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1.
Protein Sci ; 33(1): e4824, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37945533

ABSTRACT

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.


Subject(s)
Antigen-Antibody Complex , Immunoglobulin Fab Fragments , Humans , Crystallization/methods , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/chemistry , Antigen-Antibody Complex/chemistry , Antigens/chemistry , Crystallography, X-Ray , Protein Conformation
2.
J Cell Biol ; 222(5)2023 05 01.
Article in English | MEDLINE | ID: mdl-37071483

ABSTRACT

The Hippo pathway is a conserved and critical regulator of tissue growth. The FERM protein Expanded is a key signaling hub that promotes activation of the Hippo pathway, thereby inhibiting the transcriptional co-activator Yorkie. Previous work identified the polarity determinant Crumbs as a primary regulator of Expanded. Here, we show that the giant cadherin Fat also regulates Expanded directly and independently of Crumbs. We show that direct binding between Expanded and a highly conserved region of the Fat cytoplasmic domain recruits Expanded to the apicolateral junctional zone and stabilizes Expanded. In vivo deletion of Expanded binding regions in Fat causes loss of apical Expanded and promotes tissue overgrowth. Unexpectedly, we find Fat can bind its ligand Dachsous via interactions of their cytoplasmic domains, in addition to the known extracellular interactions. Importantly, Expanded is stabilized by Fat independently of Dachsous binding. These data provide new mechanistic insights into how Fat regulates Expanded, and how Hippo signaling is regulated during organ growth.


Subject(s)
Cell Adhesion Molecules , Drosophila Proteins , Drosophila melanogaster , Hippo Signaling Pathway , Membrane Proteins , Animals , Drosophila melanogaster/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/metabolism , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism
3.
MAbs ; 13(1): 1933690, 2021.
Article in English | MEDLINE | ID: mdl-34190031

ABSTRACT

In order to direct T cells to specific features of solid cancer cells, we engineered a bispecific antibody format, named Dual Antigen T cell Engager (DATE), by fusing a single-chain variable fragment targeting CD3 to a tumor-targeting antigen-binding fragment. In this format, multiple novel paratopes against different tumor antigens were able to recruit T-cell cytotoxicity to tumor cells in vitro and in an in vivo pancreatic ductal adenocarcinoma xenograft model. Since unique surface antigens in solid tumors are limited, in order to enhance selectivity, we further engineered "double-DATEs" targeting two tumor antigens simultaneously. The double-DATE contains an additional autonomous variable heavy-chain domain, which binds a second tumor antigen without itself eliciting a cytotoxic response. This novel modality provides a strategy to enhance the selectivity of immune redirection through binary targeting of native tumor antigens. The modularity and use of a common, stable human framework for all components enables a pipeline approach to rapidly develop a broad repertoire of tailored DATEs and double-DATEs with favorable biophysical properties and high potencies and selectivities.


Subject(s)
Antibodies, Bispecific/pharmacology , Antigens, Neoplasm/immunology , Antineoplastic Agents/pharmacology , Immunotherapy/methods , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal, Humanized/pharmacology , CD3 Complex/immunology , Carcinoma, Pancreatic Ductal/immunology , Humans , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , Pancreatic Neoplasms/immunology , Xenograft Model Antitumor Assays
4.
Skelet Muscle ; 9(1): 30, 2019 12 02.
Article in English | MEDLINE | ID: mdl-31791403

ABSTRACT

BACKGROUND: The mammalian target of rapamycin complex 2 (mTORC2), containing the essential protein rictor, regulates cellular metabolism and cytoskeletal organization by phosphorylating protein kinases, such as PKB/Akt, PKC, and SGK. Inactivation of mTORC2 signaling in adult skeletal muscle affects its metabolism, but not muscle morphology and function. However, the role of mTORC2 in adult muscle stem cells (MuSCs) has not been investigated. METHOD: Using histological, biochemical, and molecular biological methods, we characterized the muscle phenotype of mice depleted for rictor in the Myf5-lineage (RImyfKO) and of mice depleted for rictor in skeletal muscle fibers (RImKO). The proliferative and myogenic potential of MuSCs was analyzed upon cardiotoxin-induced injury in vivo and in isolated myofibers in vitro. RESULTS: Skeletal muscle of young and 14-month-old RImyfKO mice appeared normal in composition and function. MuSCs from young RImyfKO mice exhibited a similar capacity to proliferate, differentiate, and fuse as controls. In contrast, the number of MuSCs was lower in young RImyfKO mice than in controls after two consecutive rounds of cardiotoxin-induced muscle regeneration. Similarly, the number of MuSCs in RImyfKO mice decreased with age, which correlated with a decline in the regenerative capacity of mutant muscle. Interestingly, reduction in the number of MuSCs was also observed in 14-month-old RImKO muscle. CONCLUSIONS: Our study shows that mTORC2 signaling is dispensable for myofiber formation, but contributes to the homeostasis of MuSCs. Loss of mTORC2 does not affect their myogenic function, but impairs the replenishment of MuSCs after repeated injuries and their maintenance during aging. These results point to an important role of mTORC2 signaling in MuSC for muscle homeostasis.


