A small TAT-TrkB peptide prevents BDNF receptor cleavage and restores synaptic physiology in Alzheimer's disease.

In Alzheimer's disease (AD), amyloid ß (Aß)-triggered cleavage of TrkB-FL impairs brain-derived neurotrophic factor (BDNF) signaling, thereby compromising neuronal survival, differentiation, and synaptic transmission and plasticity. Using cerebrospinal fluid and postmortem human brain samples, we show that TrkB-FL cleavage occurs from the early stages of the disease and increases as a function of pathology severity. To explore the therapeutic potential of this disease mechanism, we designed small TAT-fused peptides and screened their ability to prevent TrkB-FL receptor cleavage. Among these, a TAT-TrkB peptide with a lysine-lysine linker prevented TrkB-FL cleavage both in vitro and in vivo and rescued synaptic deficits induced by oligomeric Aß in hippocampal slices. Furthermore, this TAT-TrkB peptide improved the cognitive performance, ameliorated synaptic plasticity deficits and prevented Tau pathology progression in vivo in the 5XFAD mouse model of AD. No evidence of liver or kidney toxicity was found. We provide proof-of-concept evidence for the efficacy and safety of this therapeutic strategy and anticipate that this TAT-TrkB peptide has the potential to be a disease-modifying drug that can prevent and/or reverse cognitive deficits in patients with AD.

Zika virus capsid protein closed structure modulates binding to host lipid systems.

Zika virus (ZIKV), a mosquito-borne Flavivirus of international concern, causes congenital microcephaly in newborns and Guillain-Barré syndrome in adults. ZIKV capsid (C) protein, one of three key structural proteins, is essential for viral assembly and encapsidation. In dengue virus, a closely related flavivirus, the homologous C protein interacts with host lipid systems, namely intracellular lipid droplets, for successful viral replication. Here, we investigate ZIKV C interaction with host lipid systems, showing that it binds host lipid droplets but, contrary to expected, in an unspecific manner. Contrasting with other flaviviruses, ZIKV C also does not bind very-low density-lipoproteins. Comparing with other Flavivirus, capsid proteins show that ZIKV C structure is particularly thermostable and seems to be locked into an auto-inhibitory conformation due to a disordered N-terminal, hence blocking specific interactions and supporting the experimental differences observed. Such distinct structural features must be considered when targeting capsid proteins in drug development.

Dengue Virus Capsid Protein Facilitates Genome Compaction and Packaging.

Dengue virus (DENV) is a single-stranded (+)-sense RNA virus that infects humans and mosquitoes, posing a significant health risk in tropical and subtropical regions. Mature virions are composed of an icosahedral shell of envelope (E) and membrane (M) proteins circumscribing a lipid bilayer, which in turn contains a complex of the approximately 11 kb genomic RNA with capsid (C) proteins. Whereas the structure of the envelope is clearly defined, the structure of the packaged genome in complex with C proteins remains elusive. Here, we investigated the interactions of C proteins with viral RNA, in solution and inside mature virions, via footprinting and cross-linking experiments. We demonstrated that C protein interaction with DENV genomes saturates at an RNA:C protein ratio below 1:250. Moreover, we also showed that the length of the RNA genome interaction sites varies, in a multimodal distribution, consistent with the C protein binding to each RNA site mostly in singlets or pairs (and, in some instances, higher numbers). We showed that interaction sites are preferentially sites with low base pairing, as previously measured by 2'-acetylation analyzed by primer extension (SHAPE) reactivity indicating structuredness. We found a clear association pattern emerged: RNA-C protein binding sites are strongly associated with long-range RNA-RNA interaction sites, particularly inside virions. This, in turn, explains the need for C protein in viral genome packaging: the protein has a chief role in coordinating these key interactions, promoting proper packaging of viral RNA. Such sites are, thus, highly consequential for viral assembly, and, as such, may be targeted in future drug development strategies against these and related viruses.

Nanoparticle-peptide conjugates for bacterial detection and neutralization: Potential applications in diagnostics and therapy.

