Shuja S Malik | King Abdullah International Medical Research Center (original) (raw)

Papers by Shuja S Malik

Current Issues in Molecular Biology

Placenta-derived stem cells (PDSCs), due to unique traits such as mesenchymal and embryonic chara... more Placenta-derived stem cells (PDSCs), due to unique traits such as mesenchymal and embryonic characteristics and the absence of ethical constraints, are in a clinically and therapeutically advantageous position. To aid in stemness maintenance, counter pathophysiological stresses, and withstand post-differentiation challenges, stem cells require elevated protein synthesis and consequently augmented proteostasis. Stem cells exhibit source-specific proteostasis traits, making it imperative to study them individually from different sources. These studies have implications for understanding stem cell biology and exploitation in the augmentation of therapeutic applications. Here, we aim to identify the primary determinants of proteotoxic stress response in PDSCs. We generated heat-induced dose-responsive proteotoxic stress models of three stem cell types: placental origin cells, the placenta-derived mesenchymal stem cells (pMSCs), maternal origin cells, the decidua parietalis mesenchymal s...

International Journal of Molecular Sciences, 2021

Adrenergic receptor β3 (ADRβ3) is a member of the rhodopsin-like G protein-coupled receptor famil... more Adrenergic receptor β3 (ADRβ3) is a member of the rhodopsin-like G protein-coupled receptor family. The binding of the ligand to ADRβ3 activates adenylate cyclase and increases cAMP in the cells. ADRβ3 is highly expressed in white and brown adipocytes and controls key regulatory pathways of lipid metabolism. Trp64Arg (W64R) polymorphism in the ADRβ3 is associated with the early development of type 2 diabetes mellitus, lower resting metabolic rate, abdominal obesity, and insulin resistance. It is unclear how the substitution of W64R affects the functioning of ADRβ3. This study was initiated to functionally characterize this obesity-linked variant of ADRβ3. We evaluated in detail the expression, subcellular distribution, and post-activation behavior of the WT and W64R ADRβ3 using single cell quantitative fluorescence microscopy. When expressed in HEK 293 cells, ADRβ3 shows a typical distribution displayed by other GPCRs with a predominant localization at the cell surface. Unlike adren...

Current Molecular Medicine, 2020

Background:G protein-coupled receptors (GPCRs) represent the largest family of surface proteins a... more Background:G protein-coupled receptors (GPCRs) represent the largest family of surface proteins and are involved in the regulation of key physiological processes. GPCRs are characterized by seven transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. Cellular response of these receptors to their ligands is largely determined by their surface expression and postactivation behavior including expression, desensitization and resensitization.Objective:To develop a quantitative fluorescence Microscopy assay to study β2- Adrenergic receptor expression and desensitization.Method:β2-Adrenergic receptor cDNA was engineered to put an HA tag at the extracellular N-terminus and GFP Tag at the intracellular C-terminus. GFP fluorescence serves as a measure of total cellular expression; whereas staining with CY3 conjugated anti-HA antibodies without permeabilizing the cells represents the surface expression of β2-AR. The images are quantified and amount of CY3 (surface)...

Biomedical reports, 2018

The 'Therapeutics discovery: From bench to first in-human trials' conference, held at the... more The 'Therapeutics discovery: From bench to first in-human trials' conference, held at the King Abdullah International Medical Research Center (KAIMRC), Ministry of National Guard Health Affairs (MNGHA), Kingdom of Saudi Arabia (KSA) from October 10-12, 2017, provided a unique opportunity for experts worldwide to discuss advances in drug discovery and development, focusing on phase I clinical trials. It was the first event of its kind to be hosted at the new research center, which was constructed to boost drug discovery and development in the KSA in collaboration with institutions, such as the Academic Drug Discovery Consortium in the United States of America (USA), Structural Genomics Consortium of the University of Oxford in the United Kingdom (UK), and Institute of Materia Medica of the Chinese Academy of Medical Sciences in China. The program was divided into two parts. A pre-symposium day took place on October 10, during which courses were conducted on clinical trials, p...

Current Drug Targets, 2017

Background & Objective: Thioredoxin-interacting protein (TXNIP) also known as thioredoxin binding... more Background & Objective: Thioredoxin-interacting protein (TXNIP) also known as thioredoxin binding protein-2 is a ubiquitously expressed protein that interacts and negatively regulates expression and function of Thioredoxin (TXN). Over the last few years, TXNIP has attracted considerable attention due to its wide-ranging functions impacting several aspects of energy metabolism. TXNIP acts as an important regulator of glucose and lipid metabolism through pleiotropic actions including regulation of β-cell function, hepatic glucose production, peripheral glucose uptake, adipogenesis, and substrate utilization. Overexpression of TXNIP in animal models has been shown to induce apoptosis of pancreatic β-cells, reduce insulin sensitivity in peripheral tissues like skeletal muscle and adipose, and decrease energy expenditure. On the contrary, TXNIP deficient animals are protected from diet induced insulin resistance and type 2 diabetes. Summary: Consequently, targeting TXNIP is thought to offer novel therapeutic opportunity and TXNIP inhibitors have the potential to become a powerful therapeutic tool for the treatment of diabetes mellitus. Here we summarize the current state of our understanding of TXNIP biology, highlight its role in metabolic regulation and raise critical questions that could help future research to exploit TXNIP as a therapeutic target.

