effects of testosterone (T) appear to masculinize behavior and physiology. However, it is unclear if the mechanism is simple masculinization of gene expression, or something more complex. The dark‐eyed junco (Junco hyemalis), a temperate North American sparrow with well‐characterized life history traits, was used to study the effects of experimentally elevated T on the gene expression of males and females, and to compare expression to sex differences. Testosterone levels that mimic maximum natural levels were administered to the juncos. Multiple tissues were analyzed by microarray to determine sex differences in gene expression and the transcriptional response to T in each sex. There were significant gene expression differences between T‐treated and control individuals in both males and females, as expected. T did not consistently masculinze gene expression, though the male hypothalamus expression was masculinized by T. The relationship between sex differences and hormones is far from clear. It appears that, despite the masculinizing phenotypic effects of testosterone, masculinization of gene expression by the hormone does not explain sex differences. However, by combining these outcomes with the extensive natural history knowledge of the junco, we hope to gain a deeper understanding of the mechanisms of T in this system. Further, these complex outcomes, in light of other recent discoveries, underscore the need for greater understanding of the mechanisms underlying seemingly simple traits.
BIRC6 Is Up‐Regulated Via Transcriptional and Post‐Transcriptional Mechanisms in Imatinib Resistant Chronic Myeloid Leukemia
Authors: Zhang Raymond , Okumu Denis , Levine Merlin , Graves Lee
Published: June 2021

BIRC6 is a recently discovered anti‐apoptotic protein and increased expression is correlated with adverse outcomes in childhood leukemia and other cancers. The expression of BIRC6 was examined in cell models of imatinib sensitive and resistant CML (MYL/MYL‐R). A significant increase in BIRC6 mRNA and protein expression was observed in the MYL‐R cells compared to the drug sensitive MYL cells. Knockdown of BIRC6 in MYL‐R cells increased imatinib sensitivity confirming the importance of this protein in drug resistance. In this study we investigated the mechanisms responsible for the up‐regulation of BIRC6 in MYL‐R cells. Using a novel CDK9 inhibitor (HY‐16462), we were able to identify CDK9 as a required kinase for BIRC6 mRNA transcription. In comparison with MCL‐1, BIRC6 protein levels remained stable despite inhibition of BIRC6 mRNA transcription by the CDK9 inhibitor. This data showed that the regulation of BIRC6 protein levels was post‐transcriptionally determined. A time course treatment with cyclohexamide showed a difference in BIRC6 degradation between MYL and MYL‐R cells. The half‐life of BIRC6 protein in MYL cells was around 3.5 hours compared to 6 hours, nearly double, in MYL‐R cells. Mass spectrometry experiments demonstrated that BIRC6 was differentially phosphorylated in MYL‐R cells. In summary, these studies identify BIRC6 as a key component of cell survival in drug‐resistant leukemia and show that its regulation lies with both mRNA transcription and protein stability. These studies suggest that compounds targeting BIRC6 or BIRC6 stability could be developed to treat cancers resistant to traditional therapies.
“Protein Un‐Design”: Evolutionary Origins of Native DNA‐Binding Architectures in Unfolded Polypeptides
Authors: Weiss M.A. , Racca J.D. , Chen Y‐S. , Phillips N.B.
Published: June 2021

We seek to test the hypothesis that the evolutionary origins of globular proteins, however exquisite in their structural organization, lie within the conformational repertoire of unstructured polypeptides. A model is provided by the high‐mobility‐group (HMG) box, an L‐shaped a‐helical motif of sharp DNA bending that represents a primordial innovation at the root of Eukarya. Whereas the modern HMG box exhibits conserved architectural features (such as an “aromatic buttress” underlying its angular protein surface; Racca, J., et al. J. Biol. Chem. E‐pub (2014)), we demonstrate that natively unfolded variants with defective buttresses can, as a seeming paradox, exhibit native DNA binding and DNA bending. Canonical structure is regained on specific DNA binding as inferred from CD and 1H‐15N NMR. Coupled DNA‐dependent protein folding and DNA bending suggest that primordial boxes could readily have evolved from unstructured precursors, driven by selection for architectural gene regulation. Functional “proof of principle” is provided by unfolded variants of human testis‐determining factor SRY. In a rat embryonic gonadal‐ridge cell line such variant HMG boxes are able to regulate the principal target gene in the nascent testis (Sox9), activating a male transcriptional program. We envisage that progressive stabilization of nascent folds represents a fundamental mechanism for the rapid evolution of autonomous protein structures, yielding the modern landscape of DNA‐binding architectures. In this view extant unstructured DNA‐ and RNA‐binding motifs represent relics of the transition between RNA and protein worlds.

