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Structure-Activity Relationships in the Endosomal Escape of Designed Miniature Proteins
Structure-Activity Relationships in the Endosomal Escape of Designed Miniature Proteins
상세정보
- 자료유형
- 학위논문 서양
- 최종처리일시
- 20260202104847
- ISBN
- 9798293893171
- DDC
- 540
- 서명/저자
- Structure-Activity Relationships in the Endosomal Escape of Designed Miniature Proteins
- 발행사항
- [Sl] : University of California, Berkeley, 2025
- 발행사항
- Ann Arbor : ProQuest Dissertations & Theses, 2025
- 형태사항
- 193 p
- 주기사항
- Source: Dissertations Abstracts International, Volume: 87-04, Section: B.
- 주기사항
- Advisor: Schepartz, Alanna.
- 학위논문주기
- Thesis (Ph.D.)--University of California, Berkeley, 2025.
- 초록/해제
- 요약Biologics, particularly protein-based therapeutics, offer novel modalities for treating complex diseases that are challenging to address with traditional small-molecule therapeutics. Despite their therapeutic promise, protein-based therapeutics are often hindered by their inability to translocate through biological membranes readily. If proteins are taken up into mammalian cells, they typically do so through endocytosis, an essential process through which the cell surveys the extracellular space and consequently makes decisions. In doing so, proteins pass through biochemically rich and distinct compartments defined as endosomes. However, they remain unable to translocate through hydrophobic membranes, rendering them susceptible to degradation by hydrolases, proteases, and the highly acidic environment found within the lysosome. We review the development of protein-based therapeutics, examine the biochemical and biophysical processes they face while moving through mammalian cells, and provide an overview of strategies for delivering and measuring exogenous proteins (Chapter 1).ZF5.3 is a compact, rationally designed mini-protein that escapes from the endosomes of multiple cell types. Despite its small size (27 amino acids), ZF5.3 can be isolated intact from the cytosol of treated cells and guides various classes of proteins into the cytosol and/or nucleus. In the best cases, delivery efficiencies reach or exceed 50% to establish nuclear or cytosolic concentrations of 500 nM or higher. But other than the requirement for unfoldable cargo and two subunits of the HOPS complex, there is little known about how ZF5.3 traverses the limiting endocytic membrane. In the following chapters, we dissect the structure-activity relationships that give rise to the endosomal escape of this designed mini-protein. First, we characterize the effect of pH on the structure of ZF5.3 (Chapter 2). We confirm that ZF5.3 is stable at pH values between 5.5 and 7.5, with no evidence of unfolding even at temperatures as high as 95 oC. The high-resolution NMR structure of ZF5.3 at pH 5.5, also reported here, shows a canonical ββ⍺ zinc-finger fold with the penta-arginine motif integrated seamlessly into the C-terminal ⍺ helix. At lower pH, ZF5.3 unfolds cooperatively as judged by both circular dichroism and high-resolution NMR. Unfolding occurs upon protonation of a single Zn (II)-binding His side chain whose pKa corresponds almost precisely to that of the late endolysosomal lumen. Next, we use rational protein design to engineer a ZF5.3 analog, BBA5.3, that remains folded at acidic pH (Chapter 3). Although BBA5.3 carries a penta-arginine motif, traffics through the endocytic pathway in a manner that resembles ZF5.3, is not degraded, and partitions comparably within LAMP1+ vesicles when visualized using STED, its ability to reach the cytosol is only about 10% that of ZF5.3. Thus, the key difference between the two molecules is that ZF5.3 unfolds at low pH, while BBA5.3 does not. This suggests that pH- dependent unfolding is necessary for ZF5.3 to access the cytosol. To investigate how the unfolding of ZF5.3 is necessary for its cytosolic access, we next utilized the recent discovery of a high- affinity interaction between ZF5.3 and a specific lipid, BMP, which is selectively enriched in the inner leaflet of late endolysosomal membranes. We examined the basis of this interaction on the delivery of ZF5.3 in cellulo (Chapter 4) and found that the genetic and chemical perturbation of BMP has an insignificant effect on the concentration of ZF5.3 measured in the cytosol. Using a FRET-based assay, we comment on the ability of ZF5.3 to translocate through synthetic liposomes of varying lipid compositions in a minimally reconstituted in vitro assay (Chapter 4). Lastly, we use an immobilized biochemical pulldown assay to identify mammalian cell surface receptor interactors with penta-arginine containing mini-proteins in vitro (Chapter 5). We report the identification of an in vitro interaction between BBA5.3 and three types of receptors in the NOTCH receptor family. The requirements for endocytic uptake and endosomal escape identified here will aid and inform the future design of proteins, peptidomimetics, and other macromolecules that reach cytosolic or nuclear targets intact and at therapeutically relevant concentration.
- 일반주제명
- Chemistry
- 일반주제명
- Cellular biology
- 일반주제명
- Molecular biology
- 일반주제명
- Biochemistry
- 키워드
- Mammalian cells
- 기타저자
- University of California, Berkeley Chemistry
- 기본자료저록
- Dissertations Abstracts International. 87-04B.