Infections caused by antibiotic-resistant bacteria continue to challenge the medical field, mostly due to conventional treatments inefficiency after years of overuse and misuse in clinics. Cases of multiresistant bacterial infections are increasing every year. This led the World Health Organization (WHO) to update the list of resistant micro-organisms that represent greatest threat to human health. To stop the growing of the global resistance to antimicrobial drugs, new alternatives are necessary to fight these pathogens. In this context, antimicrobials peptides (AMPs) emerge as a new alternative to the current antibiotics in the pharmaceutical market. To improve their antimicrobial activity, different strategies are being developed to overcome antibiotic-resistant bacteria. Nanotechnology can be used to further potentiate antimicrobials, by increasing their activity or assisting in their delivering, frequently using nanostructured materials. There are already several antimicrobial peptides used in therapeutics, some of them coupled to nanoparticles. Additionally, detection strategies taking advantage of peptides as recognition agents are also being explored. Several examples are detailed of peptides that are specific to bacterial targets, and how that specificity can be used in diagnostics systems, coupled with nanoparticles-based signal detection approaches. Thus, the same properties of AMPs that enable specific neutralization can be harnessed to detect the very same bacteria they target. Overall, this review is focused on current research on nanoparticles coupled to antimicrobial peptides and how they can be used against multidrug-resistant bacteria as antimicrobials and/or as detection system. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Dengue, West Nile, and Zika Viruses: Potential Novel Antiviral Biologics Drugs Currently at Discovery and Preclinical Development Stages.

Dengue, West Nile and Zika viruses are vector-borne flaviviruses responsible for numerous disease outbreaks in both Hemispheres. Despite relatively low mortality, infection may lead to potentially severe situations such as (depending on the virus): hypovolemic shock, encephalitis, acute flaccid paralysis, Guillain-Barré syndrome, congenital malformations (e.g., microcephaly) and, in some situations, death. Moreover, outbreaks also have major socioeconomic repercussions, especially in already vulnerable societies. Thus far, only generic symptoms relief is possible, as there are no specific treatments available yet. Dengvaxia was the world's first dengue vaccine. However, it is not fully effective. Prophylactic approaches against West Nile and Zika viruses are even more limited. Therefore, therapeutic strategies are required and will be discussed hereafter. We will first briefly present these viruses' epidemiology, life cycle and structure. Then, we introduce the clinical presentation, diagnosis approaches and available vaccines. Finally, we list and discuss promising compounds at discovery and preclinical development stages already deposited at the GlobalData database and divided into three main types, according to therapeutic molecule: antibody-based, peptide-based molecules and, other compounds. To conclude, we discuss and compare promising developments, useful for future therapies against these three flaviviruses of major concern to human health.

Lipid membrane-based therapeutics and diagnostics.

Success rates in drug discovery are extremely low, and the imbalance between new drugs entering clinical research and their approval is steadily widening. Among the causes of the failure of new therapeutic agents are the lack of safety and insufficient efficacy. On the other hand, timely disease diagnosis may enable an early management of the disease, generally leading to better and less costly outcomes. Several strategies have been explored to overcome the barriers for drug development and facilitate diagnosis. Using lipid membranes as platforms for drug delivery or as biosensors are promising strategies, due to their biocompatibility and unique physicochemical properties. We examine some of the lipid membrane-based strategies for drug delivery and diagnostics, including their advantages and shortcomings. Regarding synthetic lipid membrane-based strategies for drug delivery, liposomes are the archetypic example of a successful approach, already with a long period of well-succeeded clinical application. The use of lipid membrane-based structures from biological sources as drug carriers, currently under clinical evaluation, is also discussed. These biomimetic strategies can enhance the in vivo lifetime of drug and delivery system by avoiding fast clearance, consequently increasing their therapeutic window. The strategies under development using lipid membranes for diagnostic purposes are also reviewed.

The Pseudo-Circular Genomes of Flaviviruses: Structures, Mechanisms, and Functions of Circularization.

The circularization of viral genomes fulfills various functions, from evading host defense mechanisms to promoting specific replication and translation patterns supporting viral proliferation. Here, we describe the genomic structures and associated host factors important for flaviviruses genome circularization and summarize their functional roles. Flaviviruses are relatively small, single-stranded, positive-sense RNA viruses with genomes of approximately 11 kb in length. These genomes contain motifs at their 5' and 3' ends, as well as in other regions, that are involved in circularization. These motifs are highly conserved throughout the Flavivirus genus and occur both in mature virions and within infected cells. We provide an overview of these sequence motifs and RNA structures involved in circularization, describe their linear and circularized structures, and discuss the proteins that interact with these circular structures and that promote and regulate their formation, aiming to clarify the key features of genome circularization and understand how these affect the flaviviruses life cycle.

