First vertebrate BRICHOS antimicrobial peptides: ß-hairpin host defense peptides in limbless amphibia lung resemble those of marine worms.

Innate immunity of invertebrates offers potent antimicrobial peptides (AMPs) against drug-resistant infections. To identify new worm ß-hairpin AMPs, we explored the sequence diversity of proteins with a BRICHOS domain, which comprises worm AMP precursors. Strikingly, we discovered new BRICHOS AMPs not in worms, but in caecilians, the least studied clade of vertebrates. Two precursor proteins from Microcaecilia unicolor and Rhinatrema bivittatum resemble SP-C lung surfactants and bear worm AMP-like peptides at C-termini. The analysis of M. unicolor tissue transcriptomes shows that the AMP precursor is highly expressed in the lung along with regular SP-C, suggesting a different, protective function. The peptides form right-twisted ß-hairpins, change conformation upon lipid binding, and rapidly disrupt bacterial membranes. Both peptides exhibit broad-spectrum activity against multidrug-resistant ESKAPE pathogens with 1-4 µM MICs and remarkably low toxicity, giving 40-70-fold selectivity towards bacteria. These BRICHOS AMPs, previously unseen in vertebrates, reveal a novel lung innate immunity mechanism and offer a promising antibiotics template.

Intravenous treatment with a molecular chaperone designed against ß-amyloid toxicity improves Alzheimer's disease pathology in mouse models.

Attempts to treat Alzheimer's disease with immunotherapy against the ß-amyloid (Aß) peptide or with enzyme inhibitors to reduce Aß production have not yet resulted in effective treatment, suggesting that alternative strategies may be useful. Here we explore the possibility of targeting the toxicity associated with Aß aggregation by using the recombinant human (rh) Bri2 BRICHOS chaperone domain, mutated to act selectively against Aß42 oligomer generation and neurotoxicity in vitro. We find that treatment of Aß precursor protein (App) knockin mice with repeated intravenous injections of rh Bri2 BRICHOS R221E, from an age close to the start of development of Alzheimer's disease-like pathology, improves recognition and working memory, as assessed using novel object recognition and Y maze tests, and reduces Aß plaque deposition and activation of astrocytes and microglia. When treatment was started about 4 months after Alzheimer's disease-like pathology was already established, memory improvement was not detected, but Aß plaque deposition and gliosis were reduced, and substantially reduced astrocyte accumulation in the vicinity of Aß plaques was observed. The degrees of treatment effects observed in the App knockin mouse models apparently correlate with the amounts of Bri2 BRICHOS detected in brain sections after the end of the treatment period.

Identification of cytoskeletal proteins as binding partners of Bri2 BRICHOS domain.

Proteins must fold into three-dimensional structures to execute their biological functions. Therefore, maintenance of protein homeostasis, proteostasis, including prevention of protein misfolding is essential for cellular activity and health. Molecular chaperones are key actors in proteostasis. BRICHOS domain is an intramolecular chaperone that also interferes with several aggregation-prone proteins including amyloid ß (Aß), involved in Alzheimer's disease (AD). To extend the knowledge about Bri2 BRICHOS interactome we here used recombinant human (rh) Bri2 BRICHOS-mCherry fusion protein to probe for potential binding partners. Firstly, exogenously added Bri2 BRICHOS-mCherry was used to stain brain sections of wildtype and amyloid precursor protein (App) knock-in AD mice exhibiting robust Aß pathology. Unexpectedly, we found that rh Bri2 BRICHOS-mCherry stained the cytoplasm of neurons which are devoid of Aß deposits. To identify these intraneuronal proteins that bind to the rh Bri2 BRICHOS domain, we performed co-immunoprecipitation (co-IP) of mouse brain hippocampi homogenates using the Bri2 BRICHOS-mCherry probe and analyzed co-IP proteins by LC-MS/MS. This identified several cytoskeletal proteins including spectrin alpha and beta chain, drebrin, tubulin ß3, and ß-actin as binding partners. The interactions were confirmed by a second round of pulldown experiments using rh Bri2 BRICHOS linked to magnetic beads. The interaction of rh Bri2 BRICHOS and tubulin ß3 was further investigated by staining both mouse brain sections and SH-SY5Y neuroblastoma cells with rh Bri2 BRICHOS-mCherry and tubulin ß3 immunostaining, which revealed partial co-localization. These data suggest a possible interplay of extracellular chaperone Bri2 BRICHOS domain in the intracellular space including the cytoskeleton.

