Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA

Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies

Effects of Cisplatin in Neuroblastoma Rat Cells: Damage to Cellular Organelles

Cisplatin (cisPt) is a chemotherapy agent used as a treatment for several types of cancer. The main cytotoxic effect of cisplatin is generally accepted to be DNA damage. Recently, the mechanism by which cisPt generates the cascade of events involved in the apoptotic process has been demonstrated. In particular it has been shown that some organelles are cisPt target and are involved in cell death. This paper aims to describe the morphological and functional changes of the Golgi apparatus and lysosomes during apoptosis induced in neuronal rat cells (B50) by cisplatin. The results obtained show that the cellular organelles are the target of cisPt, so their damage can induce cell death

Deconstruction of Neurotrypsin Reveals a Multi-factorially Regulated Activity Affecting Myotube Formation and Neuronal Excitability

Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called "NT-mini" and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT's heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions

A Fe2+-dependent self-inhibited state influences the druggability of human collagen lysyl hydroxylase (LH/PLOD) enzymes

Multifunctional human collagen lysyl hydroxylase (LH/PLOD) enzymes catalyze post-translational hydroxylation and subsequent glycosylation of collagens, enabling their maturation and supramolecular organization in the extracellular matrix (ECM). Recently, the overexpression of LH/PLODs in the tumor microenvironment results in abnormal accumulation of these collagen post-translational modifications, which has been correlated with increased metastatic progression of a wide variety of solid tumors. These observations make LH/PLODs excellent candidates for prospective treatment of aggressive cancers. The recent years have witnessed significant research efforts to facilitate drug discovery on LH/PLODs, including molecular structure characterizations and development of reliable high-throughput enzymatic assays. Using a combination of biochemistry and in silico studies, we characterized the dual role of Fe2+ as simultaneous cofactor and inhibitor of lysyl hydroxylase activity and studied the effect of a promiscuous Fe2+ chelating agent, 2,2'-bipyridil, broadly considered a lysyl hydroxylase inhibitor. We found that at low concentrations, 2,2'-bipyridil unexpectedly enhances the LH enzymatic activity by reducing the inhibitory effect of excess Fe2+. Together, our results show a fine balance between Fe2+-dependent enzymatic activity and Fe2+-induced self-inhibited states, highlighting exquisite differences between LH/PLODs and related Fe2+, 2-oxoglutarate dioxygenases and suggesting that conventional structure-based approaches may not be suited for successful inhibitor development. These insights address outstanding questions regarding druggability of LH/PLOD lysyl hydroxylase catalytic site and provide a solid ground for upcoming drug discovery and screening campaigns

Full‐Length Human Collagen Lysyl Hydroxylases

Procollagen lysyl hydroxylases and glycosyltransferases (LH, also known as procollagen lysyl‐2‐oxoglutarate dioxygenases (PLOD)) are essential biosynthesis enzymes present in all collagen‐containing organisms, from sponges to humans. Higher vertebrates present three separate PLOD genes encoding for distinct enzyme isoforms (LH1, LH2a/b, and LH3), sharing ∼70% amino acid sequence identity. The LH1 and LH2 isoforms exclusively display Fe2+, 2‐oxoglutarate‐dependent lysyl 5‐hydroxylase activity, whereas LH3 is a multifunctional enzyme, able to further catalyze the Mn2+‐dependent β‐(1,O)‐galactosylation and the subsequent α‐(1,2)‐glucosylation of 5‐hydroxylysines. Despite exclusive selectivity for lysine residues within collagenous polypeptides, little is known about the specificity of LH enzymes for different amino acid sequences in different collagen types: LH1 and LH3 isoforms act on collagen triple‐helical regions, whereas the LH2 isoform specifically hydroxylates collagen telopeptides, yet no consensus sequences, nor minimum sequence lengths, have been proposed as requirements for catalysis. Available crystal structures of full‐length human LH3 show an elongated homodimeric quaternary structure, with three aligned domains constituting each enzyme's polypeptide: the N‐terminal glycosyltransferase (GT) domain, a central noncatalytic accessory (AC) domain, and a C‐terminal lysyl hydroxylase (LH) domain. Dimerization occurs in the C‐terminal domain, in proximity to the LH catalytic site. Dimerization is indeed essential for LH activity, but is dispensable for the glycosyltransferase activities of LH3

