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  1. Article ; Online: Split enzymes: Design principles and strategy.

    Lim, Shion A / Wells, James A

    Methods in enzymology

    2020  Volume 644, Page(s) 275–296

    Abstract: Engineering precise control of enzymatic activity provides a powerful means to manipulate and understand biological processes. One approach to achieve a switch-like control over enzyme activity is to design a split enzyme, where the protein is separated ... ...

    Abstract Engineering precise control of enzymatic activity provides a powerful means to manipulate and understand biological processes. One approach to achieve a switch-like control over enzyme activity is to design a split enzyme, where the protein is separated into two polypeptides with each inactive fragment fused to inducible dimerization domains. The activity of the enzyme can be controlled by the addition of a small molecule, which causes the inducible dimerization domains to come together and reconstitute the split enzyme and its activity. In recent years, split enzymes have been designed for a variety of enzyme classes, and these synthetic molecular tools have enabled spatial and temporal dissection of biological processes in ways that were difficult previously. Here, we summarize key design principles and strategies to guide future split enzyme engineering efforts, using split enzymes generated from our research group as examples.
    MeSH term(s) Dimerization ; Enzymes/genetics
    Chemical Substances Enzymes
    Language English
    Publishing date 2020-06-05
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ISSN 1557-7988
    ISSN (online) 1557-7988
    DOI 10.1016/bs.mie.2020.05.001
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: The importance of input sequence set to consensus-derived proteins and their relationship to reconstructed ancestral proteins.

    Nixon, Charlotte / Lim, Shion A / Sternke, Matt / Barrick, Doug / Harms, Mike / Marqusee, Susan

    bioRxiv : the preprint server for biology

    2023  

    Abstract: A protein sequence encodes its energy landscape - all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of ... ...

    Abstract A protein sequence encodes its energy landscape - all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs - questioning the differences and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the overall consensus protein is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted region is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order couplings using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.
    Language English
    Publishing date 2023-07-01
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.06.29.547063
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: The burst-phase folding intermediate of ribonuclease H changes conformation over evolutionary history.

    Lim, Shion A / Marqusee, Susan

    Biopolymers

    2017  Volume 109, Issue 8, Page(s) e23086

    Abstract: The amino acid sequence encodes the energy landscape of a protein. Therefore, we expect evolutionary mutations to change features of the protein energy landscape, including the conformations adopted by a polypeptide as it folds to its native state. ... ...

    Abstract The amino acid sequence encodes the energy landscape of a protein. Therefore, we expect evolutionary mutations to change features of the protein energy landscape, including the conformations adopted by a polypeptide as it folds to its native state. Ribonucleases H (RNase H) from Escherichia coli and Thermus thermophilus both fold via a partially folded intermediate in which the core region of the protein (helices A-D and strands 4-5) is structured. Strand 1, however, uniquely contributes to the T. thermophilus RNase H folding intermediate (I
    MeSH term(s) Escherichia coli/enzymology ; Escherichia coli/genetics ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/genetics ; Evolution, Molecular ; Protein Domains ; Protein Folding ; Ribonuclease H/chemistry ; Ribonuclease H/genetics ; Thermus thermophilus/enzymology ; Thermus thermophilus/genetics
    Chemical Substances Escherichia coli Proteins ; Ribonuclease H (EC 3.1.26.4)
    Language English
    Publishing date 2017-11-20
    Publishing country United States
    Document type Journal Article
    ZDB-ID 1123-x
    ISSN 1097-0282 ; 0006-3525
    ISSN (online) 1097-0282
    ISSN 0006-3525
    DOI 10.1002/bip.23086
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Engineering interferons and interleukins for cancer immunotherapy.

    Holder, Patrick G / Lim, Shion A / Huang, Christine S / Sharma, Preeti / Dagdas, Yavuz S / Bulutoglu, Beyza / Sockolosky, Jonathan T

    Advanced drug delivery reviews

    2022  Volume 182, Page(s) 114112

    Abstract: Cytokines are a class of potent immunoregulatory proteins that are secreted in response to various stimuli and act locally to regulate many aspects of human physiology and disease. Cytokines play important roles in cancer initiation, progression, and ... ...