Acknowledgements. We thank L. Labeots, A. Jansco‐Radek, J. Radek, and G. Chan for their participation in the early stages of this work.
Quantitative analysis of amyloid clearance in the Saccharomyces cerevisiae model system [PSI+]
Authors: May Shannon , Camberg Jodi
Published: June 2021

Protein misfolding is a hallmark of several neurodegenerative diseases, leading to the formation of non‐native amyloid deposits that correlate with disease progression. Alzheimer's disease, Parkinson's disease, Huntington's disease, and Creutzfeldt‐Jakob disease are examples of neurodegenerative diseases that share the neuropathological feature of amyloid deposits. Molecular chaperones are a class of proteins that catalyze the folding or activation of nascent proteins, the refolding and reactivation of misfolded proteins, and the disaggregation of protein aggregates. Several proteins, including the ATP‐dependent chaperone Hsp104, have been shown to disassemble amyloid fibers in vitro and in vivo. In the Saccharomyces cerevisiae model for prion propagation and inheritance, [PSI+], reduced amyloid burden correlates with the development of pigmentation attributable to an impaired adenine biosynthesis pathway. This colorimetric conversion assay has been extensively utilized to qualitatively compare the ability of chaperones to cure the cells of amyloid. We developed a quantitative assay to compare the pigmentation in yeast cell lysates, which allows for screening of mutants partially defective for amyloid curing. Using this assay, we compared the extent of amyloid clearance resulting from expression of several chaperones, including Hsp104 wild type and mutant proteins. These studies provide a platform to compare the effectiveness of different chaperone systems and partial loss of function mutants for reducing amyloid burden.
ALS‐related SIGMAR1 Missense Mutation Causes TDP‐43 Inclusion and Mitochondrial Injury
Authors: Shinoda Yasuharu , Tagashira Hideaki , Fukunaga Kohji
Published: June 2021

The dominant missense mutation (p.E102Q) by SIGMAR1 gene mutation was discovered in the patients of juvenile amyotrophic lateral sclerosis (ALS). The sigma‐1 receptor (Sig‐1R) is a chaperone protein localizing in the mitochondrial‐associated endoplasmic reticulum (ER) membrane where regulates Ca2+ transport from ER to the mitochondria through IP3 receptor (IP3R). When Sig‐1R mutant (Sig‐1RE102Q) overexpression in neuroblastoma neuro2A cells, Sig‐1RE102Q dissociated from the IP3R and formed aggregations in the cytosol (Biochem Biophys Acta 2014;1840:3320). Mitochondrial Ca2+ transport induced by IP3R stimulation was also disturbed by Sig‐1RE102Q expression, thereby reducing mitochondrial ATP production. The Sig‐1RE102Q mutant also reduced the mitochondrial membrane potential and promoted mitophagy. Moreover, the ATP reduction caused the decreased proteasome activity and in turn TAR DNA binding protein (TDP‐43) accumulation in the cytosol. These events were recapitulated by pharmacological inhibition of either proteasome or mitochondrial Ca2+ transport. In particular, decreased proteasome activity and accumulation of ubiquitinated TDP‐43 in Sig‐1RE102Q‐expressing cells were markedly aggravated under ER stress such as tunicamycin treatment. Finally, the supply of mitochondrial TCA cycle substrate, methyl pyruvate, improved the Sig‐1RE102Q‐induced reduction of ATP synthesis and proteasome activity with concomitant inhibition of cytoplasmic accumulation of TDP‐43. Taken together, mitochondrial injury associated with ALS caused by Sig‐1RE102Q‐is rescued by methyl pyruvate.
The Reversal of the Anticoagulant and Anti‐hemostatic Effect of LMWH by rLopap
Authors: de Andrade Sonia , Carrijo‐Carvalho Linda , Sato Ana , de Abreu Maffei Francisco , Chudzinski‐Tavassi Ana
Published: June 2021

Lopap, found in the bristles of the caterpillar Lonomia obliqua, is the first exogenous prothrombin activator that shows serineprotease‐like activity and only lipocalin reported to interfere with hemostasis mechanisms. The aim was to assess the effect of recombinant Lopap, obtained using an E. coli expression system, as an exogenous hemostatic factor in reversing bleeding induced by LMWH .The experimental protocol was approved by the Ethics Committee for the Use of Animals of the Teaching and Research Institute, Hospital Sírio‐Libanês. Briefly, anesthesia was induced with ketamine HCl (40 mg/kg, im) and xylazine (5 mg/kg, im) and was maintained by intermittent injections of the same mixture. Rabbits were allocated to 4 groups and received 1800IU/kg LMWH iv over 2 min, followed by iv administration of saline (SG) or rLopap at a dose of 1 μg/kg (LG1) or 10 μg/kg (LG10), 10 min after the injection of LMWH. The reversal of the anticoagulant and anti‐hemostatic effect of LMWH by rLopap were assessed in terms of bleeding time (BT) and laboratory tests; activated partial thromboplastin time (aPTT), prothrombin time (TP), prothrombin fragment F1+2, Thrombin generation, Thrombin‐Antithrombin complex (TAT) and fibrinogen. LG10 animals showed a decreased BT near normal baseline. The levels of prothrombin fragment F1+2 and measured thrombin were inversely proportional to BT. rLopap can be a possible template for the reversal of bleeding and anticoagulant events.

