Biochemical and Biophysical Research Communications
Biochemical insight into pseudouridine synthase 7 (PUS7) as a novel interactor of sirtuin, SIRT1
Introduction
Sirtuins are an NAD+-dependent class III histone deacetylases that catalyze the deacetylation of acetyl-lysine residues in multiple histone- and non-histone protein substrates. The seven members of the mammalian sirtuin family (SIRT1-7) have a conserved catalytic core flanked by additional N- and C-terminal extensions of different length [1] and are divergently distributed in cells to execute the attributed biological functions. SIRT1 is a predominantly nuclear protein which regulates several important biological processes, ranging from gene transcription and metabolism to stress tolerance, inflammatory response, and cellular senescence [2]. Although the histones are preliminary substrates for SIRT1, non-histone proteins such as transcription factors and cofactors (p53 [3], p65/NF-κB [4], FOXOs [5], and PGC-1α [6]), as well as nuclear receptors (PPAR c) [7], androgen receptor [8], and estrogen receptor [9]) have been reported as SIRT1 deacetylation substrates. Owing to these biological functions, SIRT1 modulators have assumed the immense therapeutic potential for several chronic diseases such as type 2 diabetes, cardiovascular disease, cancer, and neurodegenerative diseases [10].
SIRT1 deacetylase activity is regulated by cellular NAD+ levels that fluctuate in response to changing rates of NAD+ biosynthesis and consumption. Various posttranslational modifications on SIRT1, including phosphorylation, sumoylation, and ubiquitination are reported to regulate its localization, substrate specificity, and enzymatic activity [11]. Autoregulatory mechanisms are also exerted on SIRT1 activity by domain, which is essential for SIRT1 activity (ESA) within its C-terminal [12] and N-terminal domains [13,14]. Several protein regulators of SIRT1 have been established as positive regulators including AROS (active regulator of SIRT1) and Necdin [14,15] and an inhibitory protein DBC1 (deleted in breast cancer 1) [16,17]. Thus, protein-protein interactions play a central role in regulating SIRT1 mediated deacetylation as well as its localization and substrate specificity. A high confidence SIRT1 interactome studied using proteomic approach has recently been reported to have additional SIRT1 interacting proteins like USP22, CCDC99, and pseudouridine synthase 7 (PUS7) [18].
Pseudouridylation (Ψ) is isomerization of uridine to 5-ribosyluracil and is the most abundant and widespread type of RNA epigenetic modification in living organisms. RNA pseudouridylation increases RNA structure stability by altering its structure [19], increase base stacking [20], improve base-pairing [21], and rigidified sugar-phosphate backbone [22]. In human, pseudouridylation is catalyzed by 13 pseudouridine synthases (PUSs), which act in an RNA-independent manner or are guided to their targets via small nucleolar RNAs (snoRNAs) [23]. The stand-alone PUS enzymes are classified into six families (TruA, TruB, TruD, RsuA, RluA, and Pus10) [24]. PUS7 is the only member of the TruD family which modifies tRNAs, specifically at the position 13 in cytoplasmic tRNA, the position 35 in pre-tRNATyr and numerous nucleotides in mRNA. Besides, PUS7 is the only pseudouridine synthase to display a consensus sequence (UGUAR) for substrate recognition [25]. The heat shock studies in yeast showed re-localization of PUS7 from the nucleus to the cytoplasm and is correlated with an increase in mRNA pseudouridylation [26]. In embryonic stem cells, PUS7 inactivation is reported to impair the hematopoietic stem cell commitment, which may contribute to human myeloid malignancies [27]. Recent reports have implicated loss of PUS7 pseudouridylation results in intellectual disability with speech delay, microcephaly, short stature, and aggressive behavior [28,29]. All the findings make PUS7 a highly interesting enzyme with the potential to strongly influence cellular gene expression and development. However, the mechanisms governing PUS7 regulation remain unclear.
Though the previous proteomic study suggested the interaction of SIRT1 with PUS7 [18], it remains unclear how the interaction occurs between them. In this study, we investigated the mode of SIRT1-PUS7 interaction by in silico and biochemical approaches. Moreover, the PUS7 interaction study was also performed with SIRT2. The above studies have successfully revealed the domains of respective proteins contributing significantly to SIRT1/SIRT2 and PUS7 interaction.
Section snippets
Molecular docking
SIRT1-PUS7 interaction was investigated in silico by using the webserver GrammX [30] and PatchDock [31] to examine models for SIRT1-PUS7 complex. PatchDock [32] is a geometry-based molecular docking algorithm which finds docking transformations yielding good molecular shape complementarity to induce wide surface areas and minimize the amounts of steric clashes. GRAMM-X employs smoothed potentials, refinement stage and knowledge-based scoring in extension to the original GRAMM Fast Fourier
In-silico prediction of SIRT1-PUS7 interaction
The SIRT1-PUS7 interface was predicted by molecular docking studies using PatchDock and GrammX. The visual assessment of the top 5 ranked models in CONSRANK showed that the N-terminal region (1–255) of PUS7 forms the interface in SIRT1-PUS7 complex most consistently. The model 8 from PatchDock (Fig. 1A and B) was chosen as the best model by highest binding affinity (−29.4 KCal/mol) and Kd value (2.6 × 10−22) as analyzed by Prodigy server (Table S1) [35]. The SIRT1-PUS7 interface involves six
Author contributions
SD and BP designed the study. SD, SU, SP and BP designed the experiments. SD, PD and SU conducted the experiments. All contributed to analysis and interpretation of the data. All wrote and edited the manuscript.
Conflicts of interest
The authors declare no competing interests.
Acknowledgements
We thank Prof. Arun Malhotra (University of Miami) for the kind gift of PUS7 clone. Manjula Ramu is acknowledged for her initial work on sirtuins. This study was supported by Science and Engineering Research Board National Post-Doctoral Fellowship (N-PDF) (Grant Number: PDF/2016/002082) for SD. SU is grateful to the Council of Scientific and Industrial Research, India, Government of India, for SRF fellowship (09/490(0103)/2019). BP is grateful to the SERB-Department of Science and Technology
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