The anticancer effect of L-asparaginases (L-ASNases) is attributable to their ability to hydrolyze L-asparagine in the bloodstream and cancer cell microenvironment.
(RrA) has dual mechanism of action ...and plays a role in the suppression of telomerase activity. The aim of this work was to investigate the possible mechanism of RrA penetration into human cancer cells. Labeling of widely used L-ASNases by fluorescein isothiocyanate followed by flow cytometry and fluorescent microscopy demonstrated that only RrA can interact with cell membranes. The screening of inhibitors of receptor-mediated endocytosis demonstrated the involvement of clathrin receptors in RrA penetration into cells. Confocal microscopy confirmed the cytoplasmic and nuclear localization of RrA in human breast cancer SKBR3 cells. Two predicted nuclear localization motifs allow RrA to penetrate into the cell nucleus and inhibit telomerase. Chromatin relaxation promoted by different agents can increase the ability of RrA to suppress the expression of telomerase main catalytic subunit. Our study demonstrated for the first time the ability of RrA to penetrate into human cancer cells and the involvement of clathrin receptors in this process.
The majority of known nucleolar proteins are freely exchanged between the nucleolus and the surrounding nucleoplasm. One way proteins are retained in the nucleoli is by the presence of specific amino ...acid sequences, namely nucleolar localization signals (NoLSs). The mechanism by which NoLSs retain proteins inside the nucleoli is still unclear. Here, we present data showing that the charge-dependent (electrostatic) interactions of NoLSs with nucleolar components lead to nucleolar accumulation as follows: (i) known NoLSs are enriched in positively charged amino acids, but the NoLS structure is highly heterogeneous, and it is not possible to identify a consensus sequence for this type of signal; (ii) in two analyzed proteins (NF-κB-inducing kinase and HIV-1 Tat), the NoLS corresponds to a region that is enriched for positively charged amino acid residues; substituting charged amino acids with non-charged ones reduced the nucleolar accumulation in proportion to the charge reduction, and nucleolar accumulation efficiency was strongly correlated with the predicted charge of the tested sequences; and (iii) sequences containing only lysine or arginine residues (which were referred to as imitative NoLSs, or iNoLSs) are accumulated in the nucleoli in a charge-dependent manner. The results of experiments with iNoLSs suggested that charge-dependent accumulation inside the nucleoli was dependent on interactions with nucleolar RNAs. The results of this work are consistent with the hypothesis that nucleolar protein accumulation by NoLSs can be determined by the electrostatic interaction of positively charged regions with nucleolar RNAs rather than by any sequence-specific mechanism.
•Nucleolar localization signals (NoLSs) facilitate the accumulation of certain proteins in the nucleoli.•NoLSs promote the accumulation of proteins in the nucleoli through a charge-dependent mechanism.•Charge-dependent nucleolar accumulation is dependent on interactions with nucleolar RNAs.
The origin of the selective nuclear protein import machinery, which consists of nuclear pore complexes and adaptor molecules interacting with the nuclear localization signals (NLSs) of cargo ...molecules, is one of the most important events in the evolution of eukaryotic cells. How proteins were selected for import into the forming nucleus remains an open question.
Here, we demonstrate that functional NLSs may be integrated in the nucleotide-binding domains of both eukaryotic and prokaryotic proteins and may coevolve with these domains.
The presence of sequences similar to NLSs in the DNA-binding domains of prokaryotic proteins might have created an advantage for nuclear accumulation of these proteins during evolution of the nuclear-cytoplasmic barrier, influencing which proteins accumulated and became compartmentalized inside the forming nucleus (i.e., the content of the nuclear proteome).
This article was reviewed by Sergey Melnikov and Igor Rogozin.
Reviewed by Sergey Melnikov and Igor Rogozin. For the full reviews, please go to the Reviewers' comments section.
During evolution, viruses had to adapt to an increasingly complex environment of eukaryotic cells. Viral proteins that need to enter the cell nucleus or associate with nucleoli possess nuclear ...localization signals (NLSs) and nucleolar localization signals (NoLSs) for nuclear and nucleolar accumulation, respectively. As viral proteins are relatively small, acquisition of novel sequences seems to be a more complicated task for viruses than for eukaryotes. Here, we carried out a comprehensive analysis of the basic domain (BD) of HIV-1 Tat to show how viral proteins might evolve with NLSs and NoLSs without an increase in protein size. The HIV-1 Tat BD is involved in several functions, the most important being the transactivation of viral transcription. The BD also functions as an NLS, although it is substantially longer than a typical NLS. It seems that different regions in the BD could function as NLSs due to its enrichment with positively charged amino acids. Additionally, the high positive net charge inevitably causes the BD to function as an NoLS through a charge-specific mechanism. The integration of NLSs and NoLSs into functional domains enriched with positively charged amino acids might be a mechanism that allows the condensation of different functional sequences in small protein regions and, as a result, reduces protein size, influencing the origin and evolution of NLSs and NoLSs in viruses.