Supported by FAPESP
Expression of Sperm Acrosomal SLLP1 Binding (SAS1B) Protein in a Mammalian System for Crystallization
Authors: Stein Robert , Pires Eusebio , Shetty Jagathpala , Shumilin Igor , Herr John
Published: June 2021

SAS1B (or Ovastacin) is considered an ideal candidate for both female contraception and for cancer therapies. SAS1B first appears in oocytes of follicles at the primary‐secondary transition in pre‐pubertal rodent ovaries and beyond. Quiescent oocytes within primordial and primary follicles are SAS1B negative. In 7 eutherian adult ovaries, the precise temporal and spatial onset of SAS1B expression was conserved. Crystal studies are a vital tool for drug design and for better understanding the molecular structure of targetable proteins, such as SAS1B. Full length SAS1B and its mature form (lacking the N terminus) were PCR‐cloned using gene specific primers sequence containing a kozak sequence at the N‐terminus and six histidine tags at the C‐terminus, using the TOPO cloning strategy. The PCR products were introduced into a pcDNA 3.4 vector and transformed into chemically competent E. Coli cells. Cells were plated on ampicillin infused Luria agar plates and colonies were allowed to grow overnight. Colonies were inoculated in fresh ampicillin containing Luria broth and DNA was extracted using a miniprep kit. Eluted DNA was sent for DNA sequencing. One of the full length clones, clone #9, showed the correct insert for full length SAS1B while screening of E. Coli colonies for the correct sequence for matured SAS1B is ongoing. Subsequently, once the correct sequences have been confirmed through sequencing, transfection of the correct vector‐insert species will be conducted using Expi293 mammalian cells, followed by protein isolation, detection and purification for the purpose of obtaining soluble SAS1B to generate crystals.
c‐Src Regulates Mitochondrial Translation by Phosphorylation of Elongation Factor Tu
Authors: Hunter Caroline , Koc Hasan , Koc Emine
Published: June 2021

Mitochondrial dysfunction is involved in the development and progression of diseases including diabetes, Alzheimer's, cardiovascular disease, Parkinson's, and cancer. Therefore, regulation of mitochondrial biogenesis and energy production is important for a properly functioning cell and organism. Phosphorylation is a reversible post‐translational modification that is involved in the regulation of many cellular processes, including cell survival and proliferation. Over the last decade, our laboratory has shown the phosphorylation of mitochondrial translation components extensively. Mitochondrial translation plays a fundamental role in energy production due to its role in the synthesis of 13 subunits of the oxidative phosphorylation complexes. In the pursuit of kinases responsible for the Tyr phosphorylation of mitochondrial translation components, c‐Src, which is known to be translocated into the mitochondria, was evaluated as a possible Tyr kinase. In this study, we demonstrated that c‐Src phosphorylated mitochondrial translation elongation factor Tu (mtEF‐Tu) at a specific Tyr residue and inhibited protein synthesis in vitro. Over‐expression of c‐Src in mouse embryonic fibroblast cells resulted in impaired mitochondrial function and reduced synthesis of mitochondrial oxidative phosphorylation complexes while supporting higher cell proliferation rates. Our findings also suggest that c‐Src regulates metabolic reprogramming of cells by stimulating glycolytic energy metabolism similar to the Warburg effect observed in tumor cells.
Serine 32 to threonine mutation preserves peroxiredoxin 6 structure and enzymatic function but abolishes its trafficking to lamellar bodies in vivo
Authors: Sorokina Elena , Dodia Chandra , Tao Jian‐Qin , Zhou Suiping , Feinstein Sheldon , Fisher Aron
Published: June 2021

