Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology

The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported membrane-based electrophysiology (SSME). Using hPepT1-containing vesicles, we could not identify any peptide induced pre-steady-state currents, indicating that the recorded peak currents reflect steady-state transport. Electrogenic co-transport of H+/glycylglycine (GlyGly) was observed across a pH range of 5.0 to 9.0. The pH dependence is described by a bell-shaped activity curve and two pK values. KM and relative Vmax values of various canonical and non-canonical peptide substrates were contextualized with current mechanistic understandings of hPepT1. Finally, specific inhibition was observed for various inhibitors in a high throughput format, and IC50 values are reported. Taken together, these findings contribute to promoting the design and analysis of pharmacologically relevant substances

A comparative review on the well-studied GAT1 and the understudied BGT-1 in the brain

γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Its homeostasis is maintained by neuronal and glial GABA transporters (GATs). The four GATs identified in humans are GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11), and betaine/GABA transporter-1 BGT-1 (SLC6A12) which are all members of the solute carrier 6 (SLC6) family of sodium-dependent transporters. While GAT1 has been investigated extensively, the other GABA transporters are less studied and their role in CNS is not clearly defined. Altered GABAergic neurotransmission is involved in different diseases, but the importance of the different transporters remained understudied and limits drug targeting. In this review, the well-studied GABA transporter GAT1 is compared with the less-studied BGT-1 with the aim to leverage the knowledge on GAT1 to shed new light on the open questions concerning BGT-1. The most recent knowledge on transporter structure, functions, expression, and localization is discussed along with their specific role as drug targets for neurological and neurodegenerative disorders. We review and discuss data on the binding sites for Na+, Cl-, substrates, and inhibitors by building on the recent cryo-EM structure of GAT1 to highlight specific molecular determinants of transporter functions. The role of the two proteins in GABA homeostasis is investigated by looking at the transport coupling mechanism, as well as structural and kinetic transport models. Furthermore, we review information on selective inhibitors together with the pharmacophore hypothesis of transporter substrates

Unveiling the crucial role of betaine: modulation of GABA homeostasis via SLC6A1 transporter (GAT1)

Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling.In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Here we demonstrate that betaine at low concentration modulates the GABA uptake by GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. This modulation occurs through the temporal inhibition of the transporter, wherein prolonged occupancy by betaine impedes the swift transition of the transporter to the inward conformation. Importantly, the modulatory effect of betaine on GAT1 is reversible, as the blocking of GAT1 disappears with increased extracellular GABA. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine has a dual role in GAT1: at mM concentration acts as a slow substrate, and at μM as a temporal blocker of GABA, when it is below its K0.5. Given this unique modulatory characteristic and lack of any harmful side effects, betaine emerges as a promising neuromodulator of the inhibitory pathways improving GABA homeostasis via GAT1, thereby conferring neuroprotection against excitotoxicity

Entry by multiple picornaviruses is dependent on a pathway that includes TNK2, WASL, and NCK1

Comprehensive knowledge of the host factors required for picornavirus infection would facilitate antiviral development. Here we demonstrate roles for three human genes

Circulating cytokine levels and antibody responses to human Schistosoma haematobium: IL-5 and IL-10 levels depend upon age and infection status

Experimental schistosome infections induce strong parasite-specific Th2 responses. This study aims to relate human systemic cytokine and antibody levels to schistosome infection levels and history. Levels of anti-Schistosoma haematobium antibodies (directed against crude cercariae, egg and adult worm antigens) and plasma cytokines (IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, IL-21, and IL-23) were measured by ELISA in 227 Zimbabweans (6–60 years old) in a schistosome-endemic area and related to age and infection status. Egg-positive people had significantly higher levels of specific antibodies, IL-2, IFN-γ and IL-23. In contrast, egg-negative individuals had significantly higher circulating IL-10, IL-4, IL-13 and IL-21 that were detected with high frequency in all participants. Subjects with detectable plasma IL-17 produced few or no eggs. When analyzed by age, IL-4 and IL-10 increased significantly, as did schistosome-specific antibodies. However, when age was combined with infection status, IL-5 declined over time in egg-positive people, while increased with age in the egg-negative group. Older, lifelong residents had significantly higher IL-4 and IL-5 levels than younger egg-negative people. Thus, a mixed Th1/Th2 systemic environment occurs in people with patent schistosome infection, while a stronger Th2-dominated suite of cytokines is evident in egg-negative individuals

A comparative review on the well-studied GAT1 and the understudied BGT-1 in the brain

γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Its homeostasis is maintained by neuronal and glial GABA transporters (GATs). The four GATs identified in humans are GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11), and betaine/GABA transporter-1 BGT-1 (SLC6A12) which are all members of the solute carrier 6 (SLC6) family of sodium-dependent transporters. While GAT1 has been investigated extensively, the other GABA transporters are less studied and their role in CNS is not clearly defined. Altered GABAergic neurotransmission is involved in different diseases, but the importance of the different transporters remained understudied and limits drug targeting. In this review, the well-studied GABA transporter GAT1 is compared with the less-studied BGT-1 with the aim to leverage the knowledge on GAT1 to shed new light on the open questions concerning BGT-1. The most recent knowledge on transporter structure, functions, expression, and localization is discussed along with their specific role as drug targets for neurological and neurodegenerative disorders. We review and discuss data on the binding sites for Na+, Cl−, substrates, and inhibitors by building on the recent cryo-EM structure of GAT1 to highlight specific molecular determinants of transporter functions. The role of the two proteins in GABA homeostasis is investigated by looking at the transport coupling mechanism, as well as structural and kinetic transport models. Furthermore, we review information on selective inhibitors together with the pharmacophore hypothesis of transporter substrates

Regulation of pathogenesis and immunity in helminth infections

Helminths are multicellular eukaryotic parasites that infect over one quarter of the world’s population. Through coevolution with the human immune system, these organisms have learned to exploit immunoregulatory pathways, resulting in asymptomatic tolerance of infections in many individuals. When infections and the resulting immune responses become dysregulated, however, acute and chronic pathologies often develop. A recent international meeting focused on how these parasites modulate host immunity and how control of parasitic and immunopathological disease might be achieved

Chronic infections with viruses or parasites: breaking bad to make good

Eukaryotic forms of life have been continually invaded by microbes and larger multicellular parasites, such as helminths. Over a billion years ago bacterial endosymbionts permanently colonized eukaryotic cells leading to recognized organelles with a distinct genetic lineage, such as mitochondria and chloroplasts. Colonization of our skin and mucosal surfaces with bacterial commensals is now known to be important for host health. However, the contribution of chronic virus and parasitic infections to immune homeostasis is being increasingly questioned. Persistent infection does not necessarily equate to exhibiting a chronic illness: healthy hosts (e.g. humans) have chronic viral and parasitic infections with no evidence of disease. Indeed, there are now examples of complex interactions between these microbes and hosts that seem to confer an advantage to the host at a particular time, suggesting that the relationship has progressed along an axis from parasitic to commensal to one of a mutualistic symbiosis. This concept is explored using examples from viruses and parasites, considering how the relationships may be not only detrimental but also beneficial to the human host