Authors: Thomas M Norman, Max A Horlbeck, Joseph M Replogle...

How cellular and organismal complexity emerges from combinatorial expression of genes is a central question in biology. High-content phenotyping approaches such as Perturb-seq (single-cell RNA-sequencing pooled CRISPR screens) present an opportunity for exploring such genetic interactions (GIs) at scale. Here, we present an analytical framework for interpreting high-dimensional landscapes of cell states (manifolds) constructed from transcriptional phenotypes. We applied this approach to Perturb-seq profiling of strong GIs mined from a growth-based, gain-of-function GI map. Exploration of this manifold enabled ordering of regulatory pathways, principled classification of GIs (e.g., identifying suppressors), and mechanistic elucidation of synergistic interactions, including an unexpected synergy between and driving erythroid differentiation. Finally, we applied recommender system machine learning to predict interactions, facilitating exploration of vastly larger GI manifolds.

Topics: Apoptosis; CRISPR-Cas Systems; Calcium-Binding Proteins; Cell Cycle Checkpoints; Cell Line, Tumor; Epistasis, Genetic; Erythroid Cells; Erythropoiesis; Female; Gene Expression Profiling; Granulocytes; Humans; Microfilament Proteins; Proto-Oncogene Proteins c-cbl; Sequence Analysis, RNA; Single-Cell Analysis; Calponins

PubMed: 31395745
DOI: 10.1126/science.aax4438

Review

Authors: Tzu-Bou Hsieh, J-P Jin

Calponin and transgelin (originally named SM22) are homologous cytoskeleton proteins that regulate actin-activated myosin motor functions in smooth muscle contraction and non-muscle cell motility during adhesion, migration, proliferation, phagocytosis, wound healing, and inflammatory responses. They are abundant cytoskeleton proteins present in multiple cell types whereas their physiological functions remain to be fully established. This focused review summarizes the evolution of genes encoding calponin and transgelin and their isoforms and discusses the structural similarity and divergence in vertebrate and invertebrate species in the context of functions in regulating cell motility. As the first literature review focusing on the evolution of the calponin-transgelin family of proteins in relevance to their structure-function relationship, the goal is to outline a foundation of current knowledge for continued investigations to understand the biological functions of calponin and transgelin in various cell types during physiological and pathological processes.

PubMed: 37363722
DOI: 10.3389/fcell.2023.1206147

Authors: Yuan Gui, Zachary Palanza, Priya Gupta...

Calponin 2 (CNN2) is a prominent actin stabilizer. It regulates fatty acid oxidation (FAO) by interacting with estrogen receptor 2 (ESR2) to determine kidney fibrosis. However, whether CNN2 is actively involved in acute kidney injury (AKI) remains unclear. Here, we report that CNN2 was induced in human and animal kidneys after AKI. Knockdown of CNN2 preserved kidney function, mitigated tubular cell death and inflammation, and promoted cell proliferation. Distinct from kidney fibrosis, proteomics showed that the key elements in the FAO pathway had few changes during AKI, but we identified that 3-hydroxymethylglutaryl-CoA synthase 2 (Hmgcs2), a rate-limiting enzyme of endogenous ketogenesis that promotes cell self-renewal, was markedly increased in CNN2-knockdown kidneys. The production of ketone body β-hydroxybutyrate and ATP was increased in CNN2-knockdown mice. Mechanistically, CNN2 interacted with ESR2 to negatively regulate the activities of mitochondrial sirtuin 5. Activated sirtuin 5 subsequently desuccinylated Hmgcs2 to produce energy for mitigating AKI. Understanding CNN2-mediated discrete fine-tuning of protein posttranslational modification is critical to optimize organ performance after AKI.

Topics: Animals; Humans; Mice; Acute Kidney Injury; Fibrosis; Ketone Bodies; Sirtuins; Calponins

PubMed: 37751293
DOI: 10.1172/jci.insight.170521

Review

Authors: Monica Rasmussen, J-P Jin

It is well known that chemical energy can be converted to mechanical force in biological systems by motor proteins such as myosin ATPase. It is also broadly observed that constant/static mechanical signals potently induce cellular responses. However, the mechanisms that cells sense and convert the mechanical force into biochemical signals are not well understood. Calponin and transgelin are a family of homologous proteins that participate in the regulation of actin-activated myosin motor activity. An isoform of calponin, calponin 2, has been shown to regulate cytoskeleton-based cell motility functions under mechanical signaling. The expression of the calponin 2 gene and the turnover of calponin 2 protein are both under mechanoregulation. The regulation and function of calponin 2 has physiological and pathological significance, as shown in platelet adhesion, inflammatory arthritis, arterial atherosclerosis, calcific aortic valve disease, post-surgical fibrotic peritoneal adhesion, chronic proteinuria, ovarian insufficiency, and tumor metastasis. The levels of calponin 2 vary in different cell types, reflecting adaptations to specific tissue environments and functional states. The present review focuses on the mechanoregulation of calponin and transgelin family proteins to explore how cells sense steady tension and convert the force signal to biochemical activities. Our objective is to present a current knowledge basis for further investigations to establish the function and mechanisms of calponin and transgelin in cellular mechanoregulation.