Peroxiredoxin 6 (Prdx6), a bifunctional protein with lysosomal‐type phospholipase A2 (aiPLA2) and GSH peroxidase activities, protects lungs from oxidative stress and participates in lung surfactant phospholipid (PL) turnover. Prdx6 has been localized to both cytosol and acidic organelles (lamellar bodies [LB] and lysosomes) in lung epithelium and its organellar targeting sequence has been identified (31DSWGILFSHP40, Am.J.Physiol.297 : L871, 2009). Interestingly, an S32T mutation fully preserved secondary structure of recombinant protein and had no effect on either its peroxidase or aiPLA2 activity. In a “knock‐in” mouse model carrying the S32T mutation in the Prdx6 gene, Prdx6 protein and aiPLA2 activity were present in lung epithelial cells. However,the protein was absent from epithelial cell LB. Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by aiPLA2. The content of total phospholipid, PC, and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was increased in S32T mutant animals and degradation of internalized [3H]DPPC in isolated mouse lungs was significantly decreased. Thus, Thr can substitute for Ser for the enzymatic activities of Prdx6 but not for its targeting to LB. These results confirm an important role for lamellar body Prdx6 in the degradation and remodeling of lung surfactant phosphatidylcholine. [HL‐019737, HL102016 and HL105509]
Conserved Motifs Contribute to Conjugation and Localization of a Bacterial Mating ATPase
Authors: Murthy Anastasia , Aleksanyan Naira , Bui Minh , Berkmen Melanie
Published: June 2021

Bacterial conjugation involves the transfer of DNA from a donor to a recipient cell through a DNA translocation channel. ConE is a critical component of the DNA translocation channel encoded by ICEBs1, an integrative and conjugative element of Bacillus subtilis. Fusions of ConE to green fluorescent protein (GFP) localize to the cell membrane predominantly at the cell poles. As a member of the FtsK‐HerA superfamily of ATPases, ConE contains conserved Walker A and B boxes, required for ATP binding and hydrolysis, respectively. ConE and close homologs also have conserved motifs C, D, and E that may be involved in ATP sensing and/or cooperative hydrolysis. To determine the role of ConE's ATPase motifs, mutations were introduced into conserved residues and the effects on mating and localization were tested. Mating was down ~100‐fold for the Walker B (W706A) and motif E (Q737) mutants, ~9000‐fold for the mutation near Walker A (H465A), and at least 70,000‐fold for motif C (D498A and E502A) and motif D (R726A) mutants. To determine whether these residues were also critical for ConE localization, GFP fusions to the ConE mutants were visualized via fluorescence microscopy. We found that ConE‐GFP (H465A) partially mislocalizes to the cytoplasm, indicating that this mutant might be defective for mating, at least in part, because it is unable to localize properly. The other five ConE‐GFP mutants (D498A, E502A, W706A, R726A, and Q737A) localize similarly to wild‐type. Since these five residues are located near or in the catalytic pocket of ConE, we hypothesize that these residues are likely critical for mating because they are involved in ATP binding and/or hydrolysis. This research was funded by an NSF‐RUI grant.
Delivery of cargo molecules to cell interiors by a cell‐penetrating peptide‐tagged adaptor
Authors: McMurry Jonathan , Chrestensen Carol , Nowak Scott , Salerno John
Published: June 2021

The trans‐activating transcriptional activator (TAT) from HIV‐1 was the first cell penetrating peptide described. Other small peptides capable of crossing animal cell membranes have since been discovered or devised (e.g., Antennapedia peptide, PEP‐1). Cargo attached to CPP is also internalized and it is now clear that CPPs engage endocytosis mechanisms, delivering cargo to endosomes from which a fraction escapes to the cytoplasm. We expressed and purified a CPP‐tagged adaptor protein, TAT‐CaM, consisting of calmodulin with a TAT peptide. TAT‐CaM binds ligands as effectively as unmodified calmodulin as assessed by optical biosensing experiments. To assess the ability of TAT‐CaM to mediate protein translocation, neuronal nitric oxide synthase was labeled with DyLite550 and added to cultured BHK cells. After washing, cells were observed with confocal microscopy; in the absence of TAT‐CaM some aggregates of labeled nNOS were observed on the surfaces of the cells but not inside. In the presence of TAT‐CaM, cell exteriors were cleared of labeled nNOS, but the interiors were heavily labeled. Endosomal interiors loaded with labeled nNOS were clearly visible, but significant nNOS escaped into the cytoplasm. CPP tagged adaptors have potential advantages in convenience and safety. Calmodulin is particularly useful in that it can be used in affinity purification of tagged cargo, and releases cargo in the low Ca2+ cellinterior.
Lysosomal ATP transport mechanism and its significance
Authors: Dong Xianping , Zhu Michael , Murrell‐Lagnado Ruth , Cao Qi , Huang Peng , Zhong Xi , Zou Yuanjie
Published: June 2021

