Kynostatin and 17β-estradiol prevent the apoptotic death of human neuroblastoma cells exposed to HIV-1 protease

A significant number of adult male patients with acquired immunodeficiency syndrome develop cerebral atrophy and progressive brain disorders such as dementia complex and neuropsychiatric problems. Upon entering the brain via activated macrophages or microglias, the human immunodeficiency type 1 virus (HIV-1) may produce cytotoxic factors such as HIV-1 envelope protein (gp 120) and protease. Owing to significant proteolysis of nonviral proteins, the protease derived from HIV-1 may be detrimental to brain cells and neurons. Our results revealed that HIV-1 protease, at nanomolar concentrations, was as potent as gp 120 in causing neurotoxicity in human neuroblastoma neurotypic SH-SY5Y cells. As shown by the Oncor ApopTag staining procedure, HIV-1 protease significantly increased the number of apoptotic cells over the serum-free controls. Moreover, HIV-1 protease-induced neurotoxicity was blocked by a selective protease inhibitor, kynostatin (KNI-272). Antioxidants such as 17-estradiol, melatonin, andS-nitrosoglutathione also prevented protease-induced neurotoxicity. These findings indicate that oxidative proteolysis may mediate HIV-1 protease-induced apoptosis and the degeneration of neurons and other brain cells. Centrally active protease inhibitors and antioxidants may play an important role in preventing cerebral atrophy and associated dementia complex caused by HIV-1.     

The disulphide bridges of human cystatin C (γ-trace) and chicken cystatin

Human cystatin C, a powerful physiological protein inhibitor of cathepsins B, H and L, contains two disulphide bridges, at least one of which is essential to its inhibitory activity. The positions of these bridges in the single polypeptide chain of the protein have been determined by diagonal paper electrophoresis. The cysteine residues at positions 73 and 83 form one bridge, and those at positions 97 and 117 form the other. In the homologous cystatin of chicken egg-white, the disulphides are Cys 71–Cys 81, and Cys 95–Cys 115.     

Structure–activity relationships of heteroaromatic esters as human rhinovirus 3C protease inhibitors

Human rhinovirus 3C protease (HRV 3C^pro) is known to be a promising target for development of therapeutic agents against the common cold because of the importance of the protease in viral replication as well as its expression in a large number of serotypes. To explore non-peptidic inhibitors of HRV 3C^pro, a series of novel heteroaromatic esters was synthesized and evaluated for inhibitory activity against HRV 3C^pro, to determine the structure–activity relationships. The most potent inhibitor, 7, with a 5-bromopyridinyl group, had an IC₅₀ value of 80 nM. In addition, the binding mode of a novel analog, 19, with the 4-hydroxyquinolinone moiety, was explored by molecular docking, suggesting a new interaction in the S1 pocket.     

Heat-shock protein HSP70 reduces the secretion of TNFα by neuroblastoma cells and human monocytes induced with beta-amyloid peptides

The progress of neurodegeneration in Alzheimer’s disease is closely associated with inflammatory processes in the brain tissues induced by beta-amyloid peptides (Aβ). In this paper, we showed that Aβ(1-42) and isoAβ(1-42) in human neuroblastoma cells SK-N-SH and promonocyte THP-1 activated the production of tumor necrosis factor (TNFα). Notably, isoAβ(1-42) had the strongest effect on the increase in the level of TNFα. The addition of recombinant heat-shock protein HSP70 reduces TNFα production induced by Aβ, which leads to a decrease in neuronal cell damage at the organism level.     

α-Parvalbumin reduces depolarizationminduced elevations of cytosolic free calcium in human neuroblastoma cells

We investigated whether the expression of human α-parvalbumin affects depolarization-induced elevations of the cytosolic free calcium concentration ([Ca²⁺]_i) in human neuroblastoma SKNBE2 cells. A full length human parvalbumin cDNA was cloned by PCR from human cerebellum and transiently transfected into SKNBE2 cells. Immunofluorescence staining using an antibody raised against parvalbumin revealed a transfection efficacy of about 14%. In parvalbumin-expressing SKNBE2 cells, parvalbumin concentration determined by quantitative Western blotting amounted to 0.42 mM.Transfected SKNBE2 cells were depolarized for 2 min by 50 mM K⁺. During this period, [Ca²⁺]_i was monitored by video microfluorimetry using the Ca²⁺ indicator Fura-2. In a fraction of cells, depolarization induced a transient elevation in [Ca²⁺]_i The size of this elevation was compared with the immunofluorimetrically determined expression of parvalbumin on a cell-to-cell basis. Cells with a significant parvalbumin immunofluorescence responded to depolarization with smaller elevations in [Ca²⁺]_i than non-parvalbumin-expressing cells. Resting [Ca 2+], did not differ between parvalbumin-expressing and control cells. These observations indicate that large depolarization-induced transient elevations of [Ca²⁺]_i in neuroblastoma cells can be suppressed by parvalbumin.     