Nucleic Acids Research, 2016

Thymine DNA Glycosylase (TDG) is a base excision repair enzyme functioning in DNA repair and epig... more Thymine DNA Glycosylase (TDG) is a base excision repair enzyme functioning in DNA repair and epigenetic regulation. TDG removes thymine from mutagenic G•T mispairs arising from deamination of 5-methylcytosine (mC), and it processes other deamination-derived lesions including uracil (U). Essential for DNA demethylation, TDG excises 5formylcytosine and 5-carboxylcytosine, derivatives of mC generated by Tet (ten-eleven translocation) enzymes. Here, we report structural and functional studies of TDG 82-308 , a new construct containing 29 more N-terminal residues than TDG 111-308 , the construct used for previous structures of DNAbound TDG. Crystal structures and NMR experiments demonstrate that most of these N-terminal residues are disordered, for substrate-or productbound TDG 82-308. Nevertheless, G•T substrate affinity and glycosylase activity of TDG 82-308 greatly exceeds that of TDG 111-308 and is equivalent to full-length TDG. We report the first high-resolution structures of TDG in an enzyme-substrate complex, for G•U bound to TDG 82-308 (1.54Å) and TDG 111-308 (1.71Å), revealing new enzyme-substrate contacts, direct and watermediated. We also report a structure of the TDG 82-308 product complex (1.70Å). TDG 82-308 forms unique enzyme-DNA interactions, supporting its value for structure-function studies. The results advance understanding of how TDG recognizes and removes modified bases from DNA, particularly those resulting from deamination.

Nucleic Acids Research, 2015

Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and e... more Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G•T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5carboxylcytosine (caC), which are generated from mC by Tet (ten-eleven translocation) enzymes. Using improved crystallization conditions, we solved highresolution (up to 1.45Å) structures of TDG enzymeproduct complexes generated from substrates including G•U, G•T, G•hmU, G•fC and G•caC. The structures reveal many new features, including key watermediated enzyme-substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNAfree TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a K d >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme-product complex.

Biochemistry, Jan 15, 2014

The regulation of chromatin structure is controlled by a family of molecular motors called chroma... more The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilib...

Biochemistry, 2014

The chromatin remodeler ISWI is capable of repositioning clusters of nucleosomes to create well-o... more The chromatin remodeler ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. Using standard electrophoresis assays, we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing a different length of DNA symmetrically flanking the initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a spectrophotometric assay for nucleosome repositioning, we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed at various ISWI concentrations revealed that a monomeric ISWI is sufficient to obtain the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning, suggesting that DNA translocation by ISWI is not energetically rate-limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction.

Proteins: Structure, Function, and Bioinformatics, 2013

Home > The crystal structure of human quinolinic acid phosphoribosyltransferase in complex with i... more Home > The crystal structure of human quinolinic acid phosphoribosyltransferase in complex with its inhibitor phthalic acid. The crystal structure of human quinolinic acid phosphoribosyltransferase in complex with its inhibitor phthalic acid.

Journal of Molecular Biology, 2010

The NS3 helicase (NS3h) of hepatitis C virus (HCV) is a 3′ to 5′ SF2 RNA and DNA helicase that is... more The NS3 helicase (NS3h) of hepatitis C virus (HCV) is a 3′ to 5′ SF2 RNA and DNA helicase that is essential for the replication of HCV. We have examined the kinetic mechanism of translocation of NS3h along single-stranded nucleic acid with bases rU, dU and dT and have found that the macroscopic rate of translocation is dependent upon both the base and sugar moieties of the nucleic acid, with approximate macroscopic translocation rates of 3 nt/s (oligo-dT), 35 nt/s (oligo-dU), and 42 nt/s (oligo-rU), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein to the respective substrates such that weaker affinity corresponded to faster translocation. The values of K 0.5 for NS3h translocation at a saturating ATP concentration are: (3.3 ± 0.4) μM nucleotide (poly-dT), (27 ± 2) μM nucleotide (poly-dU), and (36 ± 2) μM nucleotide (poly-rU). Furthermore, the results of isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS° are the principal source of the differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometry for NS3h translocation was identical for all three substrates, ~0.5 ATP molecules consumed per nucleotide translocated. This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates.