Lysosomes contain abundant ATP, which is released through lysosomal exocytosis following exposure to various stimuli. However, molecular mechanisms underlying lysosomal ATP accumulation and its physiological significance remain unknown. In this study, we report that SLC17A9 (also called VNUT) is highly enriched in lysosomes and functions as an ATP transporter in those organelles. SLC17A9 deficiency reduced lysosome ATP accumulation and compromised lysosome function, resulting in cell death. Interestingly, Cathepsin D, a lysosomal aspartyl protease, is inhibited by SLC17A9 deficiency. Heterologous expression of Cathepsin D successfully rescues lysosome dysfunction and cell death induced by SLC17A9 deficiency. We also show that P2X4 proteins are highly expressed in lysosomes of mammalian cells. By directly recording membrane currents from enlarged lysosomal vacuoles, we demonstrate that lysosomal P2X4 proteins form channels activated by ATP from the luminal side in a pH dependent manner. While the acidic pH at the luminal side inhibits P2X4 activity, increasing the luminal pH in the presence of ATP causes P2X4 activation. Our data suggests that SLC17A9 transports ATP into lysosomes to regulate Cathepsin D and P2X4 activity, playing an important role in lysosomal physiology and cell viability.
Ferroportin‐mediated cellular iron efflux requires extracellular calcium
Authors: Shawki Ali , Ruwe T Alex , Mitchell Colin , Prakash Shahana , Nemeth Elizabeta , Ganz Tomas , Mackenzie Bryan
Published: June 2021

Ferroportin—the only known cellular iron exporter—is responsible for iron efflux from enterocytes and macrophages to the blood plasma. This transporter, under the control of hepcidin, serves as a key point of regulation in human iron homeostasis. The thermodynamic driving force for ferroportin‐mediated iron efflux is not known at present. To test the hypothesis that ferroportin is an iron/calcium antiporter, we expressed human ferroportin in RNA‐injected Xenopus oocytes and examined its activity by using radiotracer assays as described (Mitchell et al, Am J Physiol Cell Physiol 306: C450–C459, 2014). Ferroportin expression stimulated 55Fe efflux in the presence of 2 mM extracellular Ca2+ [mean (SD) rate constant, k = 3.5 (1.8) × 10−3 min−1; P < 0.001] but not in its absence (0 mM Ca2+, 1 mM EGTA) [k = 0.45 (0.60) × 10−3 min−1], the latter no different from control. Mg2+ could not substitute for Ca2+. Whereas extracellular Ca2+ stimulated ferroportin‐mediated 55Fe efflux (with half‐maximal [Ca], K0.5 ∼ 0.6 mM), microinjection of iron did not stimulate the uptake of 100 μM 45Ca2+ in oocytes expressing ferroportin (P = 0.7). We conclude that calcium is a required cofactor—but not a transported substrate—of ferroportin. Our study suggests that ferroportin‐mediated iron efflux may be limited in conditions of hypocalcemia. DK080047, DK082717
Improving function assignment for metalloenzymes through active site alignment using ProMOL/PyMOL
Authors: Kovuri Venkata , Bryan Michael , Bernstein Herbert , Craig Paul
Published: June 2021

Metal ions are an integral part of many proteins, contributing to both the structure and function of the enzyme. A full representation of Metal Macie catalytic site motif set has been created and tested as 3‐dimensional distance‐based motifs using ProMOL, a plugin for the PyMOL molecular graphics environment that utilizes the 3‐dimensional visualization and measurement capacities of PyMOL to align enzyme active sites with a collection of annotated active sites found in our motif template library. ProMOL uses active site alignment to predict enzyme function for proteins of unknown function. To date our group has focused on active sites consisting solely of the 20 canonical amino acids. With this effort, we have expanded our library of motifs to include metalloenzymes, as nearly 40% of all the PDB structures contain at least one metal ion. The purpose for this initiative can be understood by looking at the number of structures in the Protein Data Bank that are labeled as having “unknown function”; to date, there are 3945 such structures in the Protein Data Bank. More than 50 motif templates of enzyme active sites that contain metal ions have been created and tested with ProMOL. Supported by NIGMS 2R15GM078077‐02, 3R15GM078077‐02S1 and 3R15GM078077‐02S2.
A Chemical Glycoproteomics Platform Applied to Human Prostate Cancer
Authors: Spiciarich David , Maund Sophia , Peehl Donna , Bertozzi Carolyn
Published: June 2021