PubMed: 38515654
DOI: 10.1063/5.0176784

Authors: Matej Horvath, Adam Schrofel, Karolina Kowalska...

MICAL proteins play a crucial role in cellular dynamics by binding and disassembling actin filaments, impacting processes like axon guidance, cytokinesis, and cell morphology. Their cellular activity is tightly controlled, as dysregulation can lead to detrimental effects on cellular morphology. Although previous studies have suggested that MICALs are autoinhibited, and require Rab proteins to become active, the detailed molecular mechanisms remained unclear. Here, we report the cryo-EM structure of human MICAL1 at a nominal resolution of 3.1 Å. Structural analyses, alongside biochemical and functional studies, show that MICAL1 autoinhibition is mediated by an intramolecular interaction between its N-terminal catalytic and C-terminal coiled-coil domains, blocking F-actin interaction. Moreover, we demonstrate that allosteric changes in the coiled-coil domain and the binding of the tripartite assembly of CH-L2α1-LIM domains to the coiled-coil domain are crucial for MICAL activation and autoinhibition. These mechanisms appear to be evolutionarily conserved, suggesting a potential universality across the MICAL family.

Topics: Humans; Cryoelectron Microscopy; Protein Binding; LIM Domain Proteins; Actins; Models, Molecular; Protein Domains; Microfilament Proteins; Allosteric Regulation; Actin Cytoskeleton; Calponins; Mixed Function Oxygenases

PubMed: 39532862
DOI: 10.1038/s41467-024-54131-2

Review

Authors: T Stradal, W Kranewitter, S J Winder...

A sequence motif of about 100 amino acids, termed the 'calponin homology domain' has been suggested to confer actin binding to a variety of cytoskeletal and signalling molecules. Here we analyse and compare the sequences of all calponin homology domain-containing proteins identified to date. We propose that single calponin homology domains do not confer actin-binding per se and that the actin-binding motifs of cross-linking proteins, which comprise two disparate calponin homology domains, represent a unique protein module.

Topics: Actins; Amino Acid Sequence; Animals; Calcium-Binding Proteins; Databases, Factual; Humans; Microfilament Proteins; Molecular Sequence Data; Phylogeny; Protein Conformation; Sequence Homology, Amino Acid; Calponins

PubMed: 9708889
DOI: 10.1016/s0014-5793(98)00751-0

Authors: Yuan Gui, Yuanyuan Wang, Zachary Palanza...

OBJECTIVE

In the fibrotic kidneys, the extent of a formed deleterious microenvironment is determined by cellular mechanical forces. This process requires metabolism for energy. However, how cellular mechanics and metabolism are connected remains unclear.

METHODS

A multi-disciplinary approach was employed: the fibrotic kidney disease models were induced by renal ischemia-reperfusion injury and unilateral ureteral obstruction in Calponin 2 (CNN2) knockdown mice. Proteomics, bioinformatics, and in vivo and in vitro molecular experimental pathology studies were performed.

RESULT

Our proteomics revealed that actin filament binding and cell metabolism are the two most dysregulated events in the fibrotic kidneys. As a prominent actin stabilizer, CNN2 was predominantly expressed in fibroblasts and pericytes. In CKD patients, CNN2 levels was markedly induced in blood. In mice, CNN2 knockdown preserves kidney function and alleviates fibrosis. Global proteomics profiled that CNN2 knockdown enhanced the activities of the key rate-limiting enzymes and regulators of fatty acid oxidation (FAO) in the diseased kidneys. Inhibiting carnitine palmitoyltransferase 1α in the FAO pathway resulted in lipid accumulation and extracellular matrix deposition in the fibrotic kidneys, which were restored after CNN2 knockdown. Bioinformatics and chromatin immunoprecipitation showed that CNN2 interactor, estrogen receptor 2 (ESR2), binds peroxisome proliferator-activated receptor-α (PPARα) to transcriptionally regulate FAO downstream target genes expression amid kidney fibrosis. In vitro, ESR2 knockdown repressed the mRNA levels of PPARα and the key genes in the FAO pathway. Conversely, activation of PPARα reduced CNN2-induced matrix inductions.

CONCLUSIONS

Our results suggest that balancing cell mechanics and metabolism is crucial to develop therapeutic strategies to halt kidney fibrosis.

