Internal fragments of proteins and their interactions with biological membranes

 

Biomolecules, such as carbohydrates, lipids, and proteins, are modular in nature. Although it is known for decades that the building blocks of proteins are amino acids, regularities in the many levels of organization of protein structure suggest that repetitive motifs are ubiquitous in the structural hierarchy of proteins. Indeed, several proteins seem to be composed by the joining of multiple domains, which are structurally and functionally independent of the remaining parts of the protein.

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For decades, Dr Carlos Bloch Jr., my PhD supervisor, has emphasized that proteins are richer sources of encrypted bioactive peptides than anticipated. Indeed, some proteins might be regarded as chimaeras of multiple bioactive peptides joined head-to-tail, which can be decrypted either in vivo, by various proteases, or even in silico, taking advantage of computer softwares and large protein databases. Given the expertise of the Laboratório de Espectrometria de Massa (LEM) at Embrapa Genetic Resources and Biotechnology in the biochemical/biophysical characterization of Antimicrobial Peptides (AMPs) from the skin secretion of Brazilian hylid frogs, it was clear that internal fragments of proteins with membrane tropism should be useful as proof-of-concepts.

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While working at Embrapa, a two-component system was developed: (1) a bioinformatic tool, named Kamal, which searches protein databases for fragments using a predefined set of physicochemical properties; and (2) an experimental classification tool to identify sets of IAPs which induce comparable disturbances in model phospholipid membranes. Both “tools” are under constant development, as they complement each other. For instance, Kamal was thoroughly modified and recently released to the public with the invaluable contribution of Dr. André Melro Murad and Dr. José Cardozo-Fh, both from Embrapa. Kamal can be found in this link (http://alelobag.cenargen.embrapa.br/Kamal/).

 

This line of research stems from the work carried  by the LEM group, and the LSAB is working, along with many collaborators, on the unveiling of the biotechnological potential of membrane active fragment of proteins. We have focused in the study of Intragenic Antimicrobial Peptides (IAPs) in human proteins and their biotechnological application in human health not only as direct antimicrobial agents, but also as anti-inflammatory molecules. Please find below some of the projects under development.

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1.1. α-helical intragenic antimicrobial peptides (IAPs) of human proteins

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Many examples of human proteins containing encrypted antimicrobial peptides, which are released upon hydrolysis in physiologically relevant scenarios, can be encountered in the literature. The cathelicidin LL-37, derived from the protein hCAP18, is the best-known example. We are currently exploring the human proteome in order to uncover the biotechnological potential of protein fragments that interact with membranes and may produce membrane adsorption or even lysis. This line of research resulted in a first publication, in which IAPs were prospected using the software Kamal, synthesized by solid phase chemistry, and evaluated using biophysical assays with model membranes and antimicrobial tests. A three-dimensional solution structure of one protein internal fragment obtained from the unconventional myosin 1h, denominated Hs02, was determined by Nuclear Magnetic Resonance when bound to DPC micelles, in collaboration with the Laboratório de Ressonância Magnética Nuclear, headed by Dr. Aline Oliveira, of the University of Brasilia. Besides relevant antimicrobial activity, Hs02 was shown to be a potent inhibitor of lipopolysaccharide-induced release of Tumor Necrosis Factor-α (TNF-α). This work exemplifies how the human genome can be mined for molecules with biotechnological potential in human health and demonstrates that IAPs are actual alternatives to antimicrobial peptides as pharmaceutical agents or in their many other putative applications.

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Although IAPs are an interesting alternative to AMPs, they might also be subject to similar shortcomings when it comes to their application in vivo. We are currently developing a platform for the sustained release of IAPs in collaboration with Prof. Juliano Chaker from UnB, Faculdade de Ceilândia. Organic-inorganic hybrid materials, more specifically, ureasil-polyether hybrid films, are being loaded with and used for the release of one novel IAP uncovered from the human genome. This work is the effort of the undergraduate Gabriel Mariano and a former MsC student, Luis Guilherme, and has resulted in a dissertation and an undergraduate thesis, both available in the Publications section. 

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1.2. β-strand intragenic antimicrobial peptides (IAPs) of human proteins

 

Some proteins contain α-helical cationic amphiphilic segments as parts of their structure, constituting putative IAPs. A subset of these segments, once synthesized as individual entities, are capable of membrane adsorption and lysis, as demonstrated in previous publications. However, amphiphilic β-sheets are also encountered in proteins, and these might also be potentially membrane active, constituting what we may tentatively call β-IAPs. A manuscript is under preparation by a PhD student, Michele A dos Santos. Additionally, β-strands from several proteins have been prospected using the software Kamal and were synthesized to investigate whether the binding between these β-IAPs and LPS are capable to prevent the pro-inflammatory effects of the latter in cells. This project is under development by MsC student Fernanda Leonel in collaboration with other laboratories.