    Abstract Cytokines are a class of potent immunoregulatory proteins that are secreted in response to various stimuli and act locally to regulate many aspects of human physiology and disease. Cytokines play important roles in cancer initiation, progression, and elimination, and thus, there is a long clinical history associated with the use of recombinant cytokines to treat cancer. However, the use of cytokines as therapeutics has been limited by cytokine pleiotropy, complex biology, poor drug-like properties, and severe dose-limiting toxicities. Nevertheless, cytokines are crucial mediators of innate and adaptive antitumor immunity and have the potential to enhance immunotherapeutic approaches to treat cancer. Development of immune checkpoint inhibitors and combination immunotherapies has reinvigorated interest in cytokines as therapeutics, and a variety of engineering approaches are emerging to improve the safety and effectiveness of cytokine immunotherapy. In this review we highlight recent advances in cytokine biology and engineering for cancer immunotherapy.
    MeSH term(s) Bioengineering/methods ; Biomimetics ; Drug Delivery Systems/methods ; Genetic Engineering/methods ; Humans ; Hydrogen-Ion Concentration ; Interferons/adverse effects ; Interferons/metabolism ; Interferons/pharmacokinetics ; Interferons/pharmacology ; Interleukins/adverse effects ; Interleukins/metabolism ; Interleukins/pharmacokinetics ; Interleukins/pharmacology ; Neoplasms/drug therapy ; Neoplasms/pathology
    Chemical Substances Interleukins ; Interferons (9008-11-1)
    Language English
    Publishing date 2022-01-24
    Publishing country Netherlands
    Document type Journal Article ; Review
    ZDB-ID 639113-8
    ISSN 1872-8294 ; 0169-409X
    ISSN (online) 1872-8294
    ISSN 0169-409X
    DOI 10.1016/j.addr.2022.114112
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Exploring the Evolutionary History of Kinetic Stability in the α-Lytic Protease Family.

    Nixon, Charlotte F / Lim, Shion A / Sailer, Zachary R / Zheludev, Ivan N / Gee, Christine L / Kelch, Brian A / Harms, Michael J / Marqusee, Susan

    Biochemistry

    2021  Volume 60, Issue 3, Page(s) 170–181

    Abstract: In addition to encoding the tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determine the speed of folding. How these kinetic barriers are encoded is not well understood. Here, we ... ...

    Abstract In addition to encoding the tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determine the speed of folding. How these kinetic barriers are encoded is not well understood. Here, we use evolutionary sequence variation in the α-lytic protease (αLP) protein family to probe the relationship between sequence and energy landscape. αLP has an unusual energy landscape: the native state of αLP is not the most thermodynamically favored conformation and, instead, remains folded due to a large kinetic barrier preventing unfolding. To fold, αLP utilizes an N-terminal pro region similar in size to the protease itself that functions as a folding catalyst. Once folded, the pro region is removed, and the native state does not unfold on a biologically relevant time scale. Without the pro region, αLP folds on the order of millennia. A phylogenetic search uncovers αLP homologs with a wide range of pro region sizes, including some with no pro region at all. In the resulting phylogenetic tree, these homologs cluster by pro region size. By studying homologs naturally lacking a pro region, we demonstrate they can be thermodynamically stable, fold much faster than αLP, yet retain the same fold as αLP. Key amino acids thought to contribute to αLP's extreme kinetic stability are lost in these homologs, supporting their role in kinetic stability. This study highlights how the entire energy landscape plays an important role in determining the evolutionary pressures on the protein sequence.
    MeSH term(s) Bacterial Proteins/chemistry ; Bacterial Proteins/genetics ; Enzyme Stability ; Evolution, Molecular ; Kinetics ; Models, Molecular ; Phylogeny ; Protein Folding ; Serine Endopeptidases/chemistry ; Serine Endopeptidases/genetics
    Chemical Substances Bacterial Proteins ; Serine Endopeptidases (EC 3.4.21.-) ; myxobacter alpha-lytic proteinase (EC 3.4.21.12)
    Language English
    Publishing date 2021-01-12
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021/acs.biochem.0c00720
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article: Exploring the Evolutionary History of Kinetic Stability in the α-Lytic Protease Family

    Nixon, Charlotte F / Lim, Shion A / Sailer, Zachary R / Zheludev, Ivan N / Gee, Christine L / Kelch, Brian A / Harms, Michael J / Marqusee, Susan

    Biochemistry. 2021 Jan. 12, v. 60, no. 3

    2021  

    Abstract: In addition to encoding the tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determine the speed of folding. How these kinetic barriers are encoded is not well understood. Here, we ... ...