Novel BRICHOS-Related Antimicrobial Peptides from the Marine Worm Heteromastus filiformis: Transcriptome Mining, Synthesis, Biological Activities, and Therapeutic Potential.

Marine polychaetes represent an extremely rich and underexplored source of novel families of antimicrobial peptides (AMPs). The rapid development of next generation sequencing technologies and modern bioinformatics approaches allows us to apply them for characterization of AMP-derived genes and the identification of encoded immune-related peptides with the aid of genome and transcriptome mining. Here, we describe a universal bioinformatic approach based on the conserved BRICHOS domain as a search query for the identification of novel structurally unique AMP families in annelids. In this paper, we report the discovery of 13 novel BRICHOS-related peptides, ranging from 18 to 91 amino acid residues in length, in the cosmopolitan marine worm Heteromastus filiformis with the assistance of transcriptome mining. Two characteristic peptides with a low homology in relation to known AMPs-the α-helical amphiphilic linear peptide, consisting of 28 amino acid residues and designated as HfBRI-28, and the 25-mer ß-hairpin peptide, specified as HfBRI-25 and having a unique structure stabilized by two disulfide bonds-were obtained and analyzed as potential antimicrobials. Interestingly, both peptides showed the ability to kill bacteria via membrane damage, but mechanisms of their action and spectra of their activity differed significantly. Being non-cytotoxic towards mammalian cells and stable to proteolysis in the blood serum, HfBRI-25 was selected for further in vivo studies in a lethal murine model of the Escherichia coli infection, where the peptide contributed to the 100% survival rate in animals. A high activity against uropathogenic strains of E. coli (UPEC) as well as a strong ability to kill bacteria within biofilms allow us to consider the novel peptide HfBRI-25 as a promising candidate for the clinical therapy of urinary tract infections (UTI) associated with UPEC.

Novel ß-Hairpin Peptide from Marine Polychaeta with a High Efficacy against Gram-Negative Pathogens.

In recent years, new antibiotics targeting multidrug resistant Gram-negative bacteria have become urgently needed. Therefore, antimicrobial peptides are considered to be a novel perspective class of antibacterial agents. In this study, a panel of novel BRICHOS-related ß-hairpin antimicrobial peptides were identified in transcriptomes of marine polychaeta species. Two of them-abarenicin from Abarenicola pacifica and UuBRI-21 from Urechis unicinctus-possess strong antibacterial potential in vitro against a wide panel of Gram-negative bacteria including drug-resistant strains. Mechanism of action assays demonstrate that peptides disrupt bacterial and mammalian membrane integrity. Considering the stronger antibacterial potential and a low ability of abarenicin to be bound by components of serum, this peptide was selected for further modification. We conducted an alanine and arginine scanning of abarenicin by replacing individual amino acids and modulating hydrophobicity so as to improve its antibacterial potency and membrane selectivity. This design approach allowed us to obtain the Ap9 analog displaying a high efficacy in vivo in the mice septicemia and neutropenic mice peritonitis models. We demonstrated that abarenicin analogs did not significantly induce bacterial resistance after a four-week selection experiment and acted on different steps of the biofilm formation: (a) killing bacteria at their planktonic stage and preventing biofilm formation and (b) degrading pre-formed biofilm and killing embedded bacteria. The potent antibacterial and antibiofilm activity of the abarenicin analog Ap9 with its high efficacy in vivo against Gram-negative infection in mice models makes this peptide an attractive candidate for further preclinical investigation.

