Palladin is an actin binding protein that is specifically upregulated in metastatic cancer cells but also colocalizes with actin stress fibers in normal cells and is critical for embryonic ...development as well as wound healing. Of nine isoforms present in humans, only the 90 kDa isoform of palladin, comprising three immunoglobulin (Ig) domains and one proline‐rich region, is ubiquitously expressed. Previous work has established that the Ig3 domain of palladin is the minimal binding site for F‐actin. In this work, we compare functions of the 90 kDa isoform of palladin to the isolated actin binding domain. To understand the mechanism of action for how palladin can influence actin assembly, we monitored F‐actin binding and bundling as well as actin polymerization, depolymerization, and copolymerization. Together, these results demonstrate that there are key differences between the Ig3 domain and full‐length palladin in actin binding stoichiometry, polymerization, and interactions with G‐actin. Understanding the role of palladin in regulating the actin cytoskeleton may help us develop means to prevent cancer cells from reaching the metastatic stage of cancer progression.
Actin cytoskeleton remodeling requires the coordinated action of a large number of actin binding proteins that reorganize the actin cytoskeleton by promoting polymerization, stabilizing filaments, ...causing branching, or crosslinking filaments. Palladin is a key cytoskeletal actin binding protein whose normal function is to enable cell motility during development of tissues and organs of the embryo and in wound healing, but palladin is also responsible for regulating the ability of cancer cells to become invasive and metastatic. The membrane phosphoinositide phosphatidylinositol (PI) 4,5-bisphosphate PI(4,5)P2 is a well-known precursor for intracellular signaling and a bona fide regulator of actin cytoskeleton reorganization. Our results show that two palladin domains immunoglobulin (Ig) 3 and 34 interact with the head group of PI(4,5)P2 with moderate affinity (apparent Kd=17μM). Interactions with PI(4,5)P2 decrease the actin polymerizing activity of Ig domain 3 of palladin (Palld-Ig3). Furthermore, NMR titration and docking studies show that residues K38 and K51, which are present on the β-sheet C and D, form salt bridges with the head group of PI(4,5)P2. Moreover, charge neutralization at lysine 38 in the Palld-Ig3 domain severely limits the actin polymerizing and bundling activity of Palld-Ig3. Our results provide biochemical proof that PI(4,5)P2 functions as a moderator of palladin activity and have also identified residues directly involved in the crosslinking activity of palladin.
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•Actin binding and crosslinking by palladin has been linked to cellular migration.•Here, we investigated the role of phospholipids in regulating palladin function.•We found that PI(4,5)P2 interacts with palladin and decreases the rate of actin polymerization.•NMR and docking studies pinpoint electrostatic interactions.•Regulation of palladin occurs via alteration of its actin crosslinking activity.
The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential ...effect of palladin on actin dynamics has remained elusive. In the present study, we show that the actin-binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro cross-linking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes.
Abstract
Protein N-linked glycosylation is an important post-translational mechanism in Homo sapiens, playing essential roles in many vital biological processes. It occurs at the N-X-S/T sequon in ...amino acid sequences, where X can be any amino acid except proline. However, not all N-X-S/T sequons are glycosylated; thus, the N-X-S/T sequon is a necessary but not sufficient determinant for protein glycosylation. In this regard, computational prediction of N-linked glycosylation sites confined to N-X-S/T sequons is an important problem that has not been extensively addressed by the existing methods, especially in regard to the creation of negative sets and leveraging the distilled information from protein language models (pLMs). Here, we developed LMNglyPred, a deep learning-based approach, to predict N-linked glycosylated sites in human proteins using embeddings from a pre-trained pLM. LMNglyPred produces sensitivity, specificity, Matthews Correlation Coefficient, precision, and accuracy of 76.50, 75.36, 0.49, 60.99, and 75.74 percent, respectively, on a benchmark-independent test set. These results demonstrate that LMNglyPred is a robust computational tool to predict N-linked glycosylation sites confined to the N-X-S/T sequon.