    Abstract In addition to encoding the tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determine the speed of folding. How these kinetic barriers are encoded is not well understood. Here, we use evolutionary sequence variation in the α-lytic protease (αLP) protein family to probe the relationship between sequence and energy landscape. αLP has an unusual energy landscape: the native state of αLP is not the most thermodynamically favored conformation and, instead, remains folded due to a large kinetic barrier preventing unfolding. To fold, αLP utilizes an N-terminal pro region similar in size to the protease itself that functions as a folding catalyst. Once folded, the pro region is removed, and the native state does not unfold on a biologically relevant time scale. Without the pro region, αLP folds on the order of millennia. A phylogenetic search uncovers αLP homologs with a wide range of pro region sizes, including some with no pro region at all. In the resulting phylogenetic tree, these homologs cluster by pro region size. By studying homologs naturally lacking a pro region, we demonstrate they can be thermodynamically stable, fold much faster than αLP, yet retain the same fold as αLP. Key amino acids thought to contribute to αLP’s extreme kinetic stability are lost in these homologs, supporting their role in kinetic stability. This study highlights how the entire energy landscape plays an important role in determining the evolutionary pressures on the protein sequence.
    Keywords amino acid sequences ; amino acids ; catalysts ; energy ; history ; phylogeny ; proteinases ; sequence diversity ; thermodynamics ; trajectories
    Language English
    Dates of publication 2021-0112
    Size p. 170-181.
    Publishing place American Chemical Society
    Document type Article
    Note NAL-light
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021/acs.biochem.0c00720
    Database NAL-Catalogue (AGRICOLA)

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  7. Article ; Online: Targeting a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) for RAS-driven cancers.

    Lim, Shion A / Zhou, Jie / Martinko, Alexander J / Wang, Yung-Hua / Filippova, Ekaterina V / Steri, Veronica / Wang, Donghui / Remesh, Soumya G / Liu, Jia / Hann, Byron / Kossiakoff, Anthony A / Evans, Michael J / Leung, Kevin K / Wells, James A

    The Journal of clinical investigation

    2022  Volume 132, Issue 4

    Abstract: Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing ... ...

    Abstract Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.
    MeSH term(s) Animals ; Antigens, Neoplasm/genetics ; Antigens, Neoplasm/immunology ; Cell Adhesion Molecules/genetics ; Cell Adhesion Molecules/immunology ; Cell Line, Tumor ; Epitopes/genetics ; Epitopes/immunology ; Humans ; Male ; Mice ; Mice, Nude ; Neoplasm Proteins/genetics ; Neoplasm Proteins/immunology ; Neoplasms, Experimental/genetics ; Neoplasms, Experimental/immunology ; Pancreatic Neoplasms/genetics ; Pancreatic Neoplasms/immunology ; Proteolysis
    Chemical Substances Antigens, Neoplasm ; CDCP1 protein, human ; Cell Adhesion Molecules ; Epitopes ; Neoplasm Proteins
    Language English
    Publishing date 2022-01-31
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 3067-3
    ISSN 1558-8238 ; 0021-9738
    ISSN (online) 1558-8238
    ISSN 0021-9738
    DOI 10.1172/JCI154604
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: The thermostability and specificity of ancient proteins.

    Wheeler, Lucas C / Lim, Shion A / Marqusee, Susan / Harms, Michael J

    Current opinion in structural biology

    2016  Volume 38, Page(s) 37–43

    Abstract: Were ancient proteins systematically different than modern proteins? The answer to this question is profoundly important, shaping how we understand the origins of protein biochemical, biophysical, and functional properties. Ancestral sequence ... ...

    Abstract Were ancient proteins systematically different than modern proteins? The answer to this question is profoundly important, shaping how we understand the origins of protein biochemical, biophysical, and functional properties. Ancestral sequence reconstruction (ASR), a phylogenetic approach to infer the sequences of ancestral proteins, may reveal such trends. We discuss two proposed trends: a transition from higher to lower thermostability and a tendency for proteins to acquire higher specificity over time. We review the evidence for elevated ancestral thermostability and discuss its possible origins in a changing environmental temperature and/or reconstruction bias. We also conclude that there is, as yet, insufficient data to support a trend from promiscuity to specificity. Finally, we propose future work to understand these proposed evolutionary trends.
    Language English
    Publishing date 2016-06
    Publishing country England
    Document type Journal Article ; Review
    ZDB-ID 1068353-7
    ISSN 1879-033X ; 0959-440X
    ISSN (online) 1879-033X
    ISSN 0959-440X
    DOI 10.1016/j.sbi.2016.05.015
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article ; Online: Bispecific VH/Fab antibodies targeting neutralizing and non-neutralizing Spike epitopes demonstrate enhanced potency against SARS-CoV-2.

