Split NanoLuc technology allows quantitation of interactions between PII protein and its receptors with unprecedented sensitivity and reveals transient interactions

AbstractPII proteins constitute a widespread signal transduction superfamily in the prokaryotic world. The canonical PII signal proteins sense metabolic state of the cells by binding the metabolite molecules ATP, ADP and 2-oxoglutarate. Depending on bound effector molecule, PII proteins interact with and modulate the activity of multiple target proteins. To investigate the complexity of interactions of PII with target proteins, analytical methods that do not disrupt the native cellular context are required. To this purpose, split luciferase proteins have been used to develop a novel complementation reporter called NanoLuc Binary Technology (NanoBiT). The luciferase NanoLuc is divided in two subunits: a 18 kDa polypeptide termed “Large BiT” and a 1.3 kDa peptide termed “Small BiT”, which only weakly associate. When fused to proteins of interest, they reconstitute an active luciferase when the proteins of interest interact. Therefore, we set out to develop a new NanoBiT sensor based on the interaction of PII protein from Synechocystis sp. PCC6803 with PII-interacting protein X (PipX) and N-acetyl-L-glutamate kinase (NAGK). The novel NanoBiT sensor showed unprecedented sensitivity, which made it possible to detect even weak and transient interactions between PII variants and their interacting partners, thereby shedding new light in PII signalling processes.

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Tuning the in vitro sensing and signaling properties of cyanobacterial PII protein by mutation of key residues

Scientific Reports ◽

10.1038/s41598-019-55495-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Khaled A. Selim ◽

Michael Haffner ◽

Björn Watzer ◽

Karl Forchhammer

Keyword(s):

Salt Bridge ◽

Energy Status ◽

Interacting Protein ◽

Pii Protein ◽

Critical Residue ◽

Key Enzyme ◽

Domains Of Life ◽

Pii Proteins ◽

Signal Transduction Proteins

AbstractPII proteins comprise an ancient superfamily of signal transduction proteins, widely distributed among all domains of life. In general, PII proteins measure and integrate the current carbon/nitrogen/energy status of the cell through interdependent binding of ATP, ADP and 2-oxogluterate. In response to effector molecule binding, PII proteins interact with various PII-receptors to tune central carbon- and nitrogen metabolism. In cyanobacteria, PII regulates, among others, the key enzyme for nitrogen-storage, N-acetyl-glutamate kinase (NAGK), and the co-activator of the global nitrogen-trascription factor NtcA, the PII-interacting protein-X (PipX). One of the remarkable PII variants from Synechococcus elongatus PCC 7942 that yielded mechanistic insights in PII-NAGK interaction, is the NAGK-superactivating variant I86N. Here we studied its interaction with PipX. Another critical residue is Lys58, forming a salt-bridge with 2-oxoglutarate in a PII-ATP-2-oxoglutarate complex. Here, we show that Lys58 of PII protein is a key residue for mediating PII interactions. The K58N mutation not only causes the loss of 2-oxogluterate binding but also strongly impairs binding of ADP, NAGK and PipX. Remarkably, the exchange of the nearby Leu56 to Lys in the K58N variant partially compensates for the loss of K58. This study demonstrates the potential of creating custom tailored PII variants to modulate metabolism.

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Phosphorylation of KLC1 modifies interaction with JIP1 and abolishes the enhanced fast velocity of APP transport by kinesin-1

Molecular Biology of the Cell ◽

10.1091/mbc.e17-05-0303 ◽

2017 ◽

Vol 28 (26) ◽

pp. 3857-3869 ◽

Cited By ~ 4

Author(s):

Kyoko Chiba ◽

Ko-yi Chien ◽

Yuriko Sobu ◽

Saori Hata ◽

Shun Kato ◽

...

Keyword(s):

Coiled Coil ◽

Amyloid Β ◽

Tetratricopeptide Repeat ◽

Amyloid Β Protein ◽

The Novel ◽

Anterograde Transport ◽

Interacting Protein ◽

Protein Precursor ◽

Kinesin Light Chain ◽

Β Protein

In neurons, amyloid β-protein precursor (APP) is transported by binding to kinesin-1, mediated by JNK-interacting protein 1b (JIP1b), which generates the enhanced fast velocity (EFV) and efficient high frequency (EHF) of APP anterograde transport. Previously, we showed that EFV requires conventional interaction between the JIP1b C-terminal region and the kinesin light chain 1 (KLC1) tetratricopeptide repeat, whereas EHF requires a novel interaction between the central region of JIP1b and the coiled-coil domain of KLC1. We found that phosphorylatable Thr466 of KLC1 regulates the conventional interaction with JIP1b. Substitution of Glu for Thr466 abolished this interaction and EFV, but did not impair the novel interaction responsible for EHF. Phosphorylation of KLC1 at Thr466 increased in aged brains, and JIP1 binding to kinesin-1 decreased, suggesting that APP transport is impaired by aging. We conclude that phosphorylation of KLC1 at Thr466 regulates the velocity of transport of APP by kinesin-1 by modulating its interaction with JIP1b.

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Structure and Thermodynamics of Effector Molecule Binding to the Nitrogen Signal Transduction PII Protein GlnZ from Azospirillum brasilense

Journal of Molecular Biology ◽

10.1016/j.jmb.2014.05.008 ◽

2014 ◽

Vol 426 (15) ◽

pp. 2783-2799 ◽

Cited By ~ 12

Author(s):

Daphné Truan ◽

Saša Bjelić ◽

Xiao-Dan Li ◽

Fritz K. Winkler

Keyword(s):

Signal Transduction ◽

Azospirillum Brasilense ◽

Effector Molecule ◽

Pii Protein ◽

Nitrogen Signal Transduction

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Dengue virus requires apoptosis linked gene-2-interacting protein X (ALIX) for viral propagation

Virus Research ◽

10.1016/j.virusres.2018.12.015 ◽

2019 ◽

Vol 261 ◽

pp. 65-71 ◽

Cited By ~ 4

Author(s):

Chutima Thepparit ◽

Sarawut Khongwichit ◽

Kunjimas Ketsuwan ◽

Sirikwan Libsittikul ◽

Prasert Auewarakul ◽

...