A subset of actin binding proteins is able to form crosslinks between two or more actin filaments, thus producing structures of parallel or networked bundles. These actin crosslinking proteins ...interact with actin through either bivalent binding or dimerization. We recently identified two binding sites within the actin binding domain of palladin, an actin crosslinking protein that plays an important role in normal cell adhesion and motility during wound healing and embryonic development. In this study, we show that actin induces dimerization of palladin. Furthermore, the extent of dimerization reflects earlier comparisons of actin binding and bundling between different domains of palladin. On the basis of these results we hypothesized that actin binding may promote a conformational change that results in dimerization of palladin, which in turn may drive the crosslinking of actin filaments. The proximal distance between two actin binding sites on crosslinking proteins determines the ultrastructural properties of the filament network, therefore we also explored interdomain interactions using a combination of chemical crosslinking experiments and actin cosedimentation assays. Limited proteolysis data reveals that palladin is less susceptible to enzyme digestion after actin binding. Our results suggest that domain movements in palladin are necessary for interactions with actin and are induced by interactions with actin filaments. Accordingly, we put forth a model linking the structural changes to functional dynamics.
Immunoglobulin (Ig) domains are the most prevalent protein domain structure and share a highly conserved folding pattern; however, this structural family of proteins is also the most diverse in terms ...of biological roles and tissue expression. Ig domains vary significantly in amino acid sequence but share a highly conserved tryptophan in the hydrophobic core of this beta‐stranded protein. Palladin is an actin binding and bundling protein that has five Ig domains and plays an important role in normal cell adhesion and motility. Mutation of the core tryptophan in one Ig domain of palladin has been identified in a pancreatic cancer cell line, suggesting a crucial role for this sole tryptophan in palladin Ig domain structure, stability, and function. We found that actin binding and bundling was not completely abolished with removal of this tryptophan despite a partially unfolded structure and significantly reduced stability of the mutant Ig domain as shown by circular dichroism investigations. In addition, this mutant palladin domain displays a tryptophan‐like fluorescence attributed to an anomalous tyrosine emission at 341 nm. Our results indicate that this emission originates from a tyrosinate that may be formed in the excited ground state by proton transfer to a nearby aspartic acid residue. Furthermore, this study emphasizes the importance of tryptophan in protein structural stability and illustrates how tyrosinate emission contributions may be overlooked during the interpretation of the fluorescence properties of proteins.
Background
Myopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene ...mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe.
Methods
Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro.
Results
MKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway.
Conclusions
Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.
Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays
Here we examine the role of palladin during
infections in order to tease out novel ...functions of palladin. We show that palladin is co-opted by
during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells,
failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of
to generate comet tails was restored. Using purified protein components, we demonstrate that
actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility.
Structures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made by
Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex.
The interaction between α-actinin and palladin, two actin-cross-linking proteins, is essential for proper bidirectional targeting of these proteins. As a first step toward understanding the role of ...this complex in organizing cytoskeletal actin, we have characterized binding interactions between the EF-hand domain of α-actinin (Act-EF34) and peptides derived from palladin and generated an NMR-derived structural model for the Act-EF34/palladin peptide complex. The critical binding site residues are similar to an α-actinin binding motif previously suggested for the complex between Act-EF34 and titin Z-repeats. The structure-based model of the Act-EF34/palladin peptide complex expands our understanding of binding specificity between the scaffold protein α-actinin and various ligands, which appears to require an α-helical motif containing four hydrophobic residues, common to many α-actinin ligands. We also provide evidence that the Family X mutation in palladin, associated with a highly penetrant form of pancreatic cancer, does not interfere with α-actinin binding.
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► NMR-derived structural model for complex of α-actinin and palladin. ►Structural perturbations of α-actinin upon binding palladin are similar to those of titin. ►Evidence for unique binding motif present in many α-actinin ligands.
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