Prediction of human O-linked glycosylation sites using stacked generalization and embeddings from pre-trained protein language model.

Motivation: O-linked glycosylation, an essential post-translational modification process in Homo sapiens, involves attaching sugar moieties to the oxygen atoms of serine and/or threonine residues. It influences various biological and cellular functions. While threonine or serine residues within protein sequences are potential sites for O-linked glycosylation, not all serine and/or threonine residues undergo this modification, underscoring the importance of characterizing its occurrence.

Integrated structural model of the palladin-actin complex using XL-MS, docking, NMR, and SAXS.

Unlabelled: Palladin is an actin binding protein that accelerates actin polymerization and is linked to metastasis of several types of cancer. Previously, three lysine residues in an immunoglobulin-like domain of palladin have been identified as essential for actin binding. However, it is still unknown where palladin binds to F-actin.

SumoPred-PLM: human SUMOylation and SUMO2/3 sites Prediction using Pre-trained Protein Language Model.

SUMOylation is an essential post-translational modification system with the ability to regulate nearly all aspects of cellular physiology. Three major paralogues SUMO1, SUMO2 and SUMO3 form a covalent bond between the small ubiquitin-like modifier with lysine residues at consensus sites in protein substrates. Biochemical studies continue to identify unique biological functions for protein targets conjugated to SUMO1 versus the highly homologous SUMO2 and SUMO3 paralogues.

LMNglyPred: prediction of human N-linked glycosylation sites using embeddings from a pre-trained protein language model.

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.

Functional comparison of full-length palladin to isolated actin binding domain.

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.

Conserved tryptophan mutation disrupts structure and function of immunoglobulin domain revealing unusual tyrosine fluorescence.

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.

Myopalladin promotes muscle growth through modulation of the serum response factor pathway.

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.

Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Infections.

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.

A course-based undergraduate research experience investigating the consequences of nonconserved mutations in lactate dehydrogenase.

There is a growing movement to involve undergraduate students in authentic research experiences. A variety of studies have indicated the strength of this approach in developing scientific aptitude, confidence, critical thinking skills, and increasing the likelihood to become career scientists. Course-based undergraduate research experiences (CUREs) foster opportunities for students to carry out authentic research at both primarily undergraduate and large research institutions.

Phosphoinositide Binding Inhibits Actin Crosslinking and Polymerization by Palladin.

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)P] is a well-known precursor for intracellular signaling and a bona fide regulator of actin cytoskeleton reorganization.

Actin polymerization is stimulated by actin cross-linking protein palladin.

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.

Protein-protein interaction analysis by nuclear magnetic resonance spectroscopy.

Nuclear magnetic resonance (NMR) has continued to evolve as a powerful method, with an increase in the number of pulse sequences and techniques available to study protein-protein interactions. In this chapter, a straightforward method to map a protein-protein interface and design a structural model is described, using chemical shift perturbation, paramagnetic relaxation enhancement, and data-driven docking.

Actin-induced dimerization of palladin promotes actin-bundling.

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.

Structure and function of palladin's actin binding domain.

Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking.

Structural characterization of the interactions between palladin and α-actinin.

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.

NMR structure of a fungal virulence factor reveals structural homology with mammalian saposin B.

The fungal protein CBP (calcium binding protein) is a known virulence factor with an unknown virulence mechanism. The protein was identified based on its ability to bind calcium and its prevalence as Histoplasma capsulatum's most abundant secreted protein. However, CBP has no sequence homology with other CBPs and contains no known calcium binding motifs.

Structural features responsible for the biological stability of Histoplasma's virulence factor CBP.

The virulence factor CBP is the most abundant protein secreted by Histoplasma capsulatum, a pathogenic fungus that causes histoplasmosis. Although the biochemical function and pathogenic mechanism of CBP are unknown, quantitative Ca (2+) binding measurements indicate that CBP has a strong affinity for calcium ( K D = 6.45 +/- 0.

Structural and functional characterization of the VirB5 protein from the type IV secretion system encoded by the conjugative plasmid pKM101.

Type IV secretion systems mediate intercellular transfer of macro-molecules via a mechanism ancestrally related to that of bacterial conjugation machineries. TraC of the IncN plasmid pKM101 belongs to the VirB5 family of proteins, an essential component of most type IV secretion systems. Here, we present the structure of TraC.

VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion.

The coupling of ATP binding/hydrolysis to macromolecular secretion systems is crucial to the pathogenicity of Gram-negative bacteria. We reported previously the structure of the ADP-bound form of the hexameric traffic VirB11 ATPase of the Helicobacter pylori type IV secretion system (named HP0525), and proposed that it functions as a gating molecule at the inner membrane, cycling through closed and open forms regulated by ATP binding/hydrolysis. Here, we combine crystal structures with analytical ultracentrifugation experiments to show that VirB11 ATPases indeed function as dynamic hexameric assemblies.