Accurate diagnosis and treatment of prostate cancer demands a highly specific biomarker of prostate cancer, as prostate‐specific antigen (PSA), the current clinical biomarker for prostate cancer, suffers from high false positive and high false negative rates that leave many men without a conclusive diagnosis. As glycoproteins have proven to be altered in most cancers, we have focused on studying glycoproteins in human prostate tissue samples. We have developed an efficient method for labeling and enriching glycoproteins, followed by MS‐based analysis of normal and carcinoma tissue. Human prostate tissue derived from radical prostatectomies, at varying states of prostatic disease is chemically tagged with an azide functionalized monosaccharide. Labeled tissue slices are reacted with a biotin‐functionalized probe for the capture of azido‐sialylated glycoproteins. Once analysis of labeled tissue confirmed azide‐dependent labeling, glycoproteins were digested and analyzed on a linear ion trap mass spectrometer with collision‐induced dissociation (CID). Greater than 60% of the proteins identified by LC‐MS/MS were cell‐surface or secreted proteins, indicating that this strategy achieves enrichment of low abundance proteins. Additionally, a comparative proteomics analysis between diseased and healthy tissue identified proteins unique to each tissue type, which are undergoing further validation as potential biomarkers. Virtually all clinically approved cancer biomarkers are glycoproteins and this work extends the application of existing metabolic labeling techniques, while developing new methodologies for cellular labeling to discover biomarkers associated with prostate cancer.
Examination of Isoprenoid Chain‐Length Control in Corynebacterium glutamicum
Authors: Nelson Kacie , Mann Francis
Published: June 2021

Prenyltransferases catalyze the polymerization condensation reaction of isopentenyl diphosphate (IPP) and allylic acceptors of varying lengths. Throughout the prenyltransferases enzymes found in the family, Actinobacteria, there exist two catalytic DDxxD motifs in the amino acid sequence. The 4th and 5th amino acid residues before the first motif classically determine chain length of the isoprenoid product and, through manipulation of those amino acids, the resulting chain length can be altered based on the size of the amino acid residue. Corynebacterium glutamicum (C. glutamicum) contains the conserved DDxxD motifs, but the upstream amino acids vary across the three predicted prenyltransferases. Additionally, the presence of a third prenyltransferase is uncommon in the genus. The goal of the project is to explore the naturally occurring variation encoded in the C. glutamicum genome. The one specific, well‐conserved gene in C. glutamicum was cloned and manipulated using site‐directed mutagenesis to model the paralogs. The mutant sequences were confirmed, and recombinantly expressed in Escherichia coli for the determination of the isoprenoid product, compared to the wild‐type's 20‐carbon chain product, geranylgeranyl pyrophosphate, or GGPP.
Evaluation of Sample Disruption Techniques for High Throughput Extraction of Subcellular Organelles
Authors: Garrett Shari , Atwood James
Published: June 2021

Modern mass spectrometers coupled with high resolution separation strategies have enabled the quantification of thousands of proteins from minute samples. However, the large dynamic range in protein abundances, the presence of sequence variants and post‐translational modifications often require that subcellular fractionation strategies be performed in order to achieve the necessary sequence coverage and signal intensity to elucidate statistically significant changes in protein expression. Subcellular fractionation enables the organelle constituents to be separated prior to downstream enzyme digestion and proteomic analysis to increase the detection of proteins of lower abundance thus increasing proteome coverage. Subcellular fractionation encompasses at least two steps; sample disruption (homogenization) and a fractionation procedure. While many organelle fractionation procedures have been described, including density centrifugation, there is no single technique that has been demonstrated to produce an ideal homogenate containing a suspension of separated intact organelles. Here we evaluate the use of three common homogenizer technologies, dounce, rotor‐stator and bead milling for the disruption of tissues for the purpose of isolating commonly analyzed organelles, including nuclei, plasma membrane, mitochondria, endoplasmic reticulum and cytosol. Mouse brain, liver and heart tissues are processed using increasing degrees of disruption forces then homogenates were separated by density centrifugation and analyzed by microscopy, western blotting and bottom up proteomics to evaluate the degree of organelle enrichment.
Improved Accuracy of Proteome Quantification by MS/MS Fragment Intensity
Authors: Wu Qi , Shan Yichu , Zhang Lihua , Zhang Yukui , Fenton Robert
Published: June 2021

Quantitative proteomics facilitates the discovery of potential biomarkers. Development of novel algorithms to improve the accuracy of proteome quantification is of vital importance. MS/MS fragment intensity has arisen as a novel abundance feature with unique advantages.

In isobarically labelled proteome samples, the increase of MS/MS spectra complexity has adverse effect on the identification and coverage of quantification. A paired ion based scoring algorithm (PISA) based on Morpheus, was proposed to address this issue by scoring the matches using the number plus the fraction of intensity of matched paired ions. It was applied to the identification of Lys‐C digest of protein samples isobarically labelled with two different strategies involving both in‐vivo and chemical labeling. Compared to commercially available and open source software Mascot and Morpheus, more peptide spectrum matches, distinct peptides, and proteins can be identified at a 1% false discovery rate with the increase ranging from 22 % to 60 %. Moreover, 100% quantification coverage was achieved.

