Bibudhendra Sarkar , MPharm, PhD, FCIC, FRSC (UK)
The Hospital for Sick Children
Committee Member, Global Child Health
Advanced Protein Technology Centre
Senior Scientist Emeritus
Molecular Structure & Function
University of Toronto
Andrew Sass-Kortsak Award
Bibudhendra (Amu) Sarkar is an international authority on bioinorganic chemistry and its relationship to disease processes. He obtained his PhD in Biochemistry from the University of Southern California, Los Angeles, where he worked under the supervision of Paul Saltman and Bo Malmström, then a visiting professor from Sweden and later the Chairman of the Nobel Foundation Chemistry Committee in Sweden. He studied protein chemistry with Hal Dixon at the University of Cambridge, UK, and quantum biochemistry with Madame Alberte Pullman at the Université de Paris-Sorbonne, France. He joined the University of Toronto and The Hospital for Sick Children where he established his research career on metal-caused genetic diseases and the impact of heavy metals in the environment on human health. He discovered the copper-histidine treatment for Menkes disease, a devastating neurodegenerative disease in children caused by a genetic defect of copper transport. Sarkar discovered the A mino-Terminal Cu(II)-, NI(II)-binding (ATCUN) motif of proteins. His pioneering work established metalloproteomics as an effective strategy for identifying proteins whose metal-binding properties were not previously known. Since 1997 he has led an international team of scientists researching, on a volunteer basis, the major health crisis caused by multi-metal contamination of drinking water in South and South-East Asia.
Sarkar became full professor in 1978, Head of the Department of Structural Biology and Biochemistry from 1990 to 2002 and Director of the Advanced Protein Technology Centre from 1998 to 2002. He received numerous honours and awards including MRC Scholar Award of Canada; Nuffield Foundation Award of UK; member of the High Table, King’s College, University of Cambridge, UK; Visiting Professor in the Université Paris-Nord, France; Invited Speaker at the Nobel Symposium on Inorganic Biochemistry under the auspices of the Nobel Foundation in Sweden; Fellow of the Royal Society of Chemistry, UK; Fellow of IUPAC (International Union of Pure and Applied Chemistry); Fellow of the Chemical Institute of Canada; R. C. Mehrotra Award for Science, University of Delhi, India; Priaydaranjan Ray Memorial Award of the Indian Chemical Society for his outstanding contributions in Inorganic Biochemistry with special recognition for his environmental work in South Asia; Commemorative Plaque of Recognition for his work on the arsenic health crisis in Bangladesh from Faridpur Medical College and Shaheed Suhrawardy Medical College & Hospital, Dhaka, Bangladesh. In 1994, he established the International Conference on Metals and Genetics (ICMG) which is being held every three years. Sarkar has edited five books, published over 250 scientific articles, organized 22 international symposia, and he continues to serve in prominent national and international scientific and professional committees and on editorial boards. His current research focuses on genetic disorder of copper disposition and health effects of heavy metals in the environment.
The following article was published in IUBMB Life and has been reproduced with permission of the publisher Taylor & Francis Group.
How I became a biochemist by Bibudhendra Sarkar, IUBMB Life, (2003) vol. 55 no. 4-5 pp 287-289.
How I became a biochemist by Bibudhendra Sarkar
[Adobe Acrobat PDF 121 KB]
- Studies of copper-transporting ATPases
- Treatment developments for Menkes and Wilson diseases
- Intracellular disposition of copper : Role of COMMD1
- Heavy metals in the global environment
Mammalian systems are too complex to be deciphered by their genes alone. Genomic data and transcript profiling offer opportunities to identify molecular alteration in disease, but they do not specify which specific proteins interact, how these proteins occur or how long they persist in a biological system. Proteomics, the global study of proteins, encompasses protein expression and structure-function relationships both under physiological and diseased states. In defining a metalloproteome, we seek to determine the set of proteins, which have metal-binding capacity, either by virtue of being metalloproteins or having metal-binding motifs.
Our research is directed to establishing metalloproteomics, the detailed structural and functional characterization of metal-binding proteins and their structural metal-binding motifs. The establishment of the metalloproteome will provide critically important new information for understanding cellular function physiologically and in disease states arising from metal-associated cytotoxicity. We are developing a hepatic metalloproteome for copper and zinc, using hepatocyte lysates for our studies.