The use of β-amino acids in the design of protease and peptidase inhibitors

Summary The use of β-amino acids as peptidomimetics has emerged in recent years with significant potential in a number of applications. The incorporation of β-amino acids has been successful in creating peptidomimetics that not only have potent biological activity, but are also resistant to proteolysis. This article reviews the recent applications of β-amino acids in the design of protease and peptidase inhibitors. Given their structural diversity, together with the ease of synthesis and incorporation into peptide sequences using standard solid-phase peptide synthesis techniques, β-amino acids have the potential to form a new platform technology for peptidomimetic design and synthesis.     

Carbachol-stimulated calcium entry in SH-SY5Y human neuroblastoma cells: which route?

M3 muscarinic receptors expressed on SH-SY5Y human neuroblastoma cells are linked to phosphoinositide turnover and rises in [Ca2+]i. The rise in [Ca2+]i is biphasic with the peak phase being due to release from an intracellular Ins(1,4,5)P3-sensitive site and the plateau phase being due to Ca2+ entry across the plasma membrane. Ca2+ entry does not appear to involve voltage sensitive Ca2+ channels, a pertussis toxin sensitive G-protein-operated Ca2+ channel or Ins(1,4,5)P3/Ins(1,3,4,5)P4-operated Ca2+ channel. We suggest that carbachol-stimulated Ca2+ entry in SH-SY5Y human neuroblastoma cells occurs via receptor operated Ca2+ channels and through capacitive refilling.     

Assessment of α-synuclein secretion in mouse and human brain parenchyma

Genetic, biochemical, and animal model studies strongly suggest a central role for α-synuclein in the pathogenesis of Parkinson's disease. α-synuclein lacks a signal peptide sequence and has thus been considered a cytosolic protein. Recent data has suggested that the protein may be released from cells via a non-classical secretory pathway and may therefore exert paracrine effects in the extracellular environment. However, proof that α-synuclein is actually secreted into the brain extracellular space in vivo has not been obtained. We developed a novel highly sensitive ELISA in conjugation with an in vivo microdialysis technique to measure α-synuclein in brain interstitial fluid. We show for the first time that α-synuclein is readily detected in the interstitial fluid of both α-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that α-synuclein is physiologically secreted by neurons in vivo. This interstitial fluid pool of the protein may have a role in the propagation of synuclein pathology and progression of Parkinson's disease.     

Identification of small-molecule inhibitors of the human S100B–p53 interaction and evaluation of their activity in human melanoma cells

The interaction between human S100 calcium-binding protein B (S100B) and the tumor suppressor protein p53 is considered to be a possible therapeutic target for malignant melanoma. To identify potent inhibitors of this interaction, we screened a fragment library of compounds by means of a fluorescence-based competition assay involving the S100B-binding C-terminal peptide of p53. Using active compounds from the fragment library as query compounds, we constructed a focused library by means of two-dimensional similarity searching of a large database. This simple, unbiased method allowed us to identify several inhibitors of the S100B-p53 interaction, and we elucidated preliminary structure–activity relationships. One of the identified compounds had the potential to inhibit the S100B–p53 interaction in melanoma cells.     

MAPKs and NF-κB-mediated acrylamide-induced neuropathy in rat striatum and human neuroblastoma cells SY5Y

Acrylamide (ACR) is a potent neurotoxin that can be produced during high-temperature food processing, but the underlying toxicological mechanism remains unclear. In this study, the detrimental effects of ACR on the striatal dopaminergic neurons and the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) in ACR-induced neuronal apoptosis were investigated. Acute ACR exposure caused dopaminergic neurons loss and apoptosis as revealed by decreased tyrosine hydroxylase (TH)-positive cells and TH protein level and increased terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells in the striatum. ACR-decreased glutathione content, increased levels of malondialdehyde, proinflammatory cytokines tumor necrosis factor α, and interleukin 6. In addition, nuclear NF-κB and MAPKs signaling pathway with c-Jun N-terminal kinase (JNK) and p38 were activated by ACR. Specific inhibitors were used to explore the roles of MAPKs and NF-κB pathways in ACR-induced apoptosis in SH-SY5Y cells. Pretreatment with JNK-specific inhibitors SP600125 markedly upregulated the reduced B-cell lymphoma 2 (Bcl-2) content and downregulated the increased Bcl-2-associated X protein (Bax) level and thereby eventually reduced the proportions of early and late apoptotic cells induced by ACR, while p38 suppression by SB202190 only reversed the decrease in Bcl-2 expression. Inhibition of NF-κB by BAY 11-7082 markedly upregulated Bax level and decreased Bcl-2 expression, and eventually increasing the proportions of neuronal apoptosis compared with that in ACR alone. These results suggested that JNK contributed to ACR-induced apoptosis, while NF-κB acted as a protective regulator in response to ACR-induced neuropathy. This study helps to offer a deeper insight into the mechanism of ACR-induced neuropathy.     