Modeling of Protein Complexes

: The recent advances in structural biology, combined with continuously increasing computational capabilities and development of advanced softwares, have drastically simplified the workflow for protein homology modeling. Modeling of individual proteins is nowadays quick and straightforward for a large variety of protein targets, thanks to guided pipelines relying on advanced computational tools and user-friendly interfaces, which have extended and promoted the use of modeling also to scientists not focusing on molecular structures of proteins. Nevertheless, construction of models of multi-protein complexes remains quite challenging for the non-experts, often due to the usage of specific procedures depending on the system under investigation and the need for experimental validation approaches to strengthen the generated output.In this chapter, we provide a brief overview of the approaches enabling generation of multi-protein complex models starting from homology models of individual protein components. Using real-life examples, we include two examples to guide the reader in the generation of homomeric and heteromeric protein models

Collagen hydroxylysine glycosylation: non-conventional substrates for atypical glycosyltransferase enzymes

Collagen is a major constituent of the extracellular matrix (ECM) that confers fundamental mechanical properties to tissues. To allow proper folding in triple-helices and organization in quaternary super-structures, collagen molecules require essential post-translational modifications (PTMs), including hydroxylation of proline and lysine residues, and subsequent attachment of glycan moieties (galactose and glucose) to specific hydroxylysine residues on procollagen alpha chains. The resulting galactosyl-hydroxylysine (Gal-Hyl) and less abundant glucosyl-galactosyl-hydroxylysine (Glc-Gal-Hyl) are amongst the simplest glycosylation patterns found in nature and are essential for collagen and ECM homeostasis. These collagen PTMs depend on the activity of specialized glycosyltransferase enzymes. Although their biochemical reactions have been widely studied, several key biological questions about the possible functions of these essential PTMs are still missing. In addition, the lack of three-dimensional structures of collagen glycosyltransferase enzymes hinders our understanding of the catalytic mechanisms producing this modification, as well as the impact of genetic mutations causing severe connective tissue pathologies. In this mini-review, we summarize the current knowledge on the biochemical features of the enzymes involved in the production of collagen glycosylations and the current state-of-the-art methods for the identification and characterization of this important PTM

SiMPLOD, a structure-integrated database of collagen lysyl hydroxylase (LH/PLOD) enzyme variants

PLOD genes encode for procollagen lysyl hydroxylase enzymes (LH/PLOD), a family of proteins essential for collagen biosynthesis. Several mutations affect these genes causing severe disorders, such as Ehlers-Danlos and Bruck syndrome, as well a connective tissue disease with phenotype resembling osteogenesis imperfecta caused by lack of LH3 functions. The recently determined three-dimensional structures of the full-length human LH3/PLOD3 isoform, together with the structure of a fragment of a viral LH/PLOD homolog, are now allowing molecular mapping of the numerous disease-causing mutations, providing insights often suitable for the interpretation of the resulting disease phenotypes. However, the added value of molecular structure interpretation is affected by the limited accessibility of complex molecular data to scientific communities lacking direct expertise in structural biology. In this work, we present SiMPLOD (Structurally-integrated database for Mutations of PLOD genes), a publicly-available manually-curated online database with an embedded molecular viewer interface for the visualization and interpretation of LH/PLOD mutations on available molecular models. Each SiMPLOD entry is accompanied by manual annotations extrapolated from literature references and comments about the localization of the amino acid variants on the molecular structure. Additional links to the appropriate online resources for clinically-relevant as well as biochemical data are also provided in a standardized format. The web application is available at http://fornerislab.unipv.it/SiMPLOD. This article is protected by copyright. All rights reserved

Expression and purification of the heme exporter FLVCR1a

With many crucial roles in enzymatic aerobic metabolism, the concentration of the heme must be tightly regulated. The heme exporter Feline Leukemia Virus sub-group C Receptor 1a (FLVCR1a), an integral membrane protein with twelve transmembrane helices, is a key player in the maintenance of cellular heme homeostasis. It was first identified as the host receptor for the Feline Leukemia Virus sub-group C (FeLV-C), a retrovirus causing hematological abnormalities in cats and other felines. Mutations in the Flvcr1 were later identified in human patients affected by Posterior Column Ataxia and Retinitis Pigmentosa (PCARP) and Hereditary Sensory and Autonomic Neuropathies (HSANs). Despite being an essential component in heme balance, currently there is a lack in the understanding of its function at the molecular level, including the effect of disease-causing mutations on protein function and structure. Therefore, there is a need for protocols to achieve efficient recombinant production yielding milligram amounts of highly pure protein to be used for biochemical and structural studies. Here, we report the first FLVCR1a reliable protocol suitable for both antibody generation and structural characterisation