The metal-binding proteins are separated by immobilized affinity chromatography (IMAC) and subsequently isolated by ID and 2D gel electrophoresis followed by in-gel digestion. Mass finger printing (MS) and MS/MS measurements on the resulting peptides by both MALDI and ESI QqTOF mass spectrometry are used to determine protein sequences followed by protein database search for identification. Structural characterizations of metal-binding proteins are carried out by various spectroscopic techniques including fluorescence, CD, NMR, EPR and XAS.
2. Studies of copper-transporting ATPases:
Copper is an essential element, which forms an integral component of many enzymes. While trace amounts of copper are needed to sustain life, excess copper is extremely toxic. Although various aspects of copper transport and metabolism have been investigated in the past, very little is known about the specifics of intracellular copper transport. The cloning of the genes responsible for the two major genetic disorders of copper metabolism in humans, Wilson and Menkes diseases has been a major breakthrough in our understanding of intracellular copper transport.
Both genes are predicted to encode putative copper-transporting P-type ATPases similar to other cation-transporting p-type ATPases. A crucial feature of the copper-transporting ATPases is the presence of a large N-terminal segment, which contains the copper-binding domain. Other features include: eight membrane spanning domains, three intracellular loops and a short C-terminal. In the absence of any crystal structure for Wilson copper-transporting ATPase, we used comparative modeling to obtain a low-resolution model of the Wilson disease copper-transporting ATPase by homology to the X-ray structure of SERCA1 (calcium pump) which is a member of the p-type ATPase family.
Our studies are directed to a complete characterization of the structure of the N-terminal copper-binding segment by CD, XAS and NMR spectroscopy. To better understand the regulatory effect of copper binding of the N-terminal domain of ATP7B, we performed NMR characterization of WCBD 4-6 (domain 4-6 of ATP7B). 15N relaxation measurements on the apo- and Cu(II)-bound WCBD 4-6 show dynamic properties of this three domain construct. The linker between domains 4 and 5 remains flexible. The domains 5 and 6 do not form a completely rigid dimer but rather have some flexibility with respect to each other, and there is minimal change in the relative orientation of the domains in the two states. We found the protein-protein interaction between Atox1 and the copper binding domains takes place even in the absence of copper. We also carried out NMR studies with the entire N-terminal domain (WCBD 1-6). The results are consistent with our results for WCBD 4-6. Copper transfer to and between the N-terminal domains of the Wilson ATPase occurs via protein interactions that are facilitated by the flexibility of the linkers and the motional freedom of the domains with respect to each other.
3. Treatment developments for Menkes and Wilson diseases:
We pioneered the treatment of Menkes disease with copper-histidine. Although patients clearly responded well to the treatment when started very early in life, the severity of the disease in each case remained unresolved. To clarify these questions, we characterized the genetic defects in patients who were treated with copper-histidine. Results revealed severe mutations in these patients indicative of a classic form of Menkes disease. These patients would not have survived without the copper-histidine treatment.
The long-term follow-up (aged ten to 22 years) demonstrated normal or near normal intellectual development in these patients, however, they still showed problems associated with connective tissue abnormalities. We have been providing advice and our Hospital Pharmacy protocols of copper-histidine preparations and relevant information to physicians around the world who are treating Menkes disease patients.
Wilson disease is a genetic disorder of copper transport, which causes progressive hepatic or neuropsychiatric diseases in affected individuals. Without treatment, this disease is invariably lethal. Various medical treatments are available, including zinc acetate, penicillamine and other oral chelators, but each carries risks of side effects which may be severe or life threatening. A simple, effective, and non-toxic treatment would be highly desirable.
In Menkes disease, copper-histidine effectively supplies copper to cells. We hypothesize that the reverse strategy might be effective in Wilson disease: namely to supply excess histidine so that it may remove copper directly from hepatocytes. We anticipate that such treatment would be safer and more effective than existing treatment modalities. We are examining this hypothesis in a mouse model of Wilson disease. Findings from these studies are expected to improve diagnosis and treatment of Wilson disease.