Bri2 BRICHOS chaperone rescues impaired fast-spiking interneuron behavior and neuronal network dynamics in an AD mouse model in vitro.

Synchronized and properly balanced electrical activity of neurons is the basis for the brain's ability to process information, to learn, and to remember. In Alzheimer's disease (AD), which causes cognitive decline in patients, this synchronization and balance is disturbed by the accumulation of neuropathological biomarkers such as amyloid-beta peptide (Aß42). Failure of Aß42 clearance mechanisms as well as desynchronization of crucial neuronal classes such as fast-spiking interneurons (FSN) are root causes for the disruption of the cognition-relevant gamma brain rhythm (30-80 Hz) and consequent cognitive impairment observed in AD. Here we show that recombinant BRICHOS molecular chaperone domains from ProSP-C or Bri2, which interfere with Aß42 aggregation, can rescue the gamma rhythm. We demonstrate that Aß42 progressively decreases gamma oscillation power and rhythmicity, disrupts the inhibition/excitation balance in pyramidal cells, and desynchronizes FSN firing during gamma oscillations in the hippocampal CA3 network of mice. Application of the more efficacious Bri2 BRICHOS chaperone rescued the cellular and neuronal network performance from all ongoing Aß42-induced functional impairments. Collectively, our findings offer critical missing data to explain the importance of FSN for normal network function and underscore the therapeutic potential of Bri2 BRICHOS to rescue the disruption of cognition-relevant brain rhythms in AD.

Recombinant BRICHOS chaperone domains delivered to mouse brain parenchyma by focused ultrasound and microbubbles are internalized by hippocampal and cortical neurons.

The BRICHOS domain is found in human precursor proteins associated with cancer, dementia (Bri2) and amyloid lung disease (proSP-C). Recombinant human (rh) proSP-C and Bri2 BRICHOS domains delay amyloid-ß peptide (Aß) fibril formation and reduce associated toxicity in vitro and their overexpression reduces Aß neurotoxicity in animal models of Alzheimer's disease. After intravenous administration in wild-type mice, rh Bri2, but not proSP-C, BRICHOS was detected in the brain parenchyma, suggesting that Bri2 BRICHOS selectively bypasses the blood-brain barrier (BBB). Here, our objective was to increase the brain delivery of rh proSP-C (trimer of 18 kDa subunits) and Bri2 BRICHOS (monomer to oligomer of 15 kDa subunits) using focused ultrasound combined with intravenous microbubbles (FUS + MB), which enables targeted and transient opening of the BBB. FUS + MB was targeted to one hemisphere of wild type mice and BBB opening in the hippocampal region was confirmed by magnetic resonance imaging. Two hours after FUS + MB brain histology showed no signs of tissue damage and immunohistochemistry showed abundant delivery to the brain parenchyma in 13 out of 16 cases given 10 mg/kg of proSP-C or Bri2 BRICHOS domains. The Bri2, but not proSP-C BRICHOS domain was detected also in the non-targeted hemisphere. ProSP-C and Bri2 BRICHOS domains were taken up by a subset of neurons in the hippocampus and cortex, and were detected to a minor extent in early endosomes. These results indicate that rh Bri2, but not proSP-C, BRICHOS, can be efficiently delivered into the mouse brain parenchyma and that both BRICHOS domains can be internalized by cell-specific mechanisms.

BRICHOS domain of Bri2 inhibits islet amyloid polypeptide (IAPP) fibril formation and toxicity in human beta cells.

Aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils in islets of Langerhans is associated with type 2 diabetes, and formation of toxic IAPP species is believed to contribute to the loss of insulin-producing beta cells. The BRICHOS domain of integral membrane protein 2B (Bri2), a transmembrane protein expressed in several peripheral tissues and in the brain, has recently been shown to prevent fibril formation and toxicity of Aß42, an amyloid-forming peptide in Alzheimer disease. In this study, we demonstrate expression of Bri2 in human islets and in the human beta-cell line EndoC-ßH1. Bri2 colocalizes with IAPP intracellularly and is present in amyloid deposits in patients with type 2 diabetes. The BRICHOS domain of Bri2 effectively inhibits fibril formation in vitro and instead redirects IAPP into formation of amorphous aggregates. Reduction of endogenous Bri2 in EndoC-ßH1 cells with siRNA increases sensitivity to metabolic stress leading to cell death while a concomitant overexpression of Bri2 BRICHOS is protective. Also, coexpression of IAPP and Bri2 BRICHOS in lateral ventral neurons of Drosophila melanogaster results in an increased cell survival. IAPP is considered to be the most amyloidogenic peptide known, and described findings identify Bri2, or in particular its BRICHOS domain, as an important potential endogenous inhibitor of IAPP aggregation and toxicity, with the potential to be a possible target for the treatment of type 2 diabetes.

Structure Elucidation and Functional Studies of a Novel ß-hairpin Antimicrobial Peptide from the Marine Polychaeta Capitella teleta.

Endogenous antimicrobial peptides (AMPs) are evolutionary ancient molecular factors of innate immunity that play a key role in host defense. Among the most active and stable under physiological conditions AMPs are the peptides of animal origin that adopt a ß-hairpin conformation stabilized by disulfide bridges. In this study, a novel BRICHOS-domain related AMP from the marine polychaeta Capitella teleta, named capitellacin, was produced as the recombinant analogue and investigated. The mature capitellacin exhibits high homology with the known ß-hairpin AMP family-tachyplesins and polyphemusins from the horseshoe crabs. The ß-hairpin structure of the recombinant capitellacin was proved by CD and NMR spectroscopy. In aqueous solution the peptide exists as monomeric right-handed twisted ß-hairpin and its structure does not reveal significant amphipathicity. Moreover, the peptide retains this conformation in membrane environment and incorporates into lipid bilayer. Capitellacin exhibits a strong antimicrobial activity in vitro against a wide panel of bacteria including extensively drug-resistant strains. In contrast to other known ß-hairpin AMPs, this peptide acts apparently via non-lytic mechanism at concentrations inhibiting bacterial growth. The molecular mechanism of the peptide antimicrobial action does not seem to be related to the inhibition of bacterial translation therefore other molecular targets may be assumed. The reduced cytotoxicity against human cells and high antibacterial cell selectivity as compared to tachyplesin-1 make it an attractive candidate compound for an anti-infective drug design.

Novel Antimicrobial Peptides from the Arctic Polychaeta Nicomache minor Provide New Molecular Insight into Biological Role of the BRICHOS Domain.

Endogenous antimicrobial peptides (AMPs) are among the earliest molecular factors in the evolution of animal innate immunity. In this study, novel AMPs named nicomicins were identified in the small marine polychaeta Nicomache minor in the Maldanidae family. Full-length mRNA sequences encoded 239-residue prepropeptides consisting of a putative signal sequence region, the BRICHOS domain within an acidic proregion, and 33-residue mature cationic peptides. Nicomicin-1 was expressed in the bacterial system, and its spatial structure was analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. Nicomicins are unique among polychaeta AMPs scaffolds, combining an amphipathic N-terminal α-helix and C-terminal extended part with a six-residue loop stabilized by a disulfide bridge. This structural arrangement resembles the Rana-box motif observed in the α-helical host-defense peptides isolated from frog skin. Nicomicin-1 exhibited strong in vitro antimicrobial activity against Gram-positive bacteria at submicromolar concentrations. The main mechanism of nicomicin-1 action is based on membrane damage but not on the inhibition of bacterial translation. The peptide possessed cytotoxicity against cancer and normal adherent cells as well as toward human erythrocytes.