Decreased intracellular granule movement and glucagon secretion in pancreatic α cells attached to superior cervical ganglion neurites

Autonomic neurons innervate pancreatic islets of Langerhans and participate in the maintenance of blood glucose concentrations by controlling hormone levels through attachment with islet cells. We previously found that stimulated superior cervical ganglia (SCG) could induce Ca²⁺ oscillation in α cells via neuropeptide substance P using an in vitro co-culture model. In this study, we studied the effect of SCG neurite adhesion on intracellular secretory granule movement and glucagon secretion in α cells stimulated by low glucose concentration. Spinning disk microscopic analysis revealed that the mean velocity of intracellular granules was significantly lower in α cells attached to SCG neurites than that in those without neurites under low (2 mM), middle (10 mM), and high (20 mM) glucose concentrations. Stimulation by a low (2 mM) glucose concentration significantly increased glucagon secretion in α cells lacking neurites but not in those bound to neurites. These results suggest that adhesion to SCG neurites decreases low glucose-induced glucagon secretion in pancreatic α cells by attenuating intracellular granule movement activity.     

The effect of monensin on intracellular transport and secretion of α-amylase isoenzymes in barley aleurone

The effect of monensin on the secretion of -amylase and other enzymes from the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya) was studied by electrophoresis followed by fluorography and by pulse-chase and organelle-isolation experiments. Monensin markedly inhibits the secretion, but not the synthesis, of -amylase, acid phosphatase, and at least four other proteins from the aleurone layer. Monensin treatment causes -amylase to accumulate within the protoplast, but its effect on the different -amylase isoenzymes is not equal. The accumulation of isoenzyme 2 is not influenced by monensin while isoenzymes 1, 3 and 4 are not secreted but rather accumulate in the cell when monensin is included in the incubation medium. The -amylase and acid-phosphatase activities which accumulate within the aleurone cells following treatment with monensin are localized in an organelle having a buoyant density greater than that of endoplasmic reticulum and less than that of mitochondria. In pulse-chase experiments with [³⁵S]methionine, labelled proteins accumulate in this organelle in the presence of monensin and do not appear in the incubation medium. We conclude that monensin inhibits the secretion of proteins from the barley aleurone layer by influencing their intracellular transport.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium dodecyl-sulfate polyacrylamide-gel electrophoresis     

SARS-CoV-2 main protease inhibitors: What is moving in the field of peptides and peptidomimetics?

The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still affecting people worldwide. Despite the good degree of immunological protection achieved through vaccination, there are still severe cases that require effective antivirals. In this sense, two specific pharmaceutical preparations have been marketed already, the RdRp polymerase inhibitor molnupiravir and the main viral protease inhibitor nirmatrelvir (commercialized as Paxlovid, a combination with ritonavir). Nirmatrelvir is a peptidomimetic acting as orally available, covalent, and reversible inhibitor of SARS-CoV-2 main viral protease. The success of this compound has revitalized the search for new peptide and peptidomimetic protease inhibitors. This highlight collects some selected examples among those recently published in the field of SARS-CoV-2.     

Chloride uptake and base secretion in freshwater fish: a transepithelial ion-transport metabolon?

Despite all the efforts and technological advances during the last few decades, the cellular mechanisms for branchial chloride uptake in freshwater (FW) fish are still unclear. Although a tight 1 : 1 link with HCO-3 secretion has been established, not much is known about the identity of the ion-transporting proteins involved or the energizing steps that allow for the inward transport of Cl- against the concentration gradient. We propose a new model for Cl- uptake in FW fish whereby the combined action of an apical anion exchanger, cytoplasmic carbonic anhydrase, and basolateral V-type H+ -ATPase creates a local [HCO-3] high enough to energize Cl- uptake. Our model is based on analyses of structure-function relationships, reinterpretation of previous results, and novel observations about gill cell subtypes and immunolocalization of the V-H+ -ATPase.     

High-affinity uptake of γ-aminobutyric acid in cultured glial and neuronal cells

Both glial and neuronal cells maintained in primary culture were found to accumulate [³H]GABA by an efficient high-affinity uptake system (apparentK _m=9 M,V _max=0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1 mM) also strongly inhibited uptake of [³H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, -alanine, and 2,4-diaminobutyrate) inhibited uptake of [³H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine,l-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (K _m=92 M,V _max=0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [³H]GABA against a concentration gradient. ApparentK _m of this uptake was relatively high (819 M), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, none of the nontransformed continuous cell lines of either tumoral (glioma, C₆; neuroblastoma, Ml; MINN) or normal (NN; I₆) origin actively accumulated [³H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.     

Monoclonal antibodies against plasma protease inhibitors: I. Production and characterization of 23 monoclonal antibodies against human α2

23 hybridomas secreting monoclonal antibodies against human ₂ , the fast-acting inhibitor of plasmin present in plasma, have been produced by the cell-fusion technique. Isotyping of the monoclonal antibodies has revealed that 14 monoclonal antibodies belong to the class IgG1, 6 to the class IgG2a, and 3 to the class tgG2b. All light chains belong to the group. The specificity and relative avidity of these monoclonals have been determined Using an indirect enzyme-linked immunosorbent assay. 13 monoclonals exhibit a relatively high avidity for ₂ , 5 are of intermediate avidity, and 5 of low avidity. The epitope specificity of these 23 rnonoclonal antibodies, originating from a single mouse, have been examined in inhibition experiments. A group of 10 monoclonal antibodies exhibit a very similar inhibