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1.
Microb Cell ; 10(2): 18-35, 2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36789350

RESUMO

The surface proteins of parasitic protozoa mediate functions essential to survival within a host, including nutrient accumulation, environmental sensing and immune evasion. Several receptors involved in nutrient uptake and defence from the innate immune response have been described in African trypanosomes and, together with antigenic variation, contribute towards persistence within vertebrate hosts. Significantly, a superfamily of invariant surface glycoproteins (ISGs) populates the trypanosome surface, one of which, ISG75, is implicated in uptake of the century-old drug suramin. By CRISPR/Cas9 knockout and biophysical analysis, we show here that ISG75 directly binds suramin and mediates uptake of additional naphthol-related compounds, making ISG75 a conduit for entry of at least one structural class of trypanocidal compounds. However, ISG75 null cells present only modest attenuation of suramin sensitivity, have unaltered viability in vivo and in vitro and no alteration to suramin-invoked proteome responses. While ISG75 is demonstrated as a valid suramin cell entry pathway, we suggest the presence of additional mechanisms for suramin accumulation, further demonstrating the complexity of trypanosomatid drug interactions and potential for evolution of resistance.

2.
Biochem Soc Trans ; 49(4): 1601-1619, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34282823

RESUMO

The nuclear pore complex (NPC) is responsible for transport between the cytoplasm and nucleoplasm and one of the more intricate structures of eukaryotic cells. Typically composed of over 300 polypeptides, the NPC shares evolutionary origins with endo-membrane and intraflagellar transport system complexes. The modern NPC was fully established by the time of the last eukaryotic common ancestor and, hence, prior to eukaryote diversification. Despite the complexity, the NPC structure is surprisingly flexible with considerable variation between lineages. Here, we review diversification of the NPC in major taxa in view of recent advances in genomic and structural characterisation of plant, protist and nucleomorph NPCs and discuss the implications for NPC evolution. Furthermore, we highlight these changes in the context of mRNA export and consider how this process may have influenced NPC diversity. We reveal the NPC as a platform for continual evolution and adaptation.


Assuntos
Evolução Biológica , Poro Nuclear/metabolismo , Animais , Transporte Biológico , Proteínas de Membrana/metabolismo , Mitose , RNA Mensageiro/metabolismo
3.
Appl Radiat Isot ; 175: 109821, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34130170

RESUMO

The 7Li (p,p'γ)7Li reaction cross section and photon yield from a thick lithium target at proton energies from 0.7 to 1.85 MeV have been measured with a HPGe gamma-ray spectrometer. The spectrometer is calibrated on total and relative sensitivity by reference radionuclide sources of photon radiation. The measurement results are compared with those presented in the EXFOR nuclear reaction database and with other data published in open sources. The reliability of the results of previous studies is analyzed.

4.
Nucleus ; 12(1): 21-41, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33435791

RESUMO

Eukaryotic cells arose ~1.5 billion years ago, with the endomembrane system a central feature, facilitating evolution of intracellular compartments. Endomembranes include the nuclear envelope (NE) dividing the cytoplasm and nucleoplasm. The NE possesses universal features: a double lipid bilayer membrane, nuclear pore complexes (NPCs), and continuity with the endoplasmic reticulum, indicating common evolutionary origin. However, levels of specialization between lineages remains unclear, despite distinct mechanisms underpinning various nuclear activities. Several distinct modes of molecular evolution facilitate organellar diversification and  to understand which apply to the NE, we exploited proteomic datasets of purified nuclear envelopes from model systems for comparative analysis. We find enrichment of core nuclear functions amongst the widely conserved proteins to be less numerous than lineage-specific cohorts, but enriched in core nuclear functions. This, together with consideration of additional evidence, suggests that, despite a common origin, the NE has evolved as a highly diverse organelle with significant lineage-specific functionality.


Assuntos
Membrana Nuclear , Proteômica , Núcleo Celular/genética , Retículo Endoplasmático , Poro Nuclear/genética
5.
Open Biol ; 7(9)2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28903997

RESUMO

How eukaryotic chromosomes are compacted during mitosis has been a leading question in cell biology since the nineteenth century. Non-histone proteins such as condensin complexes contribute to chromosome shaping, but appear not to be necessary for mitotic chromatin compaction. Histone modifications are known to affect chromatin structure. As histones undergo major changes in their post-translational modifications during mitotic entry, we speculated that the spectrum of cell-cycle-specific histone modifications might contribute to chromosome compaction during mitosis. To test this hypothesis, we isolated core histones from interphase and mitotic cells and reconstituted chromatin with them. We used mass spectrometry to show that key post-translational modifications remained intact during our isolation procedure. Light, atomic force and transmission electron microscopy analysis showed that chromatin assembled from mitotic histones has a much greater tendency to aggregate than chromatin assembled from interphase histones, even under low magnesium conditions where interphase chromatin remains as separate beads-on-a-string structures. These observations are consistent with the hypothesis that mitotic chromosome formation is a two-stage process with changes in the spectrum of histone post-translational modifications driving mitotic chromatin compaction, while the action of non-histone proteins such as condensin may then shape the condensed chromosomes into their classic mitotic morphology.


Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Histonas/metabolismo , Linfócitos/metabolismo , Processamento de Proteína Pós-Traducional , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Cátions Bivalentes , Linhagem Celular Tumoral , Galinhas , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Humanos , Interfase/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/ultraestrutura , Magnésio/farmacologia , Microscopia de Força Atômica , Mitose/efeitos dos fármacos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Nocodazol/farmacologia , Fosforilação , Moduladores de Tubulina/farmacologia
6.
Proc Natl Acad Sci U S A ; 114(12): 3133-3138, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28265097

RESUMO

During cell division, interactions between microtubules and chromosomes are mediated by the kinetochore, a proteinaceous structure located at the primary constriction of chromosomes. In addition to the centromere histone centromere protein A (CENP-A), 15 other members of the constitutive centromere associated network (CCAN) participate in the formation of a chromatin-associated scaffold that supports kinetochore structure. We performed a targeted screen analyzing unfolded centrochromatin from CENP-depleted chromosomes. Our results revealed that CENP-C and CENP-S are critical for the stable folding of mitotic kinetochore chromatin. Multipeak fitting algorithms revealed the presence of an organized pattern of centrochromatin packing consistent with arrangement of CENP-A-containing nucleosomes into up to five chromatin "subunits"-each containing roughly 20-30 nucleosomes. These subunits could be either layers of a boustrophedon or small loops of centromeric chromatin.


Assuntos
Cinetocoros/química , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Desdobramento de Proteína , Animais , Linhagem Celular , Centrômero/química , Proteína Centromérica A/química , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Galinhas , Cromatina/química , Imunofluorescência , Expressão Gênica , Técnicas de Silenciamento de Genes , Cinetocoros/metabolismo , Microscopia de Fluorescência , Mitose , Mutação , Proteínas Recombinantes de Fusão , Vertebrados
7.
Cells ; 6(2)2017 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-28346356

RESUMO

Lamins form a scaffold lining the nucleus that binds chromatin and contributes to spatial genome organization; however, due to the many other functions of lamins, studies knocking out or altering the lamin polymer cannot clearly distinguish between direct and indirect effects. To overcome this obstacle, we specifically targeted the mapped histone-binding site of A/C lamins by microinjecting antibodies specific to this region predicting that this would make the genome more mobile. No increase in chromatin mobility was observed; however, interestingly, injected cells failed to go through mitosis, while control antibody-injected cells did. This effect was not due to crosslinking of the lamin polymer, as Fab fragments also blocked mitosis. The lack of genome mobility suggested other lamin-chromatin interactions. To determine what these might be, mini-lamin A constructs were expressed with or without the histone-binding site that assembled into independent intranuclear structures. HP1, CenpB and PML proteins accumulated at these structures for both constructs, indicating that other sites supporting chromatin interactions exist on lamin A. Together, these results indicate that lamin A-chromatin interactions are highly redundant and more diverse than generally acknowledged and highlight the importance of trying to experimentally separate their individual functions.

8.
Methods Enzymol ; 569: 79-100, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26778554

RESUMO

Lamins and associated nuclear envelope transmembrane proteins (NETs) present unique problems for biochemical studies. Lamins form insoluble intermediate filament networks, associate with chromatin, and are also connected via specific NETs to the cytoskeleton, thus further complicating their isolation and purification from mammalian cells. Adding to this complexity, NETs at the inner nuclear membrane function in three distinct environments: (a) their nucleoplasmic domain(s) can bind lamins, chromatin, and transcriptional regulators; (b) they possess one or more integral transmembrane domains; and (c) their lumenal domain(s) function in the unique reducing environment of the nuclear envelope/ER lumen. This chapter describes strategic considerations and protocols to facilitate biochemical studies of lamins and NET proteins in vitro. Studying these proteins in vitro typically involves first expressing specific polypeptide fragments in bacteria and optimizing conditions to purify each fragment. We describe parameters for choosing specific fragments and designing purification strategies and provide detailed purification protocols. Biochemical studies can provide fundamental knowledge including binding strengths and the molecular consequences of disease-causing mutations that will be essential to understand nuclear envelope-genome interactions and nuclear envelope linked disease mechanisms.


Assuntos
Laminas/isolamento & purificação , Complexo de Proteínas Formadoras de Poros Nucleares/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Escherichia coli , Solubilidade
9.
PLoS One ; 9(11): e111851, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25386906

RESUMO

Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture.


Assuntos
Cromatina/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Acetilação , Histonas/metabolismo , Humanos , Imunidade Inata , Metilação
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