Michael  Maurizi, Ph.D.
Michael Maurizi, Ph.D.
Senior Investigator
Head, Biochemistry of Proteins Section

Center for Cancer Research
National Cancer Institute

Building 37, Room 2128B
Bethesda, MD 20892-4256
301-496-7961

Dr. Maurizi conducted the seminal studies revealing the existence of multi-component ATP-dependent proteases in bacterial cells. His structural and biochemical studies of Clp protease complexes underlie current models of the mechanism by which these and other molecular machines unfold and degrade intracellular proteins. Recent research has focused on proteome-wide N-end rule degradation as a major pathway for protein quality control.

As Chief of the Biochemistry of Proteins Section Dr. Maurizi directs a multi-disciplinary research program that combines genetics, biochemistry, and high-resolution structural analysis to study the regulatory pathways in which intracellular proteolysis plays a major role and the properties and behavior of the molecular machines that carry out ATP-dependent proteolysis.

Areas of Expertise
1) protein biochemistry, 2) ATP-dependent protein degradation, 3) post-translational regulation, 4) protein quality control, 5) bacterial stress responses, 6) mitochondrial stress responses

Structure and Function of ATP-Dependent Proteases Involved in Rapid Intracellular Protein Degradation in Prokaryotes and Eukaryotes

Intracellular protein degradation is a major post-translational regulatory mechanism that comes into play in virtually every aspect of the biology of a cell. Crucial roles for protein degradation include timing of the cell division cycle, heat shock and other stress responses, adaptation to changes in nutrient availability, cell signaling mechanisms and developmental changes, response to DNA damage, viral infections, and cell death pathways. Protein degradation also serves a vital function in protein quality control, removing mistranslated, damaged, denatured, and otherwise conformationally abnormal proteins from cells. Protein quality control by proteases is accomplished in partnership with molecular chaperones. Together, the chaperones and proteases constitute a triage system that recognizes abnormal proteins and shuttles them into repair or destruction pathways.

Most intracellular proteolysis is performed by ATP-dependent proteases, which unite a chaperone and a protease activity in a single enzyme complex. Our studies have focused on the ATP-dependent proteases from E. coli and their close homologs from mammalian mitochondria. Our studies have revealed remarkable parallels in composition and structure between the E. coli ATP-dependent proteases and those in higher organisms, including the 26S proteasome, the major ATP-dependent protease in the cytosol of eukaryotic cells. These parallels underlie a set of mechanistic principles that is common to all ATP-dependent proteases. ATP-dependent proteases are high molecular weight, multimeric proteins containing at least two types of enzymatic functions, an ATP-dependent protein unfoldase, referred to as the chaperone component, and an endopeptidase, referred to as the protease component. In addition, the complexes contain one or more auxiliary components, either as intrinsic domains or as components that associate with the complex or its substrates. The chaperone and protease functions are contributed by separate domains, which may be connected tandemly in a single polypeptide, as in the E. coli proteases, Lon and FtsH, or non-covalently assembled from different polypeptides, as in the Clp proteases and the proteasomes. The protease domains are arranged so that the proteolytic active sites are sequestered within the oligomeric assembly and are inaccessible to folded polypeptides and proteins. In a reaction cycle, protein substrates bind to the chaperone component, undergo some degree of unfolding, and are either released and allowed to refold or are translocated into the protease component and degraded. Thus, substrates are kinetically partitioned between degradation and release depending either on intrinsic properties of the substrate or on extrinsic factors that favor one or the other pathway. Binding and release of substrates are controlled by cycles of ATP binding and hydrolysis. Additional regulatory components, adaptor proteins, affect substrate binding or retention and thus influence coupling between changes in nucleotide state and binding or release of substrates.