Here, we investigated the molecular mechanism of nuclear localization signal (NLS) and nucleolar localization signal (NoLS) integration into the basic domain of HIV-1 Tat (
RKKRRQRRR
) and found that these two supplementary functions (i.e., function of NLS and function of NoLS) are embedded in the basic domain amino acid sequence. The integration of NLSs and NoLSs into functional domains of viral proteins enriched with positively charged amino acids is a mechanism that allows the concentration of different functions within small protein regions. Integration of NLS and NoLS into functional protein domains might have influenced the viral evolution, as this could prevent an increase in the protein size.
The nuclear accumulation of proteins may depend on the presence of short targeting sequences, which are known as nuclear localization signals (NLSs). Here, we found that NLSs are predicted in some ...cytosolic proteins and examined the hypothesis that these NLSs may be functional under certain conditions. As a model, human cardiac troponin I (hcTnI) was used. After expression in cultured non-muscle or undifferentiated muscle cells, hcTnI accumulated inside nuclei. Several NLSs were predicted and confirmed by site-directed mutagenesis in hcTnI. Nuclear import occurred via the classical karyopherin-α/β nuclear import pathway. However, hcTnI expressed in cultured myoblasts redistributed from the nucleus to the cytoplasm, where it was integrated into forming myofibrils after the induction of muscle differentiation. It appears that the dynamic retention of proteins inside cytoplasmic structures can lead to switching between nuclear and cytoplasmic localization.
•Nuclear localization signals (NLSs) are predicted within cytoplasmic proteins.•Human cardiac Troponin I accumulated inside nuclei of nondifferentiated myoblast or nonmuscle cells•Human cardiac Troponin I is imported into nuclei via the classical karyopherin-α/β nuclear import pathway.•Human cardiac Troponin I relocalized from the nucleus to the cytoplasm during myogenic differentiation.•The switch from nuclear to cytoplasmic localization may be driven by the retention of proteins inside cytoplasmic structures.
The majority of known nuclear proteins are highly mobile. The molecular mechanisms by which they accumulate inside stable compartments that are not separated from the nucleoplasm by membranes are ...obscure. The compartmental retention of some proteins is associated with their biological function; however, some protein interactions within distinct nuclear structures may be non-specific. The non-specific retention may lead to the accumulation of proteins in distinct structural domains, even if the protein does not function inside this domain. In this study, we have shown that histone H2B-EGFP initially accumulated in the nucleolus after ectopic expression, and then gradually incorporated into the chromatin to leave only a small amount of nucleolus-bound histone that was revealed by removing chromatin-bound proteins with DNase I treatment. Nucleolar histone H2B had several characteristics: (i) it preferentially bound to granular component of the nucleolus and interacted with RNA or RNA-containing nucleolar components; (ii) it freely exchanged between the nucleolus and nucleoplasm; (iii) it associated with the nuclear matrix; and (iv) it bound to interphase prenuclear bodies that formed after hypotonic treatment. The region in histone H2B that acts as a nucleolar localization/retention signal (NoRS) was identified. This signal overlapped with a nuclear localization signal (NLS), which appears to be the primary function of this region. The NoRS activity of this region was non-specific, but the molecular mechanism was probably similar to the NoRSs of other nucleolar proteins. All known NoRSs are enriched with basic amino acids, and we demonstrated that positively charged motifs (nona-arginine (R9) and nona-lysine (K9)) were sufficient for the nucleolar accumulation of EGFP. Also, the correlation between measured NoRS activity and the predicted charge was observed. Thus, NoRSs appear to achieve their function through electrostatic interactions with the negatively charged components of the nucleolus. Though these interactions are non-specific, the functionally unrelated retention of a protein can increase the probability of its interaction with specific and functionally related binding sites.
►Histone H2B-GFP initially accumulated in the nucleolus after ectopic expression. ►The region that acts as a nucleolar retention signal (NoRS) was identified. ►This signal overlapped with a nuclear localization signal (NLS). ►The interaction of histone H2B with the nucleolus was non-specific. ►NoRSs appear to achieve their function through simple electrostatic interactions.
RNA-dependent disassembly of nuclear bodies Musinova, Yana R; Lisitsyna, Olga M; Sorokin, Dmitry V ...