Islet amyloid polypeptide & amyloid beta peptide roles in Alzheimer's disease: two triggers, one disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects millions worldwide. Due to population ageing, the incidence of AD is increasing. AD patients develop cognitive decline and dementia, features for which is known, requiring permanent care. This poses a major socio-economic burden on healthcare systems as AD patients' relatives and healthcare workers are forced to cope with rising numbers of affected people. Despite recent advances, AD pathological mechanisms are not fully understood. Nevertheless, it is clear that the amyloid beta (Aß) peptide, which forms amyloid plaques in AD patients' brains, plays a key role. Type 2 diabetes, the most common form of diabetes, affects hundreds of million people globally. Islet amyloid polypeptide (IAPP) is a hormone co-produced and secreted with insulin in pancreatic ß-cells, with a key role in diabetes, as it helps regulate glucose levels and control adiposity and satiation. Similarly to Aß, IAPP is very amyloidogenic, generating intracellular amyloid deposits that cause ß-cell dysfunction and death. It is now clear that IAPP can also have a pathological role in AD, decreasing cognitive function. IAPP harms the blood-brain barrier, directly interacts and co-deposits with Aß, promoting diabetes-associated dementia. IAPP can cause a metabolic dysfunction in the brain, leading to other diabetes-related forms of AD. Thus, here we discuss IAPP association with diabetes, Aß and dementia, in the context of what we designate a "diabetes brain phenotype" AD hypothesis. Such approach helps to set a conceptual framework for future IAPP-based drugs against AD.

Dengue and Zika Viruses: Epidemiological History, Potential Therapies, and Promising Vaccines.

Dengue virus (DENV), which can lead to fatal hemorrhagic fever, affects 390 million people worldwide. The closely related Zika virus (ZIKV) causes microcephaly in newborns and Guillain-Barré syndrome in adults. Both viruses are mostly transmitted by Aedes albopictus and Aedes aegypti mosquitoes, which, due to globalization of trade and travel alongside climate change, are spreading worldwide, paving the way to DENV and ZIKV transmission and the occurrence of new epidemics. Local outbreaks have already occurred in temperate climates, even in Europe. As there are no specific treatments, these viruses are an international public health concern. Here, we analyze and discuss DENV and ZIKV outbreaks history, clinical and pathogenesis features, and modes of transmission, supplementing with information on advances on potential therapies and restraining measures. Taking advantage of the knowledge of the structure and biological function of the capsid (C) protein, a relatively conserved protein among flaviviruses, within a genus that includes DENV and ZIKV, we designed and patented a new drug lead, pep14-23 (WO2008/028939A1). It was demonstrated that it inhibits the interaction of DENV C protein with the host lipid system, a process essential for viral replication. Such an approach can be used to develop new therapies for related viruses, such as ZIKV.

Islet Amyloid Polypeptide: A Partner in Crime With Aß in the Pathology of Alzheimer's Disease.

Diabetes affects hundreds of millions of patients worldwide. Despite the advances in understanding the disease and therapeutic options, it remains a leading cause of death and of comorbidities globally. Islet amyloid polypeptide (IAPP), or amylin, is a hormone produced by pancreatic ß-cells. It contributes to the maintenance of glucose physiological levels namely by inhibiting insulin and glucagon secretion as well as controlling adiposity and satiation. IAPP is a highly amyloidogenic polypeptide forming intracellular aggregates and amyloid structures that are associated with ß-cell death. Data also suggest the relevance of unprocessed IAPP forms as seeding for amyloid buildup. Besides the known consequences of hyperamylinemia in the pancreas, evidence has also pointed out that IAPP has a pathological role in cognitive function. More specifically, IAPP was shown to impair the blood-brain barrier; it was also seen to interact and co-deposit with amyloid beta peptide (Aß), and possibly with Tau, within the brain of Alzheimer's disease (AD) patients, thereby contributing to diabetes-associated dementia. In fact, it has been suggested that AD results from a metabolic dysfunction in the brain, leading to its proposed designation as type 3 diabetes. Here, we have first provided a brief perspective on the IAPP amyloidogenic process and its role in diabetes and AD. We have then discussed the potential interventions for modulating IAPP proteotoxicity that can be explored for therapeutics. Finally, we have proposed the concept of a "diabetes brain phenotype" hypothesis in AD, which may help design future IAPP-centered drug developmentstrategies against AD.

