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Dive into the research topics where Shamaruh Mirza is active.

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Featured researches published by Shamaruh Mirza.


Journal of Biological Chemistry | 2009

A New Isoform of Interleukin-3 Receptor α with Novel Differentiation Activity and High Affinity Binding Mode

Jinglong Chen; Jane Olsen; Sally C. Ford; Shamaruh Mirza; Andrew A. Walker; James M. Murphy; Ian G. Young

Interleukin-3 (IL-3) promotes both self-renewal and differentiation of early multipotential progenitors and is involved in inducible hematopoiesis in response to infections. Here we report new insights into these processes with the identification of a new isoform (SP2) of IL-3 receptor α (IL-3Rα), present in mouse and human hematopoietic cells, which lacks domain 1 of the full-length receptor (SP1). Binding assays with βIL-3 mutants showed that mouse SP2 uses a different high affinity binding mode to SP1, although both mouse and human SP2 and SP1 can stimulate IL-3-dependent growth. In IL-3-dependent differentiation models, human SP2 and SP1 gave differential effects on lineage commitment or self-renewal dependent on the cellular context, suggesting that different modes of ectodomain binding may modulate intracellular signaling. In a multipotential factor dependent cell-Paterson mix, the transcription factors C/EBPα and PU.1 and microRNAs miRNA-15a, -223, and -181a were up-regulated in cells undergoing SP2-supported differentiation compared with SP1-supported self-renewal. Similarly in M1 cells, SP2 promoted differentiation compared with SP1 and gave up-regulation of PU.1 and miRNA-155 and -223. These findings suggest that IL-3-promoted lineage commitment uses similar mechanisms to those of steady-state hematopoiesis. Both the SP1 and SP2 isoforms activated the Jak2/STAT5, Akt, and Erk1/2 signaling pathways in M1 cells, although the activation was more prolonged for the SP2 isoform.


Biochemical Journal | 2010

The Ig-like domain of human GM-CSF receptor α plays a critical role in cytokine binding and receptor activation

Shamaruh Mirza; Andrew A. Walker; Jinglong Chen; James M. Murphy; Ian G. Young

GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific alpha receptor (GM-CSFRalpha) and the shared hbetac (human common beta-subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRalpha in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of hbetac. To locate the specific residues in the Ig-like domain of GM-CSFRalpha involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Ralpha1 (interleukin-13 receptor alpha1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val51 and Cys60 as having critical roles in binding to the alpha receptor, with Arg54 and Leu55 also being important. High-affinity binding in the presence of hbetac was strongly affected by mutation of Cys60 and was also reduced by mutation of Val51, Arg54 and Leu55. Of the four key residues, growth signalling was most severely affected by mutation of Cys60. The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys60, of GM-CSFRalpha in the binding of GM-CSF and subsequent activation of cellular signalling.


Clinical and Experimental Pharmacology and Physiology | 2012

Proteins within the intracellular calcium store determine cardiac RyR channel activity and cardiac output

Angela F. Dulhunty; Elize Wium; Linwei Li; Amy D. Hanna; Shamaruh Mirza; Sadik Talukder; Nuur A.A. Ghazali; Nicole A. Beard

The contractile function of the heart requires the release of Ca2+ from intracellular Ca2+ stores in the sarcoplasmic reticulum (SR) of cardiac muscle cells. The efficacy of Ca2+ release depends on the amount of Ca2+ loaded into the Ca2+ store and the way in which this ‘Ca2+ load’ influences the activity of the cardiac ryanodine receptor Ca2+ release channel (RyR2). The effects of the Ca2+ load on Ca2+ release through RyR2 are facilitated by: (i) the sensitivity of RyR2 itself to luminal Ca2+ concentrations; and (ii) interactions between the cardiac Ca2+‐binding protein calsequestrin (CSQ) 2 and RyR2, transmitted through the ‘anchoring’ proteins junctin and/or triadin. Mutations in RyR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden cardiac death. The tachycardia is associated with changes in the sensitivity of RyR2 to luminal Ca2+. Triadin‐, junctin‐ or CSQ‐null animals survive, but their longevity and ability to tolerate stress is compromised. These studies reveal the importance of the proteins in normal muscle function, but do not reveal the molecular nature of their functional interactions, which must be defined before changes in the proteins leading to CPVT and heart disease can be understood. Herein, we discuss known interactions between the RyR, triadin, junctin and CSQ with emphasis on the cardiac isoforms of the proteins. Where there is little known about the cardiac isoforms, we discuss evidence from skeletal isoforms.


Channels | 2011

The elusive role of the SPRY2 domain in RyR1

Han-Shen Tae; Lan Wei; Hermia Willemse; Shamaruh Mirza; Esther M. Gallant; Philip G. Board; Robert T. Dirksen; Marco G. Casarotto; Angela F. Dulhunty

The second of three SPRY domains (SPRY2, S1085 V1208) located in the skeletal muscle ryanodine receptor (RyR1) is contained within regions of RyR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RyR1 channel gating. We examined the binding of the F loop (P1107 A1121) in SPRY2 to the ASI/basic region in RyR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling. A peptide with the F loop sequence and an antibody to the SPRY2 domain each enhanced RyR1 activity at low concentrations and inhibited at higher concentrations. A peptide containing the ASI/basic sequence bound to SPRY2 and binding decreased ~10-fold following mutation or structural disruption of the basic residues. Binding was abolished by mutation of three critical acidic F loop residues. Together these results suggest that the ASI/basic and SPRY2 domains interact in an F loop regulatory module. Although a region that includes the SPRY2 domain influences EC coupling, as does the ASI/basic region, Ca2+ release during ligand- and depolarization-induced RyR1 activation were not altered by mutation of the three critical F loop residues following expression of mutant RyR1 in RyR1-null myotubes. Therefore the electrostatic regulatory interaction between the SPRY2 F loop residues (that bind to imperatoxin A) and the ASI/basic residues of RyR1 does not influence bi-directional DHPR-RyR1 signaling during skeletal EC coupling, possibly because the interaction is interrupted by the influence of factors present in intact muscle cells.