Keyword(s):

Dengue Virus ◽

Interacting Protein ◽

Linked Gene ◽

Viral Propagation ◽

Protein X

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Phosphorylation of Calcineurin B-like (CBL) Calcium Sensor Proteins by Their CBL-interacting Protein Kinases (CIPKs) Is Required for Full Activity of CBL-CIPK Complexes toward Their Target Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m111.279331 ◽

2012 ◽

Vol 287 (11) ◽

pp. 7956-7968 ◽

Cited By ~ 101

Author(s):

Kenji Hashimoto ◽

Christian Eckert ◽

Uta Anschütz ◽

Martin Scholz ◽

Katrin Held ◽

...

Keyword(s):

Protein Kinases ◽

Calcium Sensor ◽

Interacting Protein ◽

Target Proteins ◽

Full Activity ◽

Calcineurin B ◽

Sensor Proteins ◽

Calcium Sensor Proteins

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The ESCRT pathway and HIV-1 budding

Biochemical Society Transactions ◽

10.1042/bst0370181 ◽

2009 ◽

Vol 37 (1) ◽

pp. 181-184 ◽

Cited By ~ 75

Author(s):

Yoshiko Usami ◽

Sergei Popov ◽

Elena Popova ◽

Michio Inoue ◽

Winfried Weissenhorn ◽

...

Keyword(s):

Susceptibility Gene ◽

Multivesicular Body ◽

Intramolecular Interactions ◽

Transport Pathway ◽

Interacting Protein ◽

Linked Gene ◽

Endosomal Sorting ◽

Binding Partner ◽

Protein X ◽

Hiv 1

HIV-1 Gag engages components of the ESCRT (endosomal sorting complex required for transport) pathway via so-called L (late-assembly) domains to promote virus budding. Specifically, the PTAP (Pro-Thr-Ala-Pro)-type primary L domain of HIV-1 recruits ESCRT-I by binding to Tsg101 (tumour susceptibility gene 101), and an auxiliary LYPXnL (Leu-Tyr-Pro-Xaan-Leu)-type L domain recruits the ESCRT-III-binding partner Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X]. The structurally related CHMPs (charged multivesicular body proteins), which form ESCRT-III, are kept in an inactive state through intramolecular interactions, and become potent inhibitors of HIV-1 budding upon removal of an autoinhibitory region. In the absence of the primary L domain, HIV-1 budding is strongly impaired, but can be efficiently rescued through the overexpression of Alix. This effect of Alix depends on its ability to interact with CHMP4, suggesting that it is the recruitment of CHMPs that ultimately drives virus release. Surprisingly, HIV-1 budding defects can also be efficiently corrected by overexpressing Nedd (neural-precursor-cell-expressed developmentally down-regulated) 4-2s, a member of a family of ubiquitin ligases previously implicated in the function of PPXY (Pro-Pro-Xaa-Tyr)-type L domains, which are absent from HIV-1. At least under certain circumstances, Nedd4-2s stimulates the activity of PTAP-type L domains, raising the possibility that the ubiquitin ligase regulates the activity of ESCRT-I.

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Quantitative analysis of APP axonal transport in neurons: role of JIP1 in enhanced APP anterograde transport

Molecular Biology of the Cell ◽

10.1091/mbc.e14-06-1111 ◽

2014 ◽

Vol 25 (22) ◽

pp. 3569-3580 ◽

Cited By ~ 35

Author(s):

Kyoko Chiba ◽

Masahiko Araseki ◽

Keisuke Nozawa ◽

Keiko Furukori ◽

Yoichi Araki ◽

...

Keyword(s):

Quantitative Analysis ◽

Axonal Transport ◽

High Frequency ◽

Coiled Coil ◽

Tetratricopeptide Repeat ◽

The Novel ◽

Anterograde Transport ◽

Interacting Protein ◽

A Site

Alzheimer's β-amyloid precursor protein (APP) associates with kinesin-1 via JNK-interacting protein 1 (JIP1); however, the role of JIP1 in APP transport by kinesin-1 in neurons remains unclear. We performed a quantitative analysis to understand the role of JIP1 in APP axonal transport. In JIP1-deficient neurons, we find that both the fast velocity (∼2.7 μm/s) and high frequency (66%) of anterograde transport of APP cargo are impaired to a reduced velocity (∼1.83 μm/s) and a lower frequency (45%). We identified two novel elements linked to JIP1 function, located in the central region of JIP1b, that interact with the coiled-coil domain of kinesin light chain 1 (KLC1), in addition to the conventional interaction of the JIP1b 11–amino acid C-terminal (C11) region with the tetratricopeptide repeat of KLC1. High frequency of APP anterograde transport is dependent on one of the novel elements in JIP1b. Fast velocity of APP cargo transport requires the C11 domain, which is regulated by the second novel region of JIP1b. Furthermore, efficient APP axonal transport is not influenced by phosphorylation of APP at Thr-668, a site known to be phosphorylated by JNK. Our quantitative analysis indicates that enhanced fast-velocity and efficient high-frequency APP anterograde transport observed in neurons are mediated by novel roles of JIP1b.

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