Journal of cell science,
12/2016, Letnik:
129, Številka:
24
Journal Article
Recenzirano
Odprti dostop
Nuclear bodies are membraneless organelles that play important roles in genome functioning. A specific type of nuclear bodies known as interphase prenucleolar bodies (iPNBs) are formed in the ...nucleoplasm after hypotonic stress from partially disassembled nucleoli. iPNBs are then disassembled, and the nucleoli are reformed simultaneously. Here, we show that diffusion of B23 molecules (also known as nucleophosmin, NPM1) from iPNBs, but not fusion of iPNBs with the nucleoli, contributes to the transfer of B23 from iPNBs to the nucleoli. Maturation of pre-ribosomal RNAs (rRNAs) and the subsequent outflow of mature rRNAs from iPNBs led to the disassembly of iPNBs. We found that B23 transfer was dependent on the synthesis of pre-rRNA molecules in nucleoli; these pre-rRNA molecules interacted with B23 and led to its accumulation within nucleoli. The transfer of B23 between iPNBs and nucleoli was accomplished through a nucleoplasmic pool of B23, and increased nucleoplasmic B23 content retarded disassembly, whereas B23 depletion accelerated disassembly. Our results suggest that iPNB disassembly and nucleolus assembly might be coupled through RNA-dependent exchange of nucleolar proteins, creating a highly dynamic system with long-distance correlations between spatially distinct processes.
Proteins larger than ~40 kDa are selectively transfered through the nuclear pore complexes by energy-dependent mechanism that requires additional transport factors, called karyopherins, which ...recognize nuclear localization signals (NLSs) in their cargo proteins. Recent studies recognized some important events in the nuclear envelope evolution, and the possible ancestors of key elements of import machinery – nuclear pore complexes and karyopherins, were supposed. However, the origin and evolution of NLSs is obscure. Methods. To address this question, we analyzed the data on the NLSs and their localization relative to protein domains, and also studied possible NLS-containing regions inside prokaryotic proteins. Results. The result of the current work indicate that (1) NLS could pre-exist in prokaryotic proteins inside domains that bind nucleic acids or proteins; (2) during the course of evolution, some NLSs could be integrated within functional domains and co-evolve with them; (3) the structure of some functional domains of non-nuclear proteins allows them to function as an NLS, which requires the development of mechanisms to prevent the import of these proteins into the nucleus. Conclusions. Our data indicate that enriched with positively charged amino acids regions of nucleotide-binding domains can serve as genuine NLSs. These NLSs are integrated into domains, and their evolution might be depended on the evolution of the domain. Funding. This work was supported by the Russian Science Foundation (grant 18-14-00195).
We have examined the anterior and middle hooks of many specimens of 3 species of acanthocephalans from Ukraine including (1) adults of
Acanthocephalus ranae
(Schrank, 1788) Lühe, 1911 from 4 species ...of frogs in 6 geographical locations, (2) adults of
Southwellina hispida
Van Cleave, (1925) Witenberg, 1932 from 3 species of birds in 2 geographical locations, and (3) adults and cystacanths of
Sphaerirostris picae
(Rudolphi, 1819) Golvan, 1956 from 1 species of birds, 1 species of lizards, and 1 species of mammals in 2 geographical locations
,
to analyze their Ca, S, and P spectra using Energy dispersive x_ray analysis (EDXA), and account for their intraspecific variabilities. Adults of each of
A. ranae
from frogs and adults of
S. hispida
from birds each showed comparable metal spectra irrespective of host species and geography, especially when metal weight percent figures are averaged. In
S. picae
, 5 adult specimens from birds had comparable spectra but the cystacanth from hedgehog, a mammal, had particularly dissimilar pattern with much lower levels of P and Ca. We have also studied the EDXA patterns in anterior, middle and posterior hooks of cystacanths, juveniles, and adults of
Moniliformis kalahariensis
Meyer, 1931 much lower levels of P and Ca. in South Africa, Botswana, and India collected from their invertebrate intermediate and vertebrate definitive hosts to examine the EDXA profiles. Our conclusions were comparable to those reached from the Ukrainian material with the additional emphasis on the importance of using the same developmental stage in comparing interspecific EDXA profiles. The importance of the stability of the levels of S in various developmental stages across the host species barrier is exemplified by its relationship to the genetically based protein synthesis, which gives credibility to the usage of EDXA for diagnostic purposes. Our findings show that EDXA is a useful tool to characterize the taxonomic identity of species of Acanthocephala providing that (1) a population of a number of individual specimens are analyzed and average weight percent figures of metals are used for comparisons, and (2) comparisons are made using specimens of the same developmental stage and from hosts of the same class of vertebrate. When these conditions are met, the metal spectra for each species will prove valid for diagnostic purposes.
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