Cytokine | 2008

Clarification of the role of N-glycans on the common β-subunit of the human IL-3, IL-5 and GM-CSF receptors and the murine IL-3 β-receptor in ligand-binding and receptor activation

James M. Murphy; Tatiana A. Soboleva; Shamaruh Mirza; Sally C. Ford; Jane Olsen; Jinglong Chen; Ian G. Young

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating individual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).


Journal of Cell Science | 2015

A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels

Linwei Li; Shamaruh Mirza; Spencer J. Richardson; Esther M. Gallant; Suzy M. Pace; Francesco Zorzato; Dan Liu; Nicole A. Beard; Angela F. Dulhunty

ABSTRACT Junctin, a non-catalytic splice variant encoded by the aspartate-&bgr;-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca2+ store where it modifies Ca2+ signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca2+ release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca2+ signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1–S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156.


Journal of Biological Chemistry | 2010

Two Modes of β-Receptor Recognition Are Mediated by Distinct Epitopes on Mouse and Human Interleukin-3

Shamaruh Mirza; Jinglong Chen; Bin Wen; Cameron L. Ewens; Jin Dai; James M. Murphy; Ian G. Young

The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its effects on target cells via receptors comprising an IL-3-specific α-subunit and common β-subunit (βc; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a β-subunit that specifically binds IL-3 (βIL-3; present in mice but not humans). We recently identified two splice variants of the α-subunit of the IL-3 receptor (IL-3Rα) that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length (“SP1” isoform) and a novel isoform (denoted “SP2”) lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3 (mIL-3) Rα isoform can direct mIL-3 binding to two distinct sites on the βIL-3 subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of βIL-3 recognition and whether the human IL-3Rα SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human βc subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu23, in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the βIL-3 and mIL-3Rα SP2 subunits, whereas an overlapping cluster was required for binding and activation of βIL-3 in the presence of mIL-3Rα SP1. Similarly, our studies of human IL-3 indicate that two different modes of βc binding are utilized in the presence of the hIL-3Rα SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes.


Journal of Biological Chemistry | 2010

The Role of Interchain Heterodisulfide Formation in Activation of the Human Common β and Mouse βIL-3 Receptors

Shamaruh Mirza; Jinglong Chen; James M. Murphy; Ian G. Young

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common β (βc) receptor is shared by the three cytokines and functions together with cytokine-specific α subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human βc (hβc) and unidentified cysteine residues in the N-terminal domains of the α receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hβc is part of the cytokine binding epitope of this receptor and that an IL-3Rα isoform lacking the N-terminal Ig-like domain (the “SP2” isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hβc and the related mouse receptor, βIL-3, could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hβc led to both a complete loss of high affinity binding with the human IL-3Rα SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, βIL-3, not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Rα SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hβc and βIL-3 in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.


Biophysical Journal | 2013

A Cytoplasmic Interaction between Junctin and RyRs with Major Consequences for RyR1 and RyR2 Activity In Vitro

Linwei Li; Shamaruh Mirza; Nicole A. Beard; Angela F. Dulhunty

Junctin is an intrinsic sarcoplasmic reticulum (SR) membrane protein in cardiac and skeletal muscle. The bulk of the protein is within the lumen of the SR where it binds to ryanodine receptor (RyR) calcium release channels and regulates Ca2+ release from the SR. We have previously reported that junctin added to the luminal solution increases RyR1 activity in lipid bilayers [1].The contributions of the luminal and cytoplasmic domains of junctin to RyR activation have not previously been explored, although it is assumed that activation depends on interactions between their luminal domains. To explore this further, we expressed junctins C-terminal domain (Cjun), synthesised a cytoplasmic N-terminal domain peptide (Njun) and isolated full length junctin (FLjun) from skeletal muscle. Consistent with previous findings [1,2], FLjun in luminal solutions activated purified RyR1 and RyR2. We predicted that luminal Cjun would similarly activate RyRs if the proteins interact only via their luminal domains. Unexpectedly, (a) luminal Cjun strongly inhibited RyR1 and RyR2 and (b) cytoplasmic Njun activated channels to a greater degree than luminal FLjun and also bound to cytoplasmic fragments of RyR1 [3]. Neither luminal Njun, nor scrambled Njun in cytoplasmic solution influenced channel activity. To explore cytoplasmic Njun and luminal Cjun effects further, we added both domains sequentially to channels. Excess activation by Njun was reduced to FLjun levels by adding Cjun, while significant inhibition by Cjun added first was reduced by Njun, it remained significantly different from the activation by FLjun. Therefore cytoplasmic interactions between junctin and RyRs determines the overall effect of junctin on channel activity.1. Wei et al. (2009) Int J Biochem Cell Biol41;2214.2. Gyorke et al. (2004) Biophys J 86:2121.3. Mirza et al. (2013) Biophysics abstract.


Biophysical Journal | 2013

Different Modes of Interaction between Junctin and Ryanodine Receptors

Shamaruh Mirza; Linwei Li; Nicole A. Beard; Angela F. Dulhunty

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Ian G. Young

Australian National University

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Jinglong Chen

Australian National University

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Angela F. Dulhunty

Australian National University

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Linwei Li

Australian National University

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Jane Olsen

Australian National University

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Andrew A. Walker

Commonwealth Scientific and Industrial Research Organisation

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Bin Wen

Australian National University

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Esther M. Gallant

Australian National University

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