Topics: Animals; Mice; Fibrosis; Kidney; Kidney Diseases; PPAR alpha; Calmodulin-Binding Proteins; Calponins

PubMed: 36963615
DOI: 10.1016/j.molmet.2023.101712

Review

Authors: Rong Liu, J-P Jin

Calponin is an actin filament-associated regulatory protein expressed in smooth muscle and many types of non-muscle cells. Three homologous genes, CNN1, CNN2 and CNN3, encoding calponin isoforms 1, 2, and 3, respectively, are present in vertebrate species. All three calponin isoforms are actin-binding proteins with functions in inhibiting actin-activated myosin ATPase and stabilizing the actin cytoskeleton, while each isoform executes different physiological roles based on their cell type-specific expressions. Calponin 1 is specifically expressed in smooth muscle cells and plays a role in fine-tuning smooth muscle contractility. Calponin 2 is expressed in both smooth muscle and non-muscle cells and regulates multiple actin cytoskeleton-based functions. Calponin 3 participates in actin cytoskeleton-based activities in embryonic development and myogenesis. Phosphorylation has been extensively studied for the regulation of calponin functions. Cytoskeleton tension regulates the transcription of CNN2 gene and the degradation of calponin 2 protein. This review summarizes our knowledge learned from studies over the past three decades, focusing on the evolutionary lineage of calponin isoform genes, their tissue- and cell type-specific expressions, structure-function relationships, and mechanoregulation.

Topics: Actin Cytoskeleton; Calcium-Binding Proteins; Cell Movement; Humans; Mechanoreceptors; Microfilament Proteins; Muscle Contraction; Myocytes, Smooth Muscle; Myosins; Organ Specificity; Protein Isoforms; Structure-Activity Relationship; Calponins

PubMed: 26970176
DOI: 10.1016/j.gene.2016.02.040

Authors: Canxuan Li, Yunfei Xiao, Jianqiu Kong...

Molecule interacting with CasL 1 (MICAL1) is a crucial protein involved in cell motility, axon guidance, cytoskeletal dynamics, and gene transcription. This pan-cancer study analyzed MICAL1 across 33 cancer types using bioinformatics and experiments. Dysregulated expression, diagnostic potential, and prognostic value were assessed. Associations with tumor characteristics, immune factors, and drug sensitivity were explored. Enrichment analysis revealed MICAL1's involvement in metastasis, angiogenesis, metabolism, and immune pathways. Functional experiments demonstrated its impact on renal carcinoma cells. These findings position MICAL1 as a potential biomarker and therapeutic target in specific cancers, warranting further investigation into its role in cancer pathogenesis.

Topics: Humans; Carcinoma, Renal Cell; Cell Movement; Computational Biology; Cytoskeleton; Kidney Neoplasms; Calponins; Mixed Function Oxygenases; Microfilament Proteins

PubMed: 38555517
DOI: 10.1080/19336918.2024.2335682

Authors: Yifan Lu, Zhijian Jin, Junyi Hou...

The mechanical microenvironment regulated by cancer-associated fibroblasts (CAFs) influence tumor progression. Chemotherapeutic interventions including 5-Fluorouracil (5-Fu) are commonly used for primary treatment of patients with advanced gastric cancer (GC), and the development of acquired resistance to 5-Fu limits the clinical efficacy of these chemotherapies. However, if and how the interplay between CAFs and the mechanical microenvironment regulates GC response to 5-Fu is poorly understood. In this study, we demonstrate that high-level expression of calponin 1(CNN1) in gastric CAFs predicts poor clinical outcomes of GC patients, especially for those treated with 5-Fu. CNN1 knockdown in CAFs improves the effectiveness of 5-Fu in reducing tumor growth in a mouse GC model and confers increased sensitivity to 5-Fu in a 3D culture system. Furthermore, CNN1 knockdown impairs CAF contraction and reduces matrix stiffness without affecting the expression of matrix proteins. Mechanistically, CNN1 interacts with PDZ and LIM Domain 7 (PDLIM7) and prevents its degradation by the E3 ubiquitin ligase NEDD4-1, which leads to activation of the ROCK1/MLC pathway. The increased matrix stiffness, in turn, contributes to 5-Fu resistance in GC cells by activating YAP. Taken together, our data reveal a critical role of the mechanical microenvironment in 5-Fu resistance, which is modulated by CNN1 CAFs-mediated matrix stiffening, indicating that targeting CAFs may provide a novel option for overcoming drug resistance in GC.

Topics: Animals; Mice; Stomach Neoplasms; Drug Resistance, Neoplasm; Cancer-Associated Fibroblasts; Cell Line, Tumor; Fluorouracil; Tumor Microenvironment; Calponins

PubMed: 36423735
DOI: 10.1016/j.matbio.2022.11.005