Subject(s)
Cell Self Renewal , Myoblasts/metabolism , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Animals , Cells, Cultured , Female , Male , Mice , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/physiology , Myoblasts/cytology , Myoblasts/physiology , Myogenic Regulatory Factor 5/genetics , Myogenic Regulatory Factor 5/metabolism , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Signal Transduction
5.
Dev Cell ; 48(6): 780-792.e4, 2019 03 25.
Article in English | MEDLINE | ID: mdl-30853441

ABSTRACT

FAT4 mutations lead to several human diseases that disrupt the normal development of the kidney. However, the underlying mechanism remains elusive. In studying the duplex kidney phenotypes observed upon deletion of Fat4 in mice, we have uncovered an interaction between the atypical cadherin FAT4 and RET, a tyrosine kinase receptor essential for kidney development. Analysis of kidney development in Fat4-/- kidneys revealed abnormal ureteric budding and excessive RET signaling. Removal of one copy of the RET ligand Gdnf rescues Fat4-/- kidney development, supporting the proposal that loss of Fat4 hyperactivates RET signaling. Conditional knockout analyses revealed a non-autonomous role for Fat4 in regulating RET signaling. Mechanistically, we found that FAT4 interacts with RET through extracellular cadherin repeats. Importantly, expression of FAT4 perturbs the assembly of the RET-GFRA1-GDNF complex, reducing RET signaling. Thus, FAT4 interacts with RET to fine-tune RET signaling, establishing a juxtacrine mechanism controlling kidney development.


Subject(s)
Cadherins/metabolism , Kidney/embryology , Kidney/metabolism , Proto-Oncogene Proteins c-ret/metabolism , Signal Transduction , Animals , Cadherins/chemistry , Cadherins/deficiency , Gene Deletion , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Humans , Intercellular Signaling Peptides and Proteins , Kidney/abnormalities , Mice , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/metabolism , Protein Binding , Up-Regulation
6.
Development ; 146(7)2019 04 08.
Article in English | MEDLINE | ID: mdl-30872276

ABSTRACT

The formation of multi-nucleated muscle fibers from progenitors requires the fine-tuned and coordinated regulation of proliferation, differentiation and fusion, both during development and after injury in the adult. Although some of the key factors that are involved in the different steps are well known, how intracellular signals are coordinated and integrated is largely unknown. Here, we investigated the role of the cell-growth regulator mTOR by eliminating essential components of the mTOR complexes 1 (mTORC1) and 2 (mTORC2) in mouse muscle progenitors. We show that inactivation of mTORC1, but not mTORC2, in developing muscle causes perinatal death. In the adult, mTORC1 deficiency in muscle stem cells greatly impinges on injury-induced muscle regeneration. These phenotypes are because of defects in the proliferation and fusion capacity of the targeted muscle progenitors. However, mTORC1-deficient muscle progenitors partially retain their myogenic function. Hence, our results show that mTORC1 and not mTORC2 is an important regulator of embryonic and adult myogenesis, and they point to alternative pathways that partially compensate for the loss of mTORC1.This article has an associated 'The people behind the papers' interview.


Subject(s)
Mechanistic Target of Rapamycin Complex 1/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Cells, Cultured , Immunoblotting , Male , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 2/genetics , Mechanistic Target of Rapamycin Complex 2/metabolism , Mice , Mice, Knockout , Muscle Development/genetics , Muscle Development/physiology , Reverse Transcriptase Polymerase Chain Reaction , TOR Serine-Threonine Kinases/genetics
7.
Nat Commun ; 7: 12309, 2016 08 02.
Article in English | MEDLINE | ID: mdl-27480037

ABSTRACT

Branching morphogenesis is a complex biological process common to the development of most epithelial organs. Here we demonstrate that NF2, LATS1/2 and YAP play a critical role in branching morphogenesis in the mouse kidney. Removal of Nf2 or Lats1/2 from the ureteric bud (UB) lineage causes loss of branching morphogenesis that is rescued by loss of one copy of Yap and Taz, and phenocopied by YAP overexpression. Mosaic analysis demonstrates that cells with high YAP expression have reduced contribution to UB tips, similar to Ret(-/-) cells, and that YAP suppresses RET signalling and tip identity. Conversely, Yap/Taz UB-deletion leads to cyst-like branching and expansion of UB tip markers, suggesting a shift towards tip cell identity. Based on these data we propose that NF2 and the Hippo pathway locally repress YAP/TAZ activity in the UB to promote subsequent splitting of the tip to allow branching morphogenesis.


Subject(s)
Kidney/metabolism , Morphogenesis/genetics , Neurofibromin 2/genetics , Protein Serine-Threonine Kinases/genetics , Signal Transduction/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Cycle Proteins , Hippo Signaling Pathway , Kidney/embryology , Mice, Knockout , Mice, Transgenic , Neurofibromin 2/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Ureter/embryology , Ureter/metabolism , YAP-Signaling Proteins
8.
Sci Signal ; 6(296): re7, 2013 Oct 08.
Article in English | MEDLINE | ID: mdl-24106343

ABSTRACT

The Hippo pathway is a kinase cascade, formed by Hippo, Salvador, Warts, and Mats, that regulates the subcellular distribution and transcriptional activity of Yorkie. Yorkie is a transcriptional coactivator that promotes the expression of genes that inhibit apoptosis and drive cell proliferation. We review recent studies indicating that activity of the Hippo pathway is controlled by cell-cell junctions, cell adhesion molecules, scaffolding proteins, and cytoskeletal proteins, as well as by regulators of apical-basal polarity and extracellular tension.


Subject(s)
Drosophila Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/physiology , Animals , Apoptosis/physiology , Cell Adhesion Molecules/biosynthesis , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Polarity/physiology , Cell Proliferation , Drosophila Proteins/genetics , Drosophila melanogaster , Gene Expression Regulation/physiology , Intercellular Junctions/physiology , Intracellular Signaling Peptides and Proteins/genetics , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
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