DNA Repair, 2013

MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion ... more MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G°) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugarphosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5' to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9-Rad1-Hus1 (9-1-1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein-DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.

Biophysical Journal, 2010

Biochemistry, 2011

RSC, Remodel the Structure of Chromatin, is an essential chromatin remodeler of Saccharomyces cer... more RSC, Remodel the Structure of Chromatin, is an essential chromatin remodeler of Saccharomyces cerevisiae that has been shown to have DNA translocase properties. We studied the DNA binding properties of a 'trimeric minimal RSC' (RSCt) of the RSC chromatin remodeling complex and the effect of nucleotides on this interaction using fluorescence anisotropy. RSCt binds to 20 bp fluorescein labeled double stranded DNA with a K d of approximately 100 nM. The affinity of RSCt for DNA is reduced in the presence of AMP-PNP and ADP in a concentration dependent manner with the addition of AMP-PNP having the more pronounced effect. These differences in the magnitude at which the binding of ADP and AMP-PNP affect the affinity of DNA binding by RSCt suggests that the physical movement of the enzyme along DNA begins between the binding of ATP and its subsequent hydrolysis. Furthermore, the fact that the highest affinity for DNA binding by RSCt occurs in the absence of bound nucleotide offers a mechanistic explanation for the low apparent processivity of DNA translocation by the enzyme.

Biochemical and Biophysical Research Communications, 2008

The Mycobacterium tuberculosis UsfX protein is an anti-sigma factor which regulates its cognate s... more The Mycobacterium tuberculosis UsfX protein is an anti-sigma factor which regulates its cognate sigma factor SigF. UsfX shares low sequence homology with other anti-sigma factors making it difficult to identify the nucleotide binding site and characterize its properties. We have identified that the NTP binding site occurs close to Trp106 and the area around the nucleotide binding site is predominantly negatively charged. UsfX binds to a variety of nucleotides unlike other reported anti-sigma factors and exhibits an unusual dual NTPase activity. In silico computational experiments have identified a XGSFS motif close to the nucleotide binding site for metal ion binding. This motif is analogous to the DXSXS motif reported earlier in the human integrin CR3 protein superfamily. Overall, the experiments suggest that the M. tuberculosis UsfX represents a distinct anti-sigma factor family with a novel nucleotide binding motif.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2009

M. tuberculosis employs an exquisite cascade consisting of the cognate anti-sigma factor UsfX and... more M. tuberculosis employs an exquisite cascade consisting of the cognate anti-sigma factor UsfX and anti-anti sigma factors RsfA and RsfB to regulate the functions of the alternate sigma factor SigF. We have purified these proteins to characterize their molecular properties and interactions with UsfX. UsfX forms a stable complex with SigF that could be purified only after co-expressing the proteins in E.coli. Formation of the complex is nucleotide independent and apparently requires unknown in vivo factors. Fluorescence spectroscopy experiments suggest that the nucleotide binding sites of UsfX are distal to the protein-protein interaction interface. RsfA is a novel anti-anti sigma factor whose binding to UsfX is triggered by the reduction of an intrachain disulphide bond between Cys73-Cys109. The reduction is accompanied by an increase in the hydrodynamic radius of the protein. The UsfX-RsfA complex exhibits a novel stoichiometry of 2:1 compared to the 2:2 stoichiometry reported for other anti-anti-sigma factors. The role of the disulphide bond in complex formation was explored using molecular dynamics simulations. These studies support specific conformational changes that occur upon reduction of the Cys73-Cys109 bond of RsfA. This leads to a rearrangement that increases the interactions of a conserved His107 of UsfX with Cys109 of RsfA.

Archives of Biochemistry and Biophysics, 2013

ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation... more ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning.

COVID-19 pandemic in first seven months has led to more than 15 million confirmed infected cases ... more COVID-19 pandemic in first seven months has led to more than 15 million confirmed infected cases and 600,000 deaths. SARS-CoV-2, the causative agent for COVID-19 has proved a great challenge for its ability to spread in asymptomatic stages and a diverse disease spectrum it has generated. This has created a challenge of unimaginable magnitude not only affecting human health and life but also potentially generating a long-lasting socioeconomic impact. Both medical sciences and biomedical research have also been challenged consequently leading to a large number of clinical trials and vaccine initiatives. While known proteins of pathobiological importance are targets for these therapeutic approaches, it is imperative to explore other factors of viral significance. Accessory proteins are one such trait that have diverse roles in coronavirus pathobiology. Here we analyze certain genomic characteristics of SARS-CoV-2 accessory protein ORF8, predict upon its protein features and review curr...