Summed MS/MS Total ion current (SMT) cumulates all fragment intensities of all MS/MS spectra assigned to a protein. Spectral count in Absolute Protein Expression profiling (APEX) algorithm was replaced by SMT to enhance the accuracy of absolute quantification of proteomes in label‐free manner. The combination of APEX and SMT (abbreviated as APEX‐SMT) was capable of improving the accuracy of absolute quantification by reducing the average relative deviation by ~55‐85% compared to that of APEX, through a series of tests on Universal Proteomics Standard sample with a dynamic range of 5 orders of magnitude (UPS2).
The Lipid Transfer Protein STARD3: an Architect from inside the Cell
Authors: Tomasetto Catherine , Alpy Fabien , Drin Guillaume , Levine Timothy
Published: June 2021

STARD3 [(StAR)‐related lipid transfer domain protein 3] belongs to the START domain protein family, a group of protein having lipid transfer properties. STARD3 is a sterol binding protein located on the limiting membrane of late endosomes, where it is involved in cholesterol transport. Indeed in mammalian cells, high levels of STARD3 are associated with the presence of enlarged endosomes enriched in cholesterol. Using a correlative electron and light microscopy approach, we showed that STARD3 remodels the subcellular architecture. Remarkably, STARD3 ties endosomes with the endoplasmic reticulum. The molecular mechanism of this attachment was solved and relies on the specific interaction between STARD3 and VAP proteins [VAMP‐Associated Proteins (VAP‐A and VAP‐B)]. Altogether our results show that STARD3 and VAP proteins form a novel molecular machinery creating inter‐organelle membrane contacts sites (MCSs), governing the formation of a specific subcellular territory between endosomes and the endoplasmic reticulum. Using in vitro reconstitution, we are able to address the lipid transfer potential of this novel machinery. How STARD3‐made endoplasmic reticulum‐endosomes MCSs control the dynamics of the endosomal compartment will be presented.
Transbilayer Lipid Distribution in Nano Scale
Authors: Murate Motohide , Abe Mitsuhiro , Umeda Masato , Kobayashi Toshihide
Published: June 2021

There are a limited number of methods to examine transbilayer lipid distribution in biomembranes. We employed freeze‐fracture replica labeling immunoelectron microscopy in combination with multiple lipid‐binding peptide/proteins to examine both transbilayer and lateral distribution of various phospholipids in mammalian cells. Our results indicate that phospholipids are exclusively distributed either in the outer or inner leaflet of human red blood cell (RBC) membranes. In contrast, in nucleated cells such as human skin fibroblasts and neutrophils, sphingomyelin was distributed in both leaflets while exhibiting characteristic lipid domains in the inner leaflet. Similar to RBC, lipid asymmetry was maintained both in resting and thrombin‐activated platelets. However, the microparticles released from thrombin‐activated platelets lost membrane asymmetry. Our results suggest that the microparticles were shed from platelet plasma membrane domains enriched with phosphatidylserine/phosphatidylinositol at the outer leaflet. These findings underscore the strict regulation and cell‐type specificity of lipid asymmetry in the plasma membrane. This research using human blood was followed the “Ethical Regulations for Research Involving Human Subjects in RIKEN”.
DHA up‐regulated expression of lipogenic and endoplasmic reticulum stress related genes in pig adipose tissues
Authors: Huang Chao‐Wei , Chen Yu‐Jen , Mersmann Harry , Ding Shih‐Torng
Published: June 2021

Endoplasmic reticulum (ER) is the main site for protein and lipid synthesis. In our previous study, DHA promotes triacylglycerol (TAG) accumulation in pig adipose tissues, which may correlate with ER stress. Several studies have shown the link between ER stress and lipogenesis. However, the function of n‐3 or n‐6 PUFA in regulating ER stress in adipose tissues has not been demonstrated.

We determined the effect of dietary supplementation of soy bean oil (SB oil, rich in n‐6 PUFA) and docosahexaenoic acid oil (DHA oil, rich in n‐3 PUFA) on ER stress of porcine adipose tissues. 8‐week‐old weaned pigs (18.4 ± 0.3 kg) were fed with diets supplemented with 2% (as‐fed basis) of SB oil or DHA oil for 30 days with feed and water provided ad libitum. After 30 days of feeding, we measured the body compositions by EchoMRI, adipose histological section, and analyzed the expression of lipogenic and ER stress‐related genes in the adipose tissue. Body weight and body compositions were not affected by different sources of PUFA. The average size of adipocytes in DHA‐fed pigs were 1.6‐fold greater than SB‐fed pigs. Furthermore, the lipogenic related genes, CCAAT/enhancer binding protein β, hypoxia‐inducible factors and cyclooxygenase 3 were elevated by DHA treatment. Furthermore, ER‐stress related genes including activating transcription factor 6, glucose regulated protein 78 and x‐box binding protein1 are also significantly higher in DHA‐fed than SB‐fed pigs. These results indicate that DHA supplementation may increase lipogenisis and ER stress in pig adipose tissue. Whether the observed phenomena is a mechanism for animals to cope with dietary challenges awaits further investigation.
Distribution of phosphatidylethanolamine in intracellular membrane networks
Authors: Hou Songwang , Zhao Ming
Published: June 2021