    Lim, Shion A / Gramespacher, Josef A / Pance, Katarina / Rettko, Nicholas J / Solomon, Paige / Jin, Jing / Lui, Irene / Elledge, Susanna K / Liu, Jia / Bracken, Colton J / Simmons, Graham / Zhou, Xin X / Leung, Kevin K / Wells, James A

    mAbs

    2021  Volume 13, Issue 1, Page(s) 1893426

    Abstract: Numerous neutralizing antibodies that target SARS-CoV-2 have been reported, and most directly block binding of the viral Spike receptor-binding domain (RBD) to angiotensin-converting enzyme II (ACE2). Here, we deliberately exploit non-neutralizing RBD ... ...

    Abstract Numerous neutralizing antibodies that target SARS-CoV-2 have been reported, and most directly block binding of the viral Spike receptor-binding domain (RBD) to angiotensin-converting enzyme II (ACE2). Here, we deliberately exploit non-neutralizing RBD antibodies, showing they can dramatically assist in neutralization when linked to neutralizing binders. We identified antigen-binding fragments (Fabs) by phage display that bind RBD, but do not block ACE2 or neutralize virus as IgGs. When these non-neutralizing Fabs were assembled into bispecific VH/Fab IgGs with a neutralizing VH domain, we observed a ~ 25-fold potency improvement in neutralizing SARS-CoV-2 compared to the mono-specific bi-valent VH-Fc alone or the cocktail of the VH-Fc and IgG. This effect was epitope-dependent, reflecting the unique geometry of the bispecific antibody toward Spike. Our results show that a bispecific antibody that combines both neutralizing and non-neutralizing epitopes on Spike-RBD is a promising and rapid engineering strategy to improve the potency of SARS-CoV-2 antibodies.
    MeSH term(s) Antibodies, Bispecific/genetics ; Antibodies, Bispecific/immunology ; Antibodies, Bispecific/therapeutic use ; Antibodies, Neutralizing/genetics ; Antibodies, Neutralizing/immunology ; Antibodies, Neutralizing/therapeutic use ; Antibodies, Viral/genetics ; Antibodies, Viral/immunology ; Antibodies, Viral/therapeutic use ; COVID-19/genetics ; COVID-19/immunology ; Epitopes/genetics ; Epitopes/immunology ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments/genetics ; Immunoglobulin Fab Fragments/immunology ; Immunoglobulin Fab Fragments/therapeutic use ; SARS-CoV-2/genetics ; SARS-CoV-2/immunology ; Spike Glycoprotein, Coronavirus/genetics ; Spike Glycoprotein, Coronavirus/immunology ; COVID-19 Drug Treatment
    Chemical Substances Antibodies, Bispecific ; Antibodies, Neutralizing ; Antibodies, Viral ; Epitopes ; Immunoglobulin Fab Fragments ; Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2
    Language English
    Publishing date 2021-03-05
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2537838-7
    ISSN 1942-0870 ; 1942-0870
    ISSN (online) 1942-0870
    ISSN 1942-0870
    DOI 10.1080/19420862.2021.1893426
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: Evolutionary trend toward kinetic stability in the folding trajectory of RNases H.

    Lim, Shion A / Hart, Kathryn M / Harms, Michael J / Marqusee, Susan

    Proceedings of the National Academy of Sciences of the United States of America

    2016  Volume 113, Issue 46, Page(s) 13045–13050

    Abstract: Proper folding of proteins is critical to producing the biological machinery essential for cellular function. The rates and energetics of a protein's folding process, which is described by its energy landscape, are encoded in the amino acid sequence. ... ...

    Abstract Proper folding of proteins is critical to producing the biological machinery essential for cellular function. The rates and energetics of a protein's folding process, which is described by its energy landscape, are encoded in the amino acid sequence. Over the course of evolution, this landscape must be maintained such that the protein folds and remains folded over a biologically relevant time scale. How exactly a protein's energy landscape is maintained or altered throughout evolution is unclear. To study how a protein's energy landscape changed over time, we characterized the folding trajectories of ancestral proteins of the ribonuclease H (RNase H) family using ancestral sequence reconstruction to access the evolutionary history between RNases H from mesophilic and thermophilic bacteria. We found that despite large sequence divergence, the overall folding pathway is conserved over billions of years of evolution. There are robust trends in the rates of protein folding and unfolding; both modern RNases H evolved to be more kinetically stable than their most recent common ancestor. Finally, our study demonstrates how a partially folded intermediate provides a readily adaptable folding landscape by allowing the independent tuning of kinetics and thermodynamics.
    Language English
    Publishing date 2016-11-15
    Publishing country United States
    Document type Journal Article
    ZDB-ID 209104-5
    ISSN 1091-6490 ; 0027-8424
    ISSN (online) 1091-6490
    ISSN 0027-8424
    DOI 10.1073/pnas.1611781113
    Database MEDical Literature Analysis and Retrieval System OnLINE

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