The mechanistic details of these processes are being investigated. Crystal structure analysis of ClpA and the chaperone domains of other ATP-dependent proteases have shown that their nucleotide binding module belong to the AAA+ superfamily (ATPases associated with various cellular activities). AAA+ proteins are molecular machines involved in conformational reconfiguration of macromolecular complexes. The AAA+ module is designed to couple ATP hydrolysis to conformational changes in the chaperone, which then imparts a mechanical force to portions of a bound substrate. This force can be used to transiently disrupt internal interactions within the substrate, allowing the substrate to undergo structural rearrangement, promoting remodeling of complexes involving the substrate, or permitting the substrate to translocate along or through another macromolecule. For ATP-dependent proteolysis to occur, both the structural disruption and translocation activities are required, because the proteolytic sites lie within an internal chamber formed by association of the protease subunits.  In the ClpP protease, two rings of seven subunits interact to enclose an active site chamber accessible only through narrow channels along the seven-fold symmetry axes. ClpP associates with two related chaperones, ClpA, which has two ATPase domains, and ClpX, which has only one. ClpA and ClpX form six-membered rings and stack on both faces of the double ClpP ring. The complexes are hollow cylinder with the degradation chamber in the center. ClpAP and ClpXP complexes remain intact during multiple proteolytic cycles.

One cycle, which includes substrate binding, unfolding and translocation, and complete degradation, occurs in 5-10 sec for a 30 kDa substrate protein. Substrate recognition sites are located within the chaperone component, and the mechanism of interaction and the nature and location of the binding sites are not known. Substrate interaction occurs in several stages: initial binding interactions that bring the substrate and the chaperone together, a committed binding step that imposes some barrier to reversal, and subsequent binding interactions that occur while the substrate is in transit to the protease.

Electron microscopy has shown that substrates initially interact at the ends of complexes, indicating that at least some binding sites are located on the ring surface of the chaperone. Whether these are the sites that confer specificity for degradation is not clear. Most motifs that serve as degradation tags for substrates are located at either the N-terminus or C-terminus of the substrate. Such terminally located motifs might also have access to sites within the axial channel, on the peripheral sides of the rings, or even between the rings or between subdomains of the chaperone. Alternatively, specific binding might occur only after higher affinity sites become available due to conformational changes within the chaperone. Our laboratory is working to obtain additional high-resolution structural data to go with biochemical and mutagenesis studies aimed at a better understanding of how substrates interact with the chaperones and how bound proteins are unfolded and translocated into the proteolytic chambers of the complex.

Collaborators on this research include Di Xia, Laboratory of Cell Biology, NCI; Alex Wlodawer, Macromolecular Crystallography Laboratory, NCI; Susan Gottesman, Laboratory of Molecular Biology, NCI; and Alasdair Steven, Laboratory of Structural Biology Research, NIAMS.

Scientific Focus Areas:
Cancer Biology, Cell Biology, Microbiology and Infectious Diseases, Molecular Biology and Biochemistry, Structural Biology
Selected Recent Publications
  1. Maurizi MR, Stan G.
    Cell. 155: 502-4, 2013. [ Journal Article ]
  2. Humbard MA, Surkov S, De Donatis GM, Jenkins L, Maurizi MR.
    J. Biol. Chem. 2013. [ Journal Article ]
  3. Kang JH, Chang YC, Maurizi MR.
    J. Biol. Chem. 287: 15661-71, 2012. [ Journal Article ]
  4. Derrien B, Majeran W, Effantin G, Ebenezer J, Friso G, van Wijk KJ, Steven AC, Maurizi MR, Vallon O.
    Plant Mol. Biol. 80: 189-202, 2012. [ Journal Article ]
  5. De Donatis GM, Singh SK, Viswanathan S, Maurizi MR.
    J. Biol. Chem. 210: 8771-81, 2010. [ Journal Article ]

Dr. Maurizi received his Ph.D. in biochemistry from the University of Illinois and completed postdoctoral studies with Earl Stadtman and Ann Ginsburg in the National Heart, Lung, and Blood Institute at the NIH. Dr. Maurizi began his work on energy-dependent proteases in collaboration with Susan Gottesman in the Laboratory of Molecular Biology, NCI, before joining the staff of the Laboratory of Cell Biology in 1991.

Name Position
Issa Bryant Summer Student
Poorva Dharkar Ph.D. Postdoctoral Fellow (Visiting)
Luke Diorio-toth Postdoctoral Fellow (CRTA)
Daniel Gutierrez Postdoctoral Fellow (CRTA)
Stephanie Hess Summer Student
Katie Hsia Special Volunteer
Matthew Humbard Ph.D. Postdoctoral Fellow (CRTA)
Jeong Han Kang Ph.D. Postdoctoral Fellow (Visiting)
Germaine Yeku Noukelak Postbaccalaureate Fellow
Serhiy Volodymirovych Surkov Ph.D. Postdoctoral Fellow (Visiting)
Yang Zhang Ph.D. Research Biologist

Summary

Dr. Maurizi conducted the seminal studies revealing the existence of multi-component ATP-dependent proteases in bacterial cells. His structural and biochemical studies of Clp protease complexes underlie current models of the mechanism by which these and other molecular machines unfold and degrade intracellular proteins. Recent research has focused on proteome-wide N-end rule degradation as a major pathway for protein quality control.

