N-acetyl-D-glucosamine kinase binds dynein light chain roadblock 1 and promotes protein aggregate clearance

Abstract

Emerging evidence indicates that neurodegenerative diseases (NDs) result from a failure to clear toxic protein aggregates rather than from their generation. We previously showed N-acetylglucosamine kinase (NAGK) promotes dynein functionality and suggested this might promote aggregate removal and effectively address proteinopathies. Here, we report NAGK interacts with dynein light chain roadblock type 1 (DYNLRB1) and efficiently suppresses mutant huntingtin (mHtt) (Q74) and α-synuclein (α-syn) A53T aggregation in mouse brain cells. A kinase-inactive NAGK D107A also efficiently cleared Q74 aggregates. Yeast two-hybrid selection and in silico protein-protein docking analysis showed the small domain of NAGK (NAGK-D S) binds to the C-terminal of DYNLRB1. Furthermore, a small peptide derived from NAGK-D S interfered with Q74 clearance. We propose binding of NAGK-D S to DYNLRB1 'pushes up' the tail of dynein light chain and confers momentum for inactive phi-to active open-dynein transition.

N-Acetyl-d-Glucosamine Kinase Interacts with NudC and Lis1 in Dynein Motor Complex and Promotes Cell Migration

“…The pull-down assay followed by immunoblotting with respective antibodies showed that exogenous NudC interacted with endogenous NAGK ( Figure 1 B). As both NAGK and NudC were reported to interact with dynein by previous studies [ 16 , 28 ], we also checked the presence of dynein components. Interestingly, both DYNLRB1 and light intermediate chain 1 (DYNC1L1) were found to interact with exogenous NudC.…”

“…Interestingly, this DYNLRB1 interaction domain is also the NAGK binding site, as indicated in our previous report [ 29 ]. Furthermore, our molecular modeling and cell biological studies showed that residues 59 to 69 in the small domain of NAGK are major hotspots for the NAGK–DYNLRB1 interaction [ 28 ]. Therefore, there seems to be binding competition between NAGK and IC for DYNLRB1.…”

“…The Institutional Animal Care and Use Committee of the College of Medicine, Dongguk University, approved all animal experiments (Approval no: IUCAC-2018-06, March 03, 2018) beforehand [ 28 ]. On the 13th day of pregnancy, time-pregnant rats (Sprague–Dawley) were ordered and housed in a pathogenic free standard laboratory condition (18–25 °C, 45–65% humidity, light/dark cycle of 12/12 h) and maintained with free access to food and water ad libitum.…”

“…Findings from our laboratory and other research group suggest a critical role of NAGK in the early development process [ 23 , 24 , 25 ]. NAGK is expressed at high levels in neurons but at low levels in astrocytes and oligodendrocytes [ 26 , 27 ], and it plays essential roles during the development of dendrites [ 26 , 27 ] and axons [ 25 ], in protein aggregates clearance [ 28 ], and during cell division [ 24 ]. NAGK interacted with dynein light chain roadblock 1 (DYNLRB1) during yeast two-hybrid selection [ 29 ].…”

“…Furthermore, immunostaining shows the two proteins colocalize in neuronal and non-neuronal cells in culture and brain tissue, and proximity ligation assays (PLAs) [ 29 ]. Interestingly, interruption of the NAGK–dynein interaction by the overexpression of NAGK small domain [ 26 ] or the introduction of a peptide derived from the C-terminal DYNLRB1 nullified the effects of NAGK on axonal [ 25 ] and dendritic outgrowths [ 29 ] as well as aggregates clearance [ 28 ]. Moreover, a kinase-deficient mutant NAGK also upregulated dendritic arborization [ 26 ], which confirmed that non-canonical, i.e., kinase-independent, and structural functions of NAGK promote multiple facets of dynein function.…”