A potential role for the COOH-terminal domain in the lateral packing of type III intermediate filaments.

To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti-idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness.

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Cryo-EM Structures of CTP Synthase Filaments Reveal Mechanism of pH-Sensitive Assembly During Budding Yeast Starvation

10.1101/2021.08.25.457724 ◽

2021 ◽

Author(s):

Jesse M Hansen ◽

Avital Horowitz ◽

Eric M Lynch ◽

Daniel P Farrell ◽

Joel Quispe ◽

...

Keyword(s):

Metabolic Enzymes ◽

Ph Sensitive ◽

Starvation Response ◽

Filament Assembly ◽

Assembly Mechanism ◽

Ctp Synthase ◽

Filament Bundles ◽

Cytoplasmic Acidification

ABSTRACTMany metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or hyper-assembly mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

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Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation

eLife ◽

10.7554/elife.73368 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jesse M Hansen ◽

Avital Horowitz ◽

Eric M Lynch ◽

Daniel P Farrell ◽

Joel Quispe ◽

...

Keyword(s):

Metabolic Enzymes ◽

Ph Sensitive ◽

Starvation Response ◽

Filament Assembly ◽

Assembly Mechanism ◽

Ctp Synthase ◽

Filament Bundles ◽

Cytoplasmic Acidification

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

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Mutational analysis of human immunodeficiency virus type 1 (HIV-1) accessory genes: requirement of a site in the nef gene for HIV-1 replication in activated CD4+ T cells in vitro and in vivo.

Journal of Virology ◽

10.1128/jvi.71.11.8456-8466.1997 ◽

1997 ◽

Vol 71 (11) ◽

pp. 8456-8466 ◽

Cited By ~ 36

Author(s):

Y Kawano ◽

Y Tanaka ◽

N Misawa ◽

R Tanaka ◽

J I Kira ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Human Immunodeficiency Virus Type ◽

Mutational Analysis ◽

Immunodeficiency Virus ◽

A Site ◽

Hiv 1

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Conifers Phytochemicals: A Valuable Forest with Therapeutic Potential

Molecules ◽

10.3390/molecules26103005 ◽

2021 ◽

Vol 26 (10) ◽

pp. 3005

Author(s):

Kanchan Bhardwaj ◽

Ana Sanches Silva ◽

Maria Atanassova ◽

Rohit Sharma ◽

Eugenie Nepovimova ◽

...

Keyword(s):

Experimental Data ◽

Potential Role ◽

Therapeutic Potential ◽

Fungal Infections ◽

Cell Damage ◽

Cancer Growth ◽

Naturally Occurring ◽

Antioxidant Effects

Conifers have long been recognized for their therapeutic potential in different disorders. Alkaloids, terpenes and polyphenols are the most abundant naturally occurring phytochemicals in these plants. Here, we provide an overview of the phytochemistry and related commercial products obtained from conifers. The pharmacological actions of different phytochemicals present in conifers against bacterial and fungal infections, cancer, diabetes and cardiovascular diseases are also reviewed. Data obtained from experimental and clinical studies performed to date clearly underline that such compounds exert promising antioxidant effects, being able to inhibit cell damage, cancer growth, inflammation and the onset of neurodegenerative diseases. Therefore, an attempt has been made with the intent to highlight the importance of conifer-derived extracts for pharmacological purposes, with the support of relevant in vitro and in vivo experimental data. In short, this review comprehends the information published to date related to conifers’ phytochemicals and illustrates their potential role as drugs.

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Molecular and Cellular Mechanisms of Metformin in Cervical Cancer

Cancers ◽

10.3390/cancers13112545 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2545

Author(s):

Ya-Hui Chen ◽

Po-Hui Wang ◽

Pei-Ni Chen ◽

Shun-Fa Yang ◽

Yi-Hsuan Hsiao

Keyword(s):

Cervical Cancer ◽

Potential Role ◽

Treatment Options ◽

Cellular Mechanisms ◽

Anticancer Effects ◽

Clinical Benefits ◽

Underlying Mechanisms

Cervical cancer is one of the major gynecologic malignancies worldwide. Treatment options include chemotherapy, surgical resection, radiotherapy, or a combination of these treatments; however, relapse and recurrence may occur, and the outcome may not be favorable. Metformin is an established, safe, well-tolerated drug used in the treatment of type 2 diabetes; it can be safely combined with other antidiabetic agents. Diabetes, possibly associated with an increased site-specific cancer risk, may relate to the progression or initiation of specific types of cancer. The potential effects of metformin in terms of cancer prevention and therapy have been widely studied, and a number of studies have indicated its potential role in cancer treatment. The most frequently proposed mechanism underlying the diabetes–cancer association is insulin resistance, which leads to secondary hyperinsulinemia; furthermore, insulin may exert mitogenic effects through the insulin-like growth factor 1 (IGF-1) receptor, and hyperglycemia may worsen carcinogenesis through the induction of oxidative stress. Evidence has suggested clinical benefits of metformin in the treatment of gynecologic cancers. Combining current anticancer drugs with metformin may increase their efficacy and diminish adverse drug reactions. Accumulating evidence is indicating that metformin exerts anticancer effects alone or in combination with other agents in cervical cancer in vitro and in vivo. Metformin might thus serve as an adjunct therapeutic agent for cervical cancer. Here, we reviewed the potential anticancer effects of metformin against cervical cancer and discussed possible underlying mechanisms.