As Chief of the Biochemistry of Proteins Section Dr. Maurizi directs a multi-disciplinary research program that combines genetics, biochemistry, and high-resolution structural analysis to study the regulatory pathways in which intracellular proteolysis plays a major role and the properties and behavior of the molecular machines that carry out ATP-dependent proteolysis.

Areas of Expertise
1) protein biochemistry, 2) ATP-dependent protein degradation, 3) post-translational regulation, 4) protein quality control, 5) bacterial stress responses, 6) mitochondrial stress responses

Research

Structure and Function of ATP-Dependent Proteases Involved in Rapid Intracellular Protein Degradation in Prokaryotes and Eukaryotes

Intracellular protein degradation is a major post-translational regulatory mechanism that comes into play in virtually every aspect of the biology of a cell. Crucial roles for protein degradation include timing of the cell division cycle, heat shock and other stress responses, adaptation to changes in nutrient availability, cell signaling mechanisms and developmental changes, response to DNA damage, viral infections, and cell death pathways. Protein degradation also serves a vital function in protein quality control, removing mistranslated, damaged, denatured, and otherwise conformationally abnormal proteins from cells. Protein quality control by proteases is accomplished in partnership with molecular chaperones. Together, the chaperones and proteases constitute a triage system that recognizes abnormal proteins and shuttles them into repair or destruction pathways.

Most intracellular proteolysis is performed by ATP-dependent proteases, which unite a chaperone and a protease activity in a single enzyme complex. Our studies have focused on the ATP-dependent proteases from E. coli and their close homologs from mammalian mitochondria. Our studies have revealed remarkable parallels in composition and structure between the E. coli ATP-dependent proteases and those in higher organisms, including the 26S proteasome, the major ATP-dependent protease in the cytosol of eukaryotic cells. These parallels underlie a set of mechanistic principles that is common to all ATP-dependent proteases. ATP-dependent proteases are high molecular weight, multimeric proteins containing at least two types of enzymatic functions, an ATP-dependent protein unfoldase, referred to as the chaperone component, and an endopeptidase, referred to as the protease component. In addition, the complexes contain one or more auxiliary components, either as intrinsic domains or as components that associate with the complex or its substrates. The chaperone and protease functions are contributed by separate domains, which may be connected tandemly in a single polypeptide, as in the E. coli proteases, Lon and FtsH, or non-covalently assembled from different polypeptides, as in the Clp proteases and the proteasomes. The protease domains are arranged so that the proteolytic active sites are sequestered within the oligomeric assembly and are inaccessible to folded polypeptides and proteins. In a reaction cycle, protein substrates bind to the chaperone component, undergo some degree of unfolding, and are either released and allowed to refold or are translocated into the protease component and degraded. Thus, substrates are kinetically partitioned between degradation and release depending either on intrinsic properties of the substrate or on extrinsic factors that favor one or the other pathway. Binding and release of substrates are controlled by cycles of ATP binding and hydrolysis. Additional regulatory components, adaptor proteins, affect substrate binding or retention and thus influence coupling between changes in nucleotide state and binding or release of substrates.

The mechanistic details of these processes are being investigated. Crystal structure analysis of ClpA and the chaperone domains of other ATP-dependent proteases have shown that their nucleotide binding module belong to the AAA+ superfamily (ATPases associated with various cellular activities). AAA+ proteins are molecular machines involved in conformational reconfiguration of macromolecular complexes. The AAA+ module is designed to couple ATP hydrolysis to conformational changes in the chaperone, which then imparts a mechanical force to portions of a bound substrate. This force can be used to transiently disrupt internal interactions within the substrate, allowing the substrate to undergo structural rearrangement, promoting remodeling of complexes involving the substrate, or permitting the substrate to translocate along or through another macromolecule. For ATP-dependent proteolysis to occur, both the structural disruption and translocation activities are required, because the proteolytic sites lie within an internal chamber formed by association of the protease subunits.  In the ClpP protease, two rings of seven subunits interact to enclose an active site chamber accessible only through narrow channels along the seven-fold symmetry axes. ClpP associates with two related chaperones, ClpA, which has two ATPase domains, and ClpX, which has only one. ClpA and ClpX form six-membered rings and stack on both faces of the double ClpP ring. The complexes are hollow cylinder with the degradation chamber in the center. ClpAP and ClpXP complexes remain intact during multiple proteolytic cycles.

