scholarly journals Identification of two functional nuclear localization signals in DNase γ and their roles in its apoptotic DNase activity

2003 ◽  
Vol 376 (2) ◽  
pp. 377-381 ◽  
Author(s):  
Daisuke SHIOKAWA ◽  
Yukari SHIKA ◽  
Sei-ichi TANUMA
Keyword(s):  
Dna Fragmentation ◽  
Nuclear Localization ◽  
Molecular Basis ◽  
Dnase I ◽  
Dnase Activity ◽  
Dna Ladder ◽  
Functional Feature ◽  
Dnase Γ ◽  
Nuclear Localization Signals ◽  
C Terminus

Among DNase I family members, only DNase γ causes DNA fragmentation during apoptosis. However, the molecular basis for this functional feature of DNase γ is poorly understood. Here we describe the identification of functional NLSs (nuclear localization signals) in DNase γ and their roles in its apoptotic function. DNase γ contains two NLSs: a classical bipartite-type NLS (NLS1) located in the N-terminal half, and a short basic domain (NLS2) at the C-terminus. No potential NLSs are found in the primary structures of other DNase I family DNases. Inactivation of either NLS1 or NLS2 causes reduced DNA ladder-producing activity in DNase γ. Disruption of NLS2 suppresses ladder formation more effectively than disruption of NLS1. DNase γ doubly mutated in both NLSs is enzymically active, but no longer catalyses apoptotic DNA fragmentation. Although DNase I fails to produce ladder formation during apoptosis, DNase I fused to NLS2 of DNase γ through its C-terminus is able to catalyse DNA fragmentation in apoptotic cells. These results indicate that the presence of either NLS1 or NLS2 is necessary for the apoptotic function of DNase γ, and that the most important domain for this function is NLS2. These findings also explain the lack of apoptotic DNase activity in the other DNase I family DNases.

scholarly journals Sequence Determinants of Human Junctophilin-2 Protein Nuclear Localization and Phase Separation

2021 ◽  
Author(s):  
Ang Guo ◽  
Wenjuan Fang ◽  
Savannah Gibson
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nuclear Localization ◽  
Transmembrane Domain ◽  
Nuclear Bodies ◽  
Full Length ◽  
Liquid Droplets ◽  
Nuclear Localization Signals ◽  
Regulatory Processes ◽  
C Terminus

Junctophilin-2 (JPH2) was conventionally considered as a structural membrane binding protein. Recently, it was shown that proteolytically truncated mouse JPH2 variants are imported into nucleus to exert alternative functions. However, the intranuclear behaviors of human JPH2 (hJPH2) and underlying molecular determinants have not been explored. Here, we demonstrate that full-length hJPH2 is imported into nucleus in human cells by two nuclear localization signals (NLSs), including a newly discovered one at the C-terminus. Importantly, unlike the JPH2 N-terminal truncation which diffuses throughout the nucleus, full-length hJPH2 forms nuclear bodies behaving like liquid-liquid phase separated droplets. The C-terminal transmembrane domain is required for the formation of hJPH2 droplets. Oxidation mimicking substitution of residues C678 and M679 augments the formation of hJPH2 nuclear droplets, suggesting nuclear hJPH2 liquid-liquid phase separation could be modulated by oxidative stress. Mutation A405D, which introduces a negatively charged residue into an intrinsic disordered region (IDR) of hJPH2, turns liquid-like droplets into amyloid-like aggregates. Depletion of an Alanine Rich Region in the IDR recapitulates the liquid-amyloid phase transition. The MORN repeat regions of hJPH2 encodes intrinsic tendency to form amyloid-like structure. Together, these data for the first time revealed the novel intrinsic properties of hJPH2 to form nuclear liquid droplets, and identified critical functional domains and residues encoding these properties. We propose that hJPH2 droplets could function as membrane-less organelles participating in nuclear regulatory processes.

scholarly journals Poly(A) polymerase contains multiple functional domains.

1994 ◽  
Vol 14 (5) ◽  
pp. 2946-2957 ◽  
Author(s):  
T Raabe ◽  
K G Murthy ◽  
J L Manley
Keyword(s):  
Nuclear Localization ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Nuclear Proteins ◽  
Directed Mutagenesis ◽  
Nuclear Targeting ◽  
Nuclear Localization Signals ◽  
C Terminus ◽  
Sequence Similarities

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.

scholarly journals The L2 Minor Capsid Protein of Low-Risk Human Papillomavirus Type 11 Interacts with Host Nuclear Import Receptors and Viral DNA

2006 ◽  
Vol 80 (16) ◽  
pp. 8259-8262 ◽  
Author(s):  
J. Bordeaux ◽  
S. Forte ◽  
E. Harding ◽  
M. S. Darshan ◽  
K. Klucevsek ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Low Risk ◽  
Classical Pathway ◽  
Viral Dna ◽  
Nuclear Localization Signals ◽  
C Terminus ◽  
Import Receptors

ABSTRACT Analysis of the interactions of low-risk human papillomavirus type 11 (HPV11) L2 with karyopherin β (Kap β) nuclear import receptors revealed that L2 interacted with Kap β1, Kap β2, and Kap β3 and formed a complex with the Kap α2β1 heterodimer. HPV11 L2 contains two nuclear localization signals (NLSs)—in the N terminus and the C terminus—that could mediate its nuclear import via a classical pathway. Each NLS was functional in vivo, and deletion of both of them abolished L2 nuclear localization. Both NLSs interacted with the viral DNA. Thus, HPV11 L2 can interact with several karyopherins and the viral DNA and may enter the nucleus via multiple pathways.