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■1001 ▼aGiudice, Jonathan.
■24510▼aStructure-Activity Relationships in the Endosomal Escape of Designed Miniature Proteins
■260 ▼a[Sl]▼bUniversity of California, Berkeley▼c2025
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2025
■300 ▼a193 p
■500 ▼aSource: Dissertations Abstracts International, Volume: 87-04, Section: B.
■500 ▼aAdvisor: Schepartz, Alanna.
■5021 ▼aThesis (Ph.D.)--University of California, Berkeley, 2025.
■520 ▼aBiologics, particularly protein-based therapeutics, offer novel modalities for treating complex diseases that are challenging to address with traditional small-molecule therapeutics. Despite their therapeutic promise, protein-based therapeutics are often hindered by their inability to translocate through biological membranes readily. If proteins are taken up into mammalian cells, they typically do so through endocytosis, an essential process through which the cell surveys the extracellular space and consequently makes decisions. In doing so, proteins pass through biochemically rich and distinct compartments defined as endosomes. However, they remain unable to translocate through hydrophobic membranes, rendering them susceptible to degradation by hydrolases, proteases, and the highly acidic environment found within the lysosome. We review the development of protein-based therapeutics, examine the biochemical and biophysical processes they face while moving through mammalian cells, and provide an overview of strategies for delivering and measuring exogenous proteins (Chapter 1).ZF5.3 is a compact, rationally designed mini-protein that escapes from the endosomes of multiple cell types. Despite its small size (27 amino acids), ZF5.3 can be isolated intact from the cytosol of treated cells and guides various classes of proteins into the cytosol and/or nucleus. In the best cases, delivery efficiencies reach or exceed 50% to establish nuclear or cytosolic concentrations of 500 nM or higher. But other than the requirement for unfoldable cargo and two subunits of the HOPS complex, there is little known about how ZF5.3 traverses the limiting endocytic membrane. In the following chapters, we dissect the structure-activity relationships that give rise to the endosomal escape of this designed mini-protein. First, we characterize the effect of pH on the structure of ZF5.3 (Chapter 2). We confirm that ZF5.3 is stable at pH values between 5.5 and 7.5, with no evidence of unfolding even at temperatures as high as 95 oC. The high-resolution NMR structure of ZF5.3 at pH 5.5, also reported here, shows a canonical ββ⍺ zinc-finger fold with the penta-arginine motif integrated seamlessly into the C-terminal ⍺ helix. At lower pH, ZF5.3 unfolds cooperatively as judged by both circular dichroism and high-resolution NMR. Unfolding occurs upon protonation of a single Zn (II)-binding His side chain whose pKa corresponds almost precisely to that of the late endolysosomal lumen. Next, we use rational protein design to engineer a ZF5.3 analog, BBA5.3, that remains folded at acidic pH (Chapter 3). Although BBA5.3 carries a penta-arginine motif, traffics through the endocytic pathway in a manner that resembles ZF5.3, is not degraded, and partitions comparably within LAMP1+ vesicles when visualized using STED, its ability to reach the cytosol is only about 10% that of ZF5.3. Thus, the key difference between the two molecules is that ZF5.3 unfolds at low pH, while BBA5.3 does not. This suggests that pH- dependent unfolding is necessary for ZF5.3 to access the cytosol. To investigate how the unfolding of ZF5.3 is necessary for its cytosolic access, we next utilized the recent discovery of a high- affinity interaction between ZF5.3 and a specific lipid, BMP, which is selectively enriched in the inner leaflet of late endolysosomal membranes. We examined the basis of this interaction on the delivery of ZF5.3 in cellulo (Chapter 4) and found that the genetic and chemical perturbation of BMP has an insignificant effect on the concentration of ZF5.3 measured in the cytosol. Using a FRET-based assay, we comment on the ability of ZF5.3 to translocate through synthetic liposomes of varying lipid compositions in a minimally reconstituted in vitro assay (Chapter 4). Lastly, we use an immobilized biochemical pulldown assay to identify mammalian cell surface receptor interactors with penta-arginine containing mini-proteins in vitro (Chapter 5). We report the identification of an in vitro interaction between BBA5.3 and three types of receptors in the NOTCH receptor family. The requirements for endocytic uptake and endosomal escape identified here will aid and inform the future design of proteins, peptidomimetics, and other macromolecules that reach cytosolic or nuclear targets intact and at therapeutically relevant concentration.
■590 ▼aSchool code: 0028.
■650 4▼aChemistry
■650 4▼aCellular biology
■650 4▼aMolecular biology
■650 4▼aBiochemistry
■653 ▼aSmall-molecule therapeutics
■653 ▼aMammalian cells
■653 ▼aEndocytic membrane
■690 ▼a0485
■690 ▼a0379
■690 ▼a0487
■690 ▼a0307
■71020▼aUniversity of California, Berkeley▼bChemistry.
■7730 ▼tDissertations Abstracts International▼g87-04B.
■790 ▼a0028
■791 ▼aPh.D.
■792 ▼a2025
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17359191▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