Phosphatidylethanolamine (PE) is a ubiquitous phospholipid in mammalian cellular membranes. We characterized PE distribution in intracellular membrane networks using exogenously delivered GFP‐tagged duramycin probes. The trans‐Golgi was the most prominently stained organelle. Positive staining was observed at the cytosolic side of the ER, cis‐Golgi, endosomes and autophagosomes, but not the mitochondria. The nuclear membrane was strongly stained from the luminal but not the cytosolic side. A distinct, granulated staining pattern was identified at the luminal surfaces of early endosomes. Dual staining for PE and phosphatidylserine (PS) revealed drastically different patterns, consistent with fundamentally distinct roles for these phospholipids in the intracellular membrane networks. The plasma membrane inner surface was negative for either probe. Overall, the data provided information on the intracellular distribution of PE, in comparison to that of PS; and were implicative of differential regulatory mechanisms for these synthetically linked but structurally and functionally distinct aminophospholipids.
Serum Fortilin is a Viable Biomarker of In Vivo Apoptosis
Authors: Pinkaew Decha , Wanachottrakul Nattaporn , Sinthujaroen Patuma , Petersen John , Phongdara Amornrat , Sheffield‐Moore Melinda , Fujise Ken
Published: June 2021

Apoptosis plays a critical role in many diseases including cancer and infectious diseases. The assessment of apoptosis occurring in the human body would allow clinicians to diagnose and treat these conditions. However, there is no sensitive and specific serum biomarker of apoptosis reported. Fortilin is a nuclear‐cytosolic shuttle protein with multifunctional implicated in various cellular functions. Since it is a potent anti‐apoptotic molecule and can be secreted into extracellular space via exosomes, we hypothesized that fortilin could be a viable serum biomarker of apoptosis occurring in vivo. Serum fortilin was quantified using ELISA in pre‐ and post‐treatment sera of patients with solid malignancies who were undergoing chemo‐ or radiation therapy, which trigger apoptosis in cancer tissue. The result showed that serum fortilin level was significantly elevated in patients who received anticancer therapy and more sensitive than cytochrome c, nucleosomal DNA, and fragmented cytokeratin‐18 biomarkers. Strikingly, serum fortilin level was elevated in the mouse model of apoptosis‐induced liver damage where Jo2 anti‐Fas antibody was administered to induce Fas‐mediated hepatocyte apoptosis. Moreover, fortilin was released from apoptosing Jurkat cell before plasma membrane integrity was compromised in a well‐defined Fas‐induced apoptosis. In conclusion, serum fortilin is a viable apoptosis biomarker which is secreted from apoptosing cells into the circulation, reflecting the degree of apoptosis occurring in vivo.
Induction of Apoptosis Over Time in DLD‐1 Colon Cancer Cells with Doxorubicin
Authors: Kondaveeti Sriharsha , Clack Beatrice
Published: June 2021

Doxorubicin (DOX) is a common chemotherapy drug used to treat a wide variety of cancer types. The drug is known as an intercalating agent that binds to DNA, inhibiting biosynthesis and ultimately resulting in apoptosis, or programmed cell death in treated cells. The exact molecular mechanisms are complex and remain unclear. This project aims to elucidate the sequence of events that occur in DOX treated cancer cells leading up to apoptosis in human colorectal adenocarcinoma (DLD‐1 ATCC® CCL‐221™). Cells were cultured in complete medium, synchronized through serum starvation, and treated with DOX at 4 hours intervals up to 24 hours. Both untreated and treated cells at each time point then underwent tests to measure cell survival rate and caspase 3/7 activity, which correlates to levels of apoptotic activity. The total RNA from the remaining untreated and treated cells was extracted and reverse transcribed into cDNA. cas 3, 7, 8, 9 and 10 expression levels were determined using real time PCR (RT‐PCR) all of which were normalized to levels of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) expression. RT² Profiler™ PCR Array plates (SA Biosciences) were used to test the expression levels of 84 apoptosis related gene products throughout the different treatment times. Results show that DOX induces extrinsic pathway due to levels of cas10 and cas8 gene expression being elevated. We present a timeline for the gene expression of 84 apoptosis related genes.