4. Intracellular disposition of Copper: Role of COMMD1
An extensive network of proteins manipulates copper disposition in hepatocytes, but comparatively little is known about this protein system. Copper exists in two oxidation states: most extracellular copper is Cu(II) and most, if not all, intracellular copper is Cu(I). The Cu-transporting P-typeATPases, ATP7B (Wilson ATPase) and ATP7A (Menkes ATPase), bind copper as Cu(I). We have recently shown the ubiquitous protein COMMD1 binds Cu(II) exclusively. This raises the question as to what role Cu(II) may play in intracellular processes. This issue is particularly important in the liver and brain. In Wilson disease, there is progressive liver damage due to copper accumulation. In some Bedlington terriers, mutations in COMMD1 are associated with chronic copper-overloaded liver disease which is clinically distinct from Wilson disease. It seems unlikely that Cu(II), which generates reactive oxygen species, has a physiological role intracellularly. However, Cu(II) might be the preferred state of copper for elimination from the cell, such as biliary excretion. It is possible that COMMD1 may contribute to the mechanism of biliary excretion of copper by virtue of binding Cu(II). Additionally, or alternatively COMMD1 may be an important component of an intracellular system for utilizing Cu(II), or for detecting and detoxifying it.
5. Heavy Metals in the Global Environment:
Our global environment consists of numerous natural and artificial metals. Metals have played a critical role in industrial development and technological advances. Most metals are not destroyed; indeed they are accumulating in the environment at an accelerated pace, due to the ever-growing demands of modern society. The widespread distribution of metals in the environment is of great concern because of the toxic properties of many of them. Furthermore, as global climate change takes effect, there will be an increased need for understanding metal contaminants of ground water and their potential health effects. A fine balance must be maintained between metals in the environment and human health.
One of our current research interests is in the area of arsenic and other toxic metals in the global environment with special emphasis in Bengal Delta. Many of these toxic metals, arsenic included, have both carcinogenic and noncarcinogenic potentials. Our interests are in the environmental behaviour of these toxic metals with special reference to their abundance and distributions in soil and drinking water. In 1997 we established a team of international volunteer scientists working in the villages of Bangladesh and West Bengal, India where millions of people are exposed to these toxic metals through naturally contaminated groundwater. We were the first to notice a small child who had telltale symptoms of arsenic poisoning, an observation that finally led us to discover that arsenic is not the only toxic metal groundwater contaminant. We produced national scale maps of arsenic and other toxic metals in groundwater, identifying areas of special concern. The team has also researched adverse pregnancy outcomes of women exposed to arsenic, defects in newborns, and the health impacts of arsenic exposure on the pediatric population. We have developed an inexpensive and accurate method for determining arsenic concentration in water and developed public health strategies based on water testing and sharing to alleviate the health crisis. These studies are conducted in a team effort involving international volunteer scientists from many disciplines who are probing the poisoning by toxic metals in our global environment to address the health crisis management efforts.
B. Sarkar. Metals and Genetics. Metallomics, 4, 589-592 (2012)
S. Frisbie, E. Mitchell, H. Dustin, D. Maynard, B. Sarkar. World Health Organization Discontinues Its Drinking-Water Guidelines for Manganese. Environmental Health Perspectives, 120, 775-778 (2012)
T. Bacquart, K. bradshaw, S. Frisbie, E. Mitchell, G. Springston, J. Defelice, H. Dustin, B. Sarkar. A survey of arsenic, manganese, boron, thorium, and other toxic metals in the groundwater of West Bengal, India neighbourhood. Metallomics, 4, 653-650 (2012)
E. Mitchell, S. Frisbie and B. Sarkar. Exposure to Multiple Metals from Groundwater—A Global Crisis: Geology, Climate Change, Health Effects, Testing, and Mitigation. Metallomics, 3, 874-908. (2011)
B. Sarkar, E.A. Roberts. The puzzle posed by COMMD1, a newly discovered protein binding Cu (II). Metallomics, 3, 20-27 (2011)
N. Fatemi, D.M. Korzhnev, A. Velyvis, B. Sarkar, J.D. Forman-Kay. NMR characterization of copper-binding domains 4-6 of ATP7B. Biochemistry, 49, 8468-8477 (2010)
R. Lobinsky, J. S. Becker, H. Haraguchi and B. Sarkar. Metallomics: Guidelines for terminology and critical evaluation of analytical chemistry approaches (IUPAC Technical Report). Pure and Applied Chemistry, 82, 493-504 (2010)
S.H. Frisbie, E.J. Mitchell, L.J. Mastera, D.M. Maynard, A.Z. Yusuf, M.Y. Siddiq, R. Ortega, R.K. Dunn, D.S. Westerman, T. Bacquart and B. Sarkar. Public Health Strategies for Western Bangladesh that Address the Arsenic, Manganese, Uranium and Other Toxic Elements in Their Drinking Water. Environmental Health Perspectives, 117, 410-416 (2009).