One cycle, which includes substrate binding, unfolding and translocation, and complete degradation, occurs in 5-10 sec for a 30 kDa substrate protein. Substrate recognition sites are located within the chaperone component, and the mechanism of interaction and the nature and location of the binding sites are not known. Substrate interaction occurs in several stages: initial binding interactions that bring the substrate and the chaperone together, a committed binding step that imposes some barrier to reversal, and subsequent binding interactions that occur while the substrate is in transit to the protease.

Electron microscopy has shown that substrates initially interact at the ends of complexes, indicating that at least some binding sites are located on the ring surface of the chaperone. Whether these are the sites that confer specificity for degradation is not clear. Most motifs that serve as degradation tags for substrates are located at either the N-terminus or C-terminus of the substrate. Such terminally located motifs might also have access to sites within the axial channel, on the peripheral sides of the rings, or even between the rings or between subdomains of the chaperone. Alternatively, specific binding might occur only after higher affinity sites become available due to conformational changes within the chaperone. Our laboratory is working to obtain additional high-resolution structural data to go with biochemical and mutagenesis studies aimed at a better understanding of how substrates interact with the chaperones and how bound proteins are unfolded and translocated into the proteolytic chambers of the complex.

Collaborators on this research include Di Xia, Laboratory of Cell Biology, NCI; Alex Wlodawer, Macromolecular Crystallography Laboratory, NCI; Susan Gottesman, Laboratory of Molecular Biology, NCI; and Alasdair Steven, Laboratory of Structural Biology Research, NIAMS.

Scientific Focus Areas:
Cancer Biology, Cell Biology, Microbiology and Infectious Diseases, Molecular Biology and Biochemistry, Structural Biology

Publications

Selected Recent Publications
  1. Maurizi MR, Stan G.
    Cell. 155: 502-4, 2013. [ Journal Article ]
  2. Humbard MA, Surkov S, De Donatis GM, Jenkins L, Maurizi MR.
    J. Biol. Chem. 2013. [ Journal Article ]
  3. Kang JH, Chang YC, Maurizi MR.
    J. Biol. Chem. 287: 15661-71, 2012. [ Journal Article ]
  4. Derrien B, Majeran W, Effantin G, Ebenezer J, Friso G, van Wijk KJ, Steven AC, Maurizi MR, Vallon O.
    Plant Mol. Biol. 80: 189-202, 2012. [ Journal Article ]
  5. De Donatis GM, Singh SK, Viswanathan S, Maurizi MR.
    J. Biol. Chem. 210: 8771-81, 2010. [ Journal Article ]

Biography

Dr. Maurizi received his Ph.D. in biochemistry from the University of Illinois and completed postdoctoral studies with Earl Stadtman and Ann Ginsburg in the National Heart, Lung, and Blood Institute at the NIH. Dr. Maurizi began his work on energy-dependent proteases in collaboration with Susan Gottesman in the Laboratory of Molecular Biology, NCI, before joining the staff of the Laboratory of Cell Biology in 1991.

Team

Name Position
Issa Bryant Summer Student
Poorva Dharkar Ph.D. Postdoctoral Fellow (Visiting)
Luke Diorio-toth Postdoctoral Fellow (CRTA)
Daniel Gutierrez Postdoctoral Fellow (CRTA)
Stephanie Hess Summer Student
Katie Hsia Special Volunteer
Matthew Humbard Ph.D. Postdoctoral Fellow (CRTA)
Jeong Han Kang Ph.D. Postdoctoral Fellow (Visiting)
Germaine Yeku Noukelak Postbaccalaureate Fellow
Serhiy Volodymirovych Surkov Ph.D. Postdoctoral Fellow (Visiting)
Yang Zhang Ph.D. Research Biologist