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Regulation of RNA helicase activity: principles and examples

Biological Chemistry ◽

10.1515/hsz-2020-0362 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Pascal Donsbach ◽

Dagmar Klostermeier

Keyword(s):

Atp Hydrolysis ◽

Wide Spectrum ◽

Mrna Translation ◽

Rna Degradation ◽

Rna Helicases ◽

Interaction Partners ◽

Rna Duplexes ◽

Self Association

Abstract RNA helicases are a ubiquitous class of enzymes involved in virtually all processes of RNA metabolism, from transcription, mRNA splicing and export, mRNA translation and RNA transport to RNA degradation. Although ATP-dependent unwinding of RNA duplexes is their hallmark reaction, not all helicases catalyze unwinding in vitro, and some in vivo functions do not depend on duplex unwinding. RNA helicases are divided into different families that share a common helicase core with a set of helicase signature motives. The core provides the active site for ATP hydrolysis, a binding site for the non-sequence-specific interactions with RNA, and in many cases a basal unwinding activity. Its activity is often regulated by flanking domains, by interaction partners, or by self-association. In this review, we summarize the regulatory mechanisms that modulate the activities of the helicase core. Case studies on selected helicases with functions in translation, splicing, and RNA sensing illustrate the various modes and layers of regulation in time and space that harness the helicase core for a wide spectrum of cellular tasks.

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In vitro and in vivo analysis of [64Cu-NO2A-8-Aoc-BBN(7–14)NH2]: a site-directed radiopharmaceutical for positron-emission tomography imaging of T-47D human breast cancer tumors

Nuclear Medicine and Biology ◽

10.1016/j.nucmedbio.2008.11.005 ◽

2009 ◽

Vol 36 (2) ◽

pp. 171-181 ◽

Cited By ~ 40

Author(s):

Adam F. Prasanphanich ◽

Lauren Retzloff ◽

Stephanie R. Lane ◽

Prasant K. Nanda ◽

Gary L. Sieckman ◽

...

Keyword(s):

Human Breast Cancer ◽

Positron Emission Tomography Imaging ◽

Emission Tomography ◽

Human Breast ◽

Tomography Imaging ◽

Positron Emission ◽

In Vivo Analysis ◽

A Site

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In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.3868 ◽

1990 ◽

Vol 10 (8) ◽

pp. 3868-3872 ◽

Cited By ~ 15

Author(s):

C M Shumard ◽

C Torres ◽

D C Eichler

Keyword(s):

18S Rrna ◽

Precise Determination ◽

In Vivo Studies ◽

Rrna Sequence ◽

S1 Nuclease ◽

In Vitro Processing ◽

Rrna Maturation ◽

A Site

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

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Contributions of the structural domains of filensin in polymer formation and filament distribution

Journal of Cell Science ◽

10.1242/jcs.109.2.447 ◽

1996 ◽

Vol 109 (2) ◽

pp. 447-456

Author(s):

G. Goulielmos ◽

S. Remington ◽

F. Schwesinger ◽

S.D. Georgatos ◽

F. Gounari

Keyword(s):

De Novo ◽

Chimeric Proteins ◽

Tail Domain ◽

Structural Domains ◽

Filament Assembly ◽

Polymer Formation ◽

Filament Bundles ◽

Specific Contribution ◽

Mcf 7

Filensin and phakinin constitute the subunits of a heteropolymeric, lens-specific intermediate filament (IF) system known as the beaded-chain filaments (BFs). Since the rod of filensin is four heptads shorter than the rods of all other IF proteins, we decided to examine the specific contribution of this protein in filament assembly. For these purposes, we constructed chimeric proteins in which regions of filensin were exchanged with the equivalent ones of vimentin, a self-polymerizing IF protein. Our in vitro studies show that the filensin rod domain does not allow homopolymeric filament elongation. However, the filensin rod is necessary for co-polymerization of filensin with phakinin and seems to counteract the inherent tendency of the latter protein to homopolymerize into large, laterally associated filament bundles. Apart from the rod domain, the presence of an authentic or substituted tail domain in filensin is also essential for co-assembly with the naturally tail-less phakinin and formation of extended filaments in vitro. Finally, transfection experiments in CHO and MCF-7 cells show that the rod domain of filensin plays an important role in de novo filament formation and distribution. The same type of analysis further suggests that the end-domains of filensin interact with cell-specific, assembly-modulating factors.

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