Bipartite Nuclear Localization Signals in the C Terminus of Human Topoisomerase IIα

1997 ◽  
Vol 237 (2) ◽  
pp. 452-455 ◽  
Author(s):  
Shelagh E.L. Mirski ◽  
James H. Gerlach ◽  
Heather J. Cummings ◽  
Ralph Zirngibl ◽  
Peter A. Greer ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Topoisomerase Iiα ◽  
Nuclear Localization Signals ◽  
C Terminus

Poly(A) polymerase contains multiple functional domains

1994 ◽  
Vol 14 (5) ◽  
pp. 2946-2957
Author(s):  
T Raabe ◽  
K G Murthy ◽  
J L Manley
Keyword(s):  
Nuclear Localization ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Nuclear Proteins ◽  
Directed Mutagenesis ◽  
Nuclear Targeting ◽  
Nuclear Localization Signals ◽  
C Terminus ◽  
Sequence Similarities

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.

scholarly journals Purification and properties of DNase γ from apoptotic rat thymocytes

1997 ◽  
Vol 326 (3) ◽  
pp. 675-681 ◽  
Author(s):  
Daisuke SHIOKAWA ◽  
Harumi OHYAMA ◽  
Takeshi YAMADA ◽  
Sei-ichi TANUMA
Keyword(s):  
Amino Acid ◽  
Metal Ions ◽  
Molecular Mass ◽  
Dna Fragmentation ◽  
Dnase I ◽  
X Rays ◽  
Bivalent Metal ◽  
Intact Cells ◽  
Dnase Γ ◽  
Bivalent Metal Ions

We previously identified three distinct DNA endonucleases, DNases α, β and γ, present in rat thymocyte nuclei. On the basis of their enzymic and biochemical properties, γ-type DNase was regarded as a candidate for the apoptotic endonuclease. Here we purified DNase γ to apparent homogeneity from apoptotic rat thymocyte nuclei induced by X-irradiation and characterized its properties in detail. The purified DNase γ exhibited one predominant protein band on SDS/PAGE and an endonuclease activity in a zymography with an estimated molecular mass of 33 kDa. The molecular mass of the native form determined by G2000SW gel-filtration HPLC was 30 kDa. Amino acid analysis showed that the amino acid composition of DNase γ was similar to that of rat DNase I (molecular mass 32 kDa) but different with regard to alanine and lysine residues. The N-terminal amino acid sequence of DNase γ was revealed to be not identical with that of rat DNase I. In accordance with previous studies, homogeneously purified DNase γ requires both Ca2+ and Mg2+ for activity. This requirement could be partially supplied by Mn2+. Of the bivalent metal ions tested, Co2+, Ni2+, Cu2+ and Zn2+ inhibited DNase γ activity. These bivalent cations also suppressed apoptotic DNA fragmentation in rat thymocytes irradiated by X-rays. The same order of inhibitory ability was observed for these bivalent metal ions in vivo(in intact cells) and in vitro, suggesting that the suppression of apoptotic DNA fragmentation at the cellular level is due to the inhibition of DNase γ. DNase γ activity was found to exist at high levels in spleen, lymph node, thymus, liver and kidney, but little was present in brain, heart or pancreas. On the basis of these findings, together with previous data, we conclude that DNase γ is a novel DNase I-like endonuclease responsible for internucleosomal cleavage of chromatin during thymic apoptosis.

scholarly journals Sumoylation of SAE2 C Terminus Regulates SAE Nuclear Localization

2012 ◽  
Vol 287 (51) ◽  
pp. 42611-42619 ◽  
Author(s):  
Khue Truong ◽  
Terry D. Lee ◽  
Baozong Li ◽  
Yuan Chen
Keyword(s):  
Nuclear Localization ◽  
C Terminus

DNase γ, DNase I and caspase-activated DNase cooperate to degrade dead cells

2016 ◽  
Vol 21 (11) ◽  
pp. 1150-1163 ◽  
Author(s):  
Ryo Koyama ◽  
Tomoya Arai ◽  
Marie Kijima ◽  
Shoko Sato ◽  
Shigetoshi Miura ◽  
...  
Keyword(s):  
Dnase I ◽  
Dnase Γ ◽  
Caspase Activated Dnase
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