S. Narindrasorasak, P. Kulkarni, P. Deschamps, Y.M. She and B. Sarkar. Characterization of Copper Binding Properties of Human COMMD1 (MURR1). Biochemistry, 46, 3116-3128 (2007).
P.P. Kulkarni, Y.-M. She, S.D. Smith, E.A. Roberts and B. Sarkar. Proteomics of Metal Transport and Metal-Associated Diseases. Chemistry - A European Journal, 12, 2410-2422 (2006).
S.H. Frisbie, E.J. Mitchell, A.Z. Yusuf, M.Y. Siddiq, R.E. Sanchez, R. Ortega, D.M. Maynard and B. Sarkar. The Development and Use of an Innovative Laboratory Method for Measuring Arsenic in Drinking Water from Western Bangladesh. Environmental Health Perspectives, 113, 1196-1204 (2005).
P. Deschamps, P. Kulkarni, M. Gautam-Basak and B. Sarkar. The Saga of Copper (II)-L-Histidine. Coordination Chemistry Reviews, 249, 895-905 (2005).
S.D. Smith, Y.-M. She, E.A. Roberts and Sarkar, B. Using Immobilized Metal Affinity Chromatography, Two-Dimensional Electrophoresis and Mass Spectrometry to Identify Hepatocellular Proteins with Copper-Binding Ability. Journal of Proteome Research, 3, 834-840 (2004).
P. Deschamps, P. P. Kulkarni and B. Sarkar. X-ray structure of physiological copper (II) -bis (L-histidinate) complex. Inorganic Chemistry, 43, 3338-3340 (2004).
Y.M. She, S. Narindrasorasak, S. Yang, N. Spitale, E.A. Roberts and B. Sarkar. Identification of proteins in human hepatoma lines by immunomobilized metal affinity chromatography and mass spectrometry. Mol. Cell. Proteomics, 2, 1306-1318 (2003).
S.H. Frisbie, R. Ortega, D.M. Maynard, B. Sarkar. The concentrations of Arsenic and other Toxic Elements in Bangladesh's Drinking Water. Environmental Health Perspectives, 110, 1147-1153 (2002).
Z. J. Hou, S. Narindrasorasak, B. Bhushan, B. Sarkar and B. Mitra. Functional analysis of chimeric proteins of the Wilson Cu(I)-ATPase (ATP7B) and ZntA, a Pb(II)/Zn(II)/Cd(II)-ATPase from Escherichia coli. J. Biol. Chem, 276, 40858-40863 (2001).
L.W. Donaldson, N.R. Skrynnikov, Wing-Yiu Choy, D. RanjithMuhandirak, B. Sarkar, J. D. Forman-Kay, and L. E. Kay. Structural characterization of Proteins with an Attached ATCUN Motif by Paramagnetic Relaxation Enhancement NMR Spectroscopy. J Am. Chem. Soc., 123, 9843-9847 (2001).
B. Sarkar. Treatment of Wilson and Menkes Diseases – Medicinal Inorganic Chemistry. Chem. Rev., 99, 2535-2544 (1999).
J. Christodoulou, D.M. Danks, B. Sarkar, K.E. Baerlocher, R. Casey, N. Horn, Z. Tümer and J.T.R. Clarke. Early Treatment of Menkes Disease with Parenteral Copper-Histidine: Long-term Follow up of Four Treated Patients. Am. J. Med. Genet., 76: 154-164 (1998).
C. Harford and B. Sarkar. Amino Terminal Cu(II) and Ni (II)-Binding (ATCUN) motif of proteins and peptides. Acc. Chem. Res., 30, 123-130 (1997).
M. DiDonato, S. Narindrasorasak, J.R. Forbes, D.W. Cox and B. Sarkar. Expression, purification and metal binding properties of the N-terminal domain from the Wilson disease putative Cu-transporting ATPase (ATP7B). J. Biol. Chem., 272, 33279-33282 (1997).
Z. Tümer, N. Horn; T. Tønnesen, J. Christodolou, J. T.R. Clarke and B. Sarkar. Efficacy of Early Copper-Histidine Treatment for Menkes Disease. Nature Genetics, 12, 11-13 (1996).