 

1.3. Intragenic Antimicrobial Peptides as a first line of defense against emergent viruses 

 

SARS-CoV-2 is a zoonotic virus that is causing considerable damage to public human health and consequently to the world economy. At the onset of the COVID-19 outbreak, no effective antiviral molecules were available, turning the attention of the scientific community to repurposed molecules with broad antiviral mechanisms, such as hydroxychloroquine. Many researchers have urged the necessity for broadly acting antiviral agents to tackle infections caused by emergent viruses, and peptides are interesting candidates. This is a new line of research that aims to investigate whether membrane disrupting IAPs can inhibit the infectivity of enveloped viruses like SARS-CoV-2 in Vero cells with little cytotoxicity. It counts on the collaboration of national and international researchers, such as Prof. José Roberto Leite from the Faculty of Medicine at UnB, to conduct in vitro assays.

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1.4. Rational design of membrane associating chimaeric peptides and the release of bioactive fragments at the membrane interface.

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Some IAPs have a strong yet superficial interaction with negatively charged phospholipid membranes. Thus, these can be used for the rational design of molecular constructs as carriers, capable of preferential accumulation in prokaryotic membranes while holding other bioactive portions to be released by the enzymatic arsenal on site. This work is currently under development by a PhD student, Thiago Vianna, as well as several collaborators.

 

The optimization of the enzyme hydrolytic site to use enzymes produced and/or secreted by the microorganisms themselves should be an interesting alternative to increase the selectivity of the on-site bioactive peptide production. This project is currently under development by the PhD student Samuel Costa.

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Mass Spectrometric analysis of N- and O-linked glycans.

 

2.1. Analysis of N- and O-glycans in Type II Congenital Disorders of Glycosylation patients

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This line of research is new to LSAB and stems from a collaboration with PhD MD Jaime Brum, the head of the Molecular Pathology laboratory at the Sarah Network of Rehabilitation Hospitals.

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Congenital disorders of glycosylation (CDG) are a rare but expanding group of diseases with challenging diagnostics, especially in Brazil. Currently, there are more than 130 disorders involving genetic defects of glycosylation, and these organized in two groups: CDG-I is caused by defects in the synthesis and/or transfer of lipid-linked oligosaccharides to polypeptide chains in the endoplasmic reticulum; and CDG-II, caused by defects in the trimming/processing reactions of glycans in the Golgi apparatus. We developed a Mass Spectrometric method to identify and quantify plasma N-glycans of CDG-II patients using a stable isotope labelling strategy (SIL). From our preliminary data, and from the literature, it became clear that most of the CDGs produce only quantitative changes in normal N-glycans, making the analysis more difficult. Therefore, we developed a quantitative SIL-based glycan-by-glycan approach, establishing the normal population variance and quantifying the deviation from normality even for underrepresented N-glycans in plasma, which are usually disconsidered in other types of analyses. This work stems from the invaluable contribution of the Laboratório de Espectrometria de Massa and Laboratório de Bioquímica Genética.

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We are currently working on the adaptation of this methodology to the analysis of O-glycans in CDG-II patients. Although some alterations in O-glycans in CDG-II patients have been described in the literature, we feel they have not been thoroughly explored in CDG-II patients. We are in the first stages of development, and one undergraduate thesis was produced in our lab so far, authored by Matheus Watanabe. Please find the link in the Publications section.

 

2.2. Analysis of plasma N- and O-glycans in Colorectal Cancer

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This line of research stems from collaboration with MD Marcelo Melo de Andrade Coura, working as a PhD student under the supervision of PhD MD João Batista Sousa and Prof Carlos Bloch Jr.

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Colorectal cancer (CRC) is the third cause of mortality by cancer in Brazil. It is known that colorectal tumor cells present different patterns of N- and O-linked oligosaccharides in relation to the normal tissue and it is presumed that these alterations indicate distinct biological behaviors between tumoral masses. The differential content of glycans observed in the tumoral tissue can also be present in the patients´ plasma with direct implications in the diagnostics, prognostics and in treatment.  We are developing this methodology further by performing additional analyses in the plasma N-glycans of CRC patients as well as expanding to O-glycans. The analysis of O-glycans in CRC patients was developed by an undergraduate student (PIBIC scholarship 2018), João Bueno, and his final report can also be encountered in the Publications section.

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23/07/2020, by GDB and the LSAB group