Intrinsically disordered protein domains in flavivirus infection.

Intrinsically disordered protein regions are at the core of biological processes and involved in key protein-ligand interactions. The Flavivirus proteins, of viruses of great biomedical importance such as Zika and dengue viruses, exemplify this. Several proteins of these viruses have disordered regions that are of the utmost importance for biological activity. Disordered proteins can adopt several conformations, each able to interact with and/or bind to different ligands. In fact, such interactions can help stabilize a particular fold. Moreover, by being promiscuous in the number of target molecules they can bind to, these protein regions increase the number of functions that their small proteome (10 proteins) can achieve. A folding energy waterfall better describes the protein folding landscape of these proteins. A disordered protein can be thought as rolling down the folding energy cascade, in order "to fall, fold and function". This is the case of many viral protein regions, as seen in the flaviviruses proteome. Given their small size, flaviviruses are a good model system for understanding the role of intrinsically disordered protein regions in viral function. Finally, studying these viruses disordered protein regions will certainly contribute to the development of therapeutic approaches against such promising (yet challenging) targets.

Structural and Functional Properties of the Capsid Protein of Dengue and Related Flavivirus.

Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.

West Nile Virus Capsid Protein Interacts With Biologically Relevant Host Lipid Systems.

West Nile and dengue viruses are closely related flaviviruses, originating mosquito-borne viral infections for which there are no effective and specific treatments. Their capsid proteins sequence and structure are particularly similar, forming highly superimposable α-helical homodimers. Measuring protein-ligand interactions at the single-molecule level yields detailed information of biological and biomedical relevance. In this work, such an approach was successfully applied on the characterization of the West Nile virus capsid protein interaction with host lipid systems, namely intracellular lipid droplets (an essential step for dengue virus replication) and blood plasma lipoproteins. Dynamic light scattering measurements show that West Nile virus capsid protein binds very low-density lipoproteins, but not low-density lipoproteins, and this interaction is dependent of potassium ions. Zeta potential experiments show that the interaction with lipid droplets is also dependent of potassium ions as well as surface proteins. The forces involved on the binding of the capsid protein with lipid droplets and lipoproteins were determined using atomic force microscopy-based force spectroscopy, proving that these interactions are K+-dependent rather than a general dependence of ionic strength. The capsid protein interaction with host lipid systems may be targeted in future therapeutic strategies against different flaviviruses. The biophysical and nanotechnology approaches employed in this study may be applied to characterize the interactions of other important proteins from different viruses, in order to understand their life cycles, as well as to find new strategies to inhibit them.

Fast NMR method to probe solvent accessibility and disordered regions in proteins.

Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established 1H-15N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method.

Impact of γ'γ' fibrinogen interaction with red blood cells on fibrin clots.

AIM: γ' fibrinogen has been associated with thrombosis. Here the interactions between γ'γ' or γAγA fibrinogen and red blood cells (RBCs), and their role on fibrin clot properties were studied. MATERIALS & METHODS: Atomic Force microscopy (AFM)-based force spectroscopy, rheological, electron and confocal microscopy, and computational approaches were conducted for both fibrinogen variants. RESULTS & CONCLUSION: AFM shows that the recombinant human (rh)γ'γ' fibrinogen increases the binding force and the frequency of the binding to RBCs compared with rhγAγA, promoting cell aggregation. Structural changes in rhγ'γ' fibrin clots, displaying a nonuniform fibrin network were shown by microscopy approaches. The presence of RBCs decreases the fibrinolysis rate and increases viscosity of rhγ'γ' fibrin clots. The full length of the γ' chain structure, revealed by computational analysis, occupies a much wider surface and is more flexible, allowing an increase of the binding between γ' fibers, and eventually with RBCs.

Methods for Lipid Droplet Biophysical Characterization in Flaviviridae Infections.

Lipid droplets (LDs) are intracellular organelles for neutral lipid storage, originated from the endoplasmic reticulum. They play an essential role in lipid metabolism and cellular homeostasis. In fact, LDs are complex organelles, involved in many more cellular processes than those initially proposed. They have been extensively studied in the context of LD-associated pathologies. In particular, LDs have emerged as critical for virus replication and assembly. Viruses from the Flaviviridae family, namely dengue virus (DENV), hepatitis C virus (HCV), West Nile virus (WNV), and Zika virus (ZIKV), interact with LDs to usurp the host lipid metabolism for their own viral replication and pathogenesis. In general, during Flaviviridae infections it is observed an increasing number of host intracellular LDs. Several viral proteins interact with LDs during different steps of the viral life cycle. The HCV core protein and DENV capsid protein, extensively interact with LDs to regulate their replication and assembly. Detailed studies of LDs in viral infections may contribute for the development of possible inhibitors of key steps of viral replication. Here, we reviewed different techniques that can be used to characterize LDs isolated from infected or non-infected cells. Microscopy studies have been commonly used to observe LDs accumulation and localization in infected cell cultures. Fluorescent dyes, which may affect LDs directly, are widely used to probe LDs but there are also approaches that do not require the use of fluorescence, namely stimulated Raman scattering, electron and atomic force microscopy-based approaches. These three are powerful techniques to characterize LDs morphology. Raman scattering microscopy allows studying LDs in a single cell. Electron and atomic force microscopies enable a better characterization of LDs in terms of structure and interaction with other organelles. Other biophysical techniques, such as dynamic light scattering and zeta potential are also excellent to characterize LDs in terms of size in a simple and fast way and test possible LDs interaction with viral proteins. These methodologies are reviewed in detail, in the context of viral studies.

Glycation potentiates α-synuclein-associated neurodegeneration in synucleinopathies.

α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.

Effects of Penicillium chrysogenum var. halophenolicum on kraft lignin: color stabilization and cytotoxicity evaluation.

Wood industries and agricultural crops generate an inexhaustible supply of by-products like lignin, which constitutes an environmental problem. Increasing efforts have been done to find new applications for lignin. One of them is as a food additive, but its chemical nature makes it sensitive to browning which constitutes a major drawback for this type of lignin application. In the present study we are documenting how color stabilization of a commercial kraft lignin was achieved after the treatment with Penicillium chrysogenum var. halophenolicum. In addition the fungal capacity to remove lignin is studied together with the effect of its treatment on cytotoxicity of lignin. P. chrysogenum var. halophenolicum was able to transform lignin, ensuring its color stability for more than 24 months. Dynamic light scattering and atomic force microscopy showed that the fungus contributed to homogenize particle size and hydrodynamic properties in lignin suspensions without increase the toxicity over HeLa cells and human primary fibroblasts. These findings suggest new uses for kraft lignin after P. chrysogenum var. halophenolicum treatment providing an effective approach for improve color stability.

Understanding Dengue Virus Capsid Protein Interaction with Key Biological Targets.

Dengue virus (DENV) causes over 500,000 hospitalizations and 20,000 deaths worldwide every year. Dengue epidemics now reach temperate regions due to globalization of trade and travel and climate changes. Currently, there are no successful therapeutic or preventive approaches. We previously developed a peptide drug lead, pep14-23, that inhibits the biologically relevant interaction of DENV capsid (C) protein with lipid droplets (LDs). Surprisingly, pep14-23 also inhibits DENV C interaction with very low-density lipoproteins (VLDL). We thus investigated the similarity between the proposed DENV C molecular targets in LDs and VLDL, respectively, the proteins perilipin 3 (PLIN3) and apolipoprotein E (APOE). APOE N-terminal and PLIN3 C-terminal regions are remarkably similar, namely APOE α-helix 4 (APOEα4) and PLIN3 α-helix 5 (PLIN3α5) sequences, which are also highly superimposable structurally. Interestingly, APOE α-helical N-terminal sequence and structure superimposes with DENV C α-helices α1 and α2. Moreover, the DENV C hydrophobic cleft can accommodate the structurally analogous APOEα4 and PLIN3α5 helical regions. Mirroring DENV C-LDs interaction (previously shown experimentally to require PLIN3), we experimentally demonstrated that DENV C-VLDL interaction requires APOE. Thus, the results fit well with previous data and suggest future drug development strategies targeting the above mentioned α-helical structures.