BRICHOS binds to a designed amyloid-forming ß-protein and reduces proteasomal inhibition and aggresome formation.

The BRICHOS domain is associated with proliferative, degenerative and amyloid diseases, and it has been shown to inhibit fibril formation and toxicity of the Alzheimer's disease-associated amyloid ß-peptide. ProSP-C (prosurfactant protein C) BRICHOS binds to stretches of hydrophobic amino acid residues, which are unfolded or in ß-strand conformation, suggesting that it may have broad anti-amyloid activity. We have studied the effect of the proSP-C BRICHOS domain on the designed amyloidogenic ß-sheet proteins ß17 and ß23. ß17 expressed in the secretory pathway of HEK (human embryonic kidney)-293 cells forms intracellular inclusions, whereas ß23 is rapidly degraded. Co-expression of BRICHOS leads to a reduction in ß17 inclusion size and increased levels of soluble ß17 and ß23. Furthermore, BRICHOS interacts with the ß-proteins intracellularly, reduces their ubiquitination and decreases aggresome formation and proteasomal inhibition. Collectively, these data suggest that BRICHOS is capable of delaying the aggregation process and toxicity of amyloidogenic proteins in a generic manner.

Specific chaperones and regulatory domains in control of amyloid formation.

Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology.

The unfolded protein response controls ER stress-induced apoptosis of lung epithelial cells through angiotensin generation.

Recent work from this laboratory showed that endoplasmic reticulum (ER) stress-induced apoptosis of alveolar epithelial cells (AECs) is regulated by the autocrine angiotensin (ANG)II/ANG1-7 system. The proteasome inhibitor MG132 or surfactant protein C (SP-C) BRICHOS domain mutation G100S induced apoptosis in human AECs by activating the proapoptotic cathepsin D and reducing antiapoptotic angiotensin converting enzyme-2 (ACE-2). This study tested the hypothesis that ER stress-induced apoptosis of human AECs might be mediated by influence of the unfolded protein response (UPR) on the autocrine ANGII/ANG1-7 system. A549 cells were challenged with MG132 or SP-C BRICHOS domain mutant G100S to induce ER stress and activation of UPR pathways. The results showed that either MG132 or G100S SP-C mutation activated all three canonical pathways of the UPR (IRE1/XBP1, ATF6, and PERK/eIF2α), which led to a significant increase in cathepsin D or in TACE (an ACE-2 ectodomain shedding enzyme) and eventually caused AEC apoptosis. However, ER stress-induced AEC apoptosis could be prevented by chemical chaperone or by UPR blockers. It is also suggested that ATF6 and IRE1 pathways might play important role in regulation of angiotensin system. These data demonstrate that ER stress induces apoptosis in human AECs through mediation of UPR pathways, which in turn regulate the autocrine ANGII/ANG1-7 system. They also demonstrated that ER stress-induced AEC apoptosis can be blocked by inhibition of UPR signaling pathways.

Amyloid-ß-induced action potential desynchronization and degradation of hippocampal gamma oscillations is prevented by interference with peptide conformation change and aggregation.

The amyloid-ß hypothesis of Alzheimer's Disease (AD) focuses on accumulation of amyloid-ß peptide (Aß) as the main culprit for the myriad physiological changes seen during development and progression of AD including desynchronization of neuronal action potentials, consequent development of aberrant brain rhythms relevant for cognition, and final emergence of cognitive deficits. The aim of this study was to elucidate the cellular and synaptic mechanisms underlying the Aß-induced degradation of gamma oscillations in AD, to identify aggregation state(s) of Aß that mediate the peptides neurotoxicity, and to test ways to prevent the neurotoxic Aß effect. We show that Aß(1-42) in physiological concentrations acutely degrades mouse hippocampal gamma oscillations in a concentration- and time-dependent manner. The underlying cause is an Aß-induced desynchronization of action potential generation in pyramidal cells and a shift of the excitatory/inhibitory equilibrium in the hippocampal network. Using purified preparations containing different aggregation states of Aß, as well as a designed ligand and a BRICHOS chaperone domain, we provide evidence that the severity of Aß neurotoxicity increases with increasing concentration of fibrillar over monomeric Aß forms, and that Aß-induced degradation of gamma oscillations and excitatory/inhibitory equilibrium is prevented by compounds that interfere with Aß aggregation. Our study provides correlative evidence for a link between Aß-induced effects on synaptic currents and AD-relevant neuronal network oscillations, identifies the responsible aggregation state of Aß and proofs that strategies preventing peptide aggregation are able to prevent the deleterious action of Aß on the excitatory/inhibitory equilibrium and on the gamma rhythm.

Molecular basis for different substrate-binding sites and chaperone functions of the BRICHOS domain.

Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.

Specific inhibition of α-synuclein oligomer generation and toxicity by the chaperone domain Bri2 BRICHOS.

Protein misfolding and aggregation are involved in several neurodegenerative disorders, such as α-synuclein (αSyn) implicated in Parkinson's disease, where new therapeutic approaches remain essential to combat these devastating diseases. Elucidating the microscopic nucleation mechanisms has opened new opportunities to develop therapeutics against toxic mechanisms and species. Here, we show that naturally occurring molecular chaperones, represented by the anti-amyloid Bri2 BRICHOS domain, can be used to target αSyn-associated nucleation processes and structural species related to neurotoxicity. Our findings revealed that BRICHOS predominantly suppresses the formation of new nucleation units on the fibrils surface (secondary nucleation), decreasing the oligomer generation rate. Further, BRICHOS directly binds to oligomeric αSyn species and effectively diminishes αSyn fibril-related toxicity. Hence, our studies show that molecular chaperones can be utilized as tools to target molecular processes and structural species related to αSyn neurotoxicity and have the potential as protein-based treatments against neurodegenerative disorders.

Evolution of the human gastrokine locus and confounding factors regarding the pseudogenicity of GKN3.

In a screen for genes expressed specifically in gastric mucous neck cells, we identified GKN3, the recently discovered third member of the gastrokine family. We present confirmatory mouse data and novel porcine data showing that mouse GKN3 expression is confined to mucous cells of the corpus neck and antrum base and is prominently expressed in metaplastic lesions. GKN3 was proposed originally to be expressed in some human populations and a pseudogene in others. To investigate that hypothesis, we studied human GKN3 evolution in the context of its paralogous genomic neighbors, GKN1 and GKN2. Haplotype analysis revealed that GKN3 mimics GKN2 in patterns of exonic SNP allocation, whereas GKN1 appeared to be more stringently selected. GKN3 showed signatures of both directional selection and population based selective sweeps in humans. One such selective sweep includes SNP rs10187256, originally identified as an ancestral tryptophan to premature STOP codon mutation. The derived (nonancestral) allele went to fixation in Asia. We show that another SNP, rs75578132, identified 5 bp downstream of rs10187256, exhibits a second selective sweep in almost all Europeans, some Latinos, and some Africans, possibly resulting from a reintroduction of European genes during African colonization. Finally, we identify a mutation that would destroy the splice donor site in the putative exon3-intron3 boundary, which occurs in all human genomes examined to date. Our results highlight a stomach-specific human genetic locus, which has undergone various selective sweeps across European, Asian, and African populations and thus reflects geographic and ethnic patterns in genome evolution.

Abrogation of ER stress-induced apoptosis of alveolar epithelial cells by angiotensin 1-7.

Earlier work showed that apoptosis of alveolar epithelial cells (AECs) in response to endogenous or xenobiotic factors is regulated by autocrine generation of angiotensin (ANG) II and its counterregulatory peptide ANG1-7. Mutations in surfactant protein C (SP-C) induce endoplasmic reticulum (ER) stress and apoptosis in AECs and cause lung fibrosis. This study tested the hypothesis that ER stress-induced apoptosis of AECs might also be regulated by the autocrine ANGII/ANG1-7 system of AECs. ER stress was induced in A549 cells or primary cultures of human AECs with the proteasome inhibitor MG132 or the SP-C BRICHOS domain mutant G100S. ER stress activated the ANGII-generating enzyme cathepsin D and simultaneously decreased the ANGII-degrading enzyme ACE-2, which normally generates the antiapoptotic peptide ANG1-7. TAPI-2, an inhibitor of ADAM17/TACE, significantly reduced both the activation of cathepsin D and the loss of ACE-2. Apoptosis of AECs induced by ER stress was measured by assays of mitochondrial function, JNK activation, caspase activation, and nuclear fragmentation. Apoptosis induced by either MG132 or the SP-C BRICHOS mutant G100S was significantly inhibited by the ANG receptor blocker saralasin and was completely abrogated by ANG1-7. Inhibition by ANG1-7 was blocked by the specific mas antagonist A779. These data show that ER stress-induced apoptosis is mediated by the autocrine ANGII/ANG1-7 system in human AECs and demonstrate effective blockade of SP-C mutation-induced apoptosis by ANG1-7. They also suggest that therapeutic strategies aimed at administering ANG1-7 or stimulating ACE-2 may hold potential for the management of ER stress-induced fibrotic lung disorders.

Functional analysis of the NH2-terminal hydrophobic region and BRICHOS domain of GKN1.

Gastrokine 1 (GKN1) protects the gastric antral mucosa and promotes healing by facilitating restitution and proliferation after injury. GKN1 is down-regulated in Helicobacter pylori-infected gastric epithelial cells and loss of GKN1 expression is tightly associated with gastric carcinogenesis. However, the underlying mechanisms as a tumor suppressor are largely unknown. Presently, the hydrophobic region and BRICHOS domain of GKN1, pGKN1(D13N), pGKN1(Δ68-199), and pGKN1(Δ1-67,165-199) were shown to suppress gastric cancer cell growth and recapitulate GKN1 functions. As well, the hydrophobic region and BRICHOS domain of GKN1 had a synergistic anti-cancer effect with 5-FU on tumor cell growth, implying that the NH2-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for tumor suppression, thereby suggesting a therapeutic intervention for gastric cancer. Also, its domain inducing endogenous miR-185 directly targeted the epigenetic effectors DNMT1 and EZH2 in gastric cancer cells. Our results suggest that the NH2-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for its tumor suppressor activities.

Updates on Aß Processing by Hsp90, BRICHOS, and Newly Reported Distinctive Chaperones.

Alzheimer's disease (AD) is an extremely devastating neurodegenerative disease, and there is no cure for it. AD is specified as the misfolding and aggregation of amyloid-ß protein (Aß) and abnormalities in hyperphosphorylated tau protein. Current approaches to treat Alzheimer's disease have had some success in slowing down the disease's progression. However, attempts to find a cure have been largely unsuccessful, most likely due to the complexity associated with AD pathogenesis. Hence, a shift in focus to better understand the molecular mechanism of Aß processing and to consider alternative options such as chaperone proteins seems promising. Chaperone proteins act as molecular caretakers to facilitate cellular homeostasis under standard conditions. Chaperone proteins like heat shock proteins (Hsps) serve a pivotal role in correctly folding amyloid peptides, inhibiting mitochondrial dysfunction, and peptide aggregation. For instance, Hsp90 plays a significant role in maintaining cellular homeostasis through its protein folding mechanisms. In this review, we analyze the most recent studies from 2020 to 2023 and provide updates on Aß regulation by Hsp90, BRICHOS domain chaperone, and distinctive newly reported chaperones.