Souvik Chakraborty

Distinct and separable activities of the endocytic clathrin coat components Fcho1/2 and AP-2 in developmental patterning

Clathrin-mediated endocytosis occurs at multiple independent import sites on the plasma membrane, but how these positions are selected and how different cargo is simultaneously recognized is obscure. FCHO1 and FCHO2 are early-arriving proteins at surface clathrin assemblies and are speculated to act as compulsory coat nucleators, preceding the core clathrin adaptor AP-2. Here, we show that the μ-homology domain of FCHO1/2 represents an endocytic interaction hub. Translational silencing of fcho1 in zebrafish embryos causes strong dorsoventral patterning defects analogous to Bmp signal failure. The Fcho1 μ-homology domain interacts with the Bmp receptor Alk8, uncovering an endocytic component that positively modulates Bmp signal transmission. Still, the fcho1 morphant phenotype is distinct from severe embryonic defects apparent when AP-2 is depleted. Our data thus challenge the primacy of FCHO1/2 in coat initiation.

E-cadherin Differentially Regulates the Assembly of Connexin43 and Connexin32 into Gap Junctions in Human Squamous Carcinoma Cells

It is as yet unknown how the assembly of connexins (Cx) into gap junctions (GJ) is initiated upon cell-cell contact. We investigated whether the trafficking and assembly of Cx43 and Cx32 into GJs were contingent upon cell-cell adhesion mediated by E-cadherin. We also examined the role of the carboxyl termini of these Cxs in initiating the formation of GJs. Using cadherin and Cx-null cells, and by introducing Cx43 and Cx32, either alone or in combination with E-cadherin, our studies demonstrated that E-cadherin-mediated cell-cell adhesion was neither essential nor sufficient to initiate GJ assembly de novo in A431D human squamous carcinoma cells. However, E-cadherin facilitated the growth and assembly of preformed GJs composed of Cx43, although the growth of cells on Transwell filters was required to initiate the assembly of Cx32. Our results also documented that the carboxyl termini of both Cxs were required in this cell type to initiate the formation of GJs de novo. Our findings also showed that GJ puncta composed of Cx43 co-localized extensively with ZO-1 and actin fibers at cell peripheries and that ZO-1 knockdown attenuated Cx43 assembly. These findings suggest that the assembly of Cx43 and Cx32 into GJs is differentially modulated by E-cadherin-mediated cell-cell adhesion and that direct or indirect cross-talk between carboxyl tails of Cxs and actin cytoskeleton via ZO-1 may regulate GJ assembly and growth.

AMN Directs Endocytosis of the Intrinsic Factor-Vitamin B12 Receptor Cubam by Engaging ARH or Dab2

Cubam is a multi‐ligand receptor involved in dietary uptake of intrinsic factor‐vitamin B12 in the small intestine and reabsorption of various low‐molecular‐weight proteins (such as albumin, transferrin, apolipoprotein A‐I and vitamin D‐binding protein) in the kidney. Cubam is composed of two proteins: cubilin and amnionless. Cubilin harbors ligand binding capabilities, while amnionless provides membrane anchorage and potential endocytic capacity via two FXNPXF signals within the cytosolic domain. These signals are similar to the FXNPXY signals found in members of the low‐density lipoprotein receptor superfamily, which associate with clathrin‐associated sorting proteins, including Disabled‐2 (Dab2) and autosomal recessive hypercholesterolemia (ARH), during endocytosis. We therefore investigated the functionality of each amnionless FXNPXF signal and their respective interaction with sorting proteins. By sequential mutation and expression of a panel of amnionless mutants combined with yeast two‐hybrid analyses, we demonstrate that the signals are functionally redundant and both are able to mediate endocytosis of cubam through interaction with Dab2 and ARH.

Assembly of connexin43 into gap junctions is regulated differentially by E-cadherin and N-cadherin in rat liver epithelial cells.

E-cadherin and N-cadherin affect the assembly of connexin43 into gap junctions differentially. N-cadherin disrupts assembly by triggering endocytosis of connexin43, whereas E-cadherin facilitates the assembly.

A balanced level of profilin-1 promotes stemness and tumor-initiating potential of breast cancer cells

ABSTRACT Profilin-1 (Pfn1) is an important actin-regulatory protein that is downregulated in human breast cancer and when forcibly elevated, it suppresses the tumor-initiating ability of triple-negative breast cancer cells. In this study, we demonstrate that Pfn1 overexpression reduces the stem-like phenotype (a key biologic feature associated with higher tumor-initiating potential) of MDA-MB-231 (MDA-231) triple-negative breast cancer cells. Interestingly, the stem-like trait of MDA-231 cells is also attenuated upon depletion of Pfn1. A comparison of cancer stem cell gene (CSC) gene expression signatures between depleted and elevated conditions of Pfn1 further suggest that Pfn1 may be somehow involved in regulating the expression of a few CSC-related genes including MUC1, STAT3, FZD7, and ITGB1. Consistent with the reduced stem-like phenotype associated with loss-of-function of Pfn1, xenograft studies showed lower tumor-initiating frequency of Pfn1-depleted MDA-231 cells compared to their control counterparts. In MMTV:PyMT mouse model, homozygous but not heterozygous deletion of Pfn1 gene leads to severe genetic mosaicism and positive selection of Pfn1-proficient tumor cells further supporting the contention that a complete lack of Pfn1 is likely not conducive for efficient tumor initiation capability of breast cancer cells. In summary, these findings suggest that the maintenance of optimal stemness and tumor-initiating ability of breast cancer cells requires a balanced expression of Pfn1.

Liver Disease in α1-Antitrypsin Deficiency

Liver disease affects a subgroup of homozygotes for the classical form of α1-antitrypsin deficiency (ATD). There are at least four forms of this disease with onset in infancy, in childhood, in adolescence, and at 50–65 years of age. The liver disease is mostly slowly progressive and characterized by fibrosis and cirrhosis with minimal inflammation. These patients also have increased susceptibility to hepatocellular carcinoma (HCC). The pathobiology of liver injury in ATD is not completely understood, but we know that it involves a gain-of-function mechanism in which accumulation of misfolded α1-antitrypsin Z (ATZ) in the early compartments of the secretory pathway is proteotoxic for the liver cells. Intracellular pathways for degradation of misfolded ATZ, particularly the autophagic pathway, are believed to play a critical role, and FDA-approved drugs that enhance autophagy and can be repurposed appear to be attractive candidates for treatment. Several other promising drug-, cell-, or gene-based therapeutic strategies are being developed for this liver disease.

A Phosphotyrosine Switch for Cargo Sequestration at Clathrin-Coated Buds

Background: The AP-2 adaptor is a central protein-protein interaction hub during clathrin-mediated endocytosis. Results: Introducing a specific phosphotyrosine mimetic on the large AP-2 β2 subunit selectively regulates protein interactions with the adaptor complex. Conclusion: A directed post-translational modification can locally rewire the AP-2 interaction network. Significance: This provides a plausible mechanism for regional cargo uncoupling at cell surface clathrin coats. The AP-2 clathrin adaptor complex oversees endocytic cargo selection in two parallel but independent manners. First, by physically engaging peptide-based endocytic sorting signals, a subset of clathrin-dependent transmembrane cargo is directly collected into assembling buds. Synchronously, by interacting with an assortment of clathrin-associated sorting proteins (CLASPs) that independently select different integral membrane cargo for inclusion within the incipient bud, AP-2 handles additional cargo capture indirectly. The distal platform subdomain of the AP-2 β2 subunit appendage is a privileged CLASP-binding surface that recognizes a cognate, short α-helical interaction motif. This signal, found in the CLASPs β-arrestin and the autosomal recessive hypercholesterolemia (ARH) protein, docks into an elongated groove on the β2 appendage platform. Tyr-888 is a critical constituent of this spatially confined β2 appendage contact interface and is phosphorylated in numerous high-throughput proteomic studies. We find that a phosphomimetic Y888E substitution does not interfere with incorporation of expressed β2-YFP subunit into AP-2 or alter AP-2 deposition at surface clathrin-coated structures. The Y888E mutation does not affect interactions involving the sandwich subdomain of the β2 appendage, indicating that the mutated appendage is folded and operational. However, the Y888E, but not Y888F, switch selectively uncouples interactions with ARH and β-arrestin. Phyogenetic conservation of Tyr-888 suggests that this residue can reversibly control occupancy of the β2 platform-binding site and, hence, cargo sorting.

Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells

BackgroundAdhesion-mediated activation of FAK/ERK signalling pathway, enabled by the formation of filopodial protrusions (FLP), has been shown to be an important event for triggering of dormancy-to-proliferation switch and metastatic outgrowth of breast cancer cells (BCC). We studied the role of actin-binding protein profilin1 (Pfn1) in these processes.MethodsQuantitative immunohistochemistry (IHC) of BC tissue microarray (TMA) and survival analyses of curated transcriptome datasets of BC patients were performed to examine Pfn1’s association with certain clinicopathological features. FLP formation and single cell outgrowth of BCC were assessed using a 3D matrigel culture that accurately predicts dormant vs metastatic outgrowth phenotypes of BCC in certain microenvironment. Gene expression studies were performed to identify potential biological pathways that are perturbed under Pfn1-depleted condition.ResultsLower Pfn1 expression is correlated with lower nuclear grade of breast tumours and longer relapse-free survival of BC patients. Pfn1 depletion leads to defects in FLP and outgrowth of BCC but without impairing either FAK or ERK activation. Guided by transcriptome analyses, we further showed that Pfn1 depletion is associated with prominent SMAD3 upregulation. Although knockdown and overexpression experiments revealed that SMAD3 has an inhibitory effect on the outgrowth of breast cancer cells, SMAD3 knockdown alone was not sufficient to enhance the outgrowth potential of Pfn1-depleted BCC suggesting that other proliferation-regulatory pathways in conjunction with SMAD3 upregulation may underlie the outgrowth-deficient phenotype of BCC cells upon depletion of Pfn1.ConclusionOverall, these data suggest that Pfn1 may be a novel biomarker for BC recurrence and a possible target to reduce metastatic outgrowth of BCC.

Alpha-1-Antitrypsin Deficiency: A Misfolded Secretory Glycoprotein Damages the Liver by Proteotoxicity and Its Reduced Secretion Predisposes to Emphysematous Lung Disease Because of Protease-Inhibitor Imbalance

In the classical form of α1-antitrypsin deficiency (ATD) a point mutation leads to protein misfolding such that the mutant protein accumulates in liver cells with a marked decrease in levels of this protein in the blood and body fluids. Because the major function of α1-antitrypsin is inhibition of neutrophil elastase and several other neutrophil proteases, individuals with ATD are susceptible to destructive lung disease/emphysema, now called chronic obstructive pulmonary disease (COPD), by a loss-of-function mechanism in which uninhibited neutrophil proteases destroy the connective tissue matrix of the lung. Homozygous individuals are also susceptible to liver disease by a gain-of-toxic function mechanism triggered by the intracellular accumulation of the misfolded protein. The only therapeutic strategies currently available are protein replacement therapy and lung transplantation for COPD and liver transplantation for the subgroup with progressive liver involvement. New therapeutic strategies that mitigate the intracellular accumulation/proteotoxicity have advanced to clinical trials and corrective genetic strategies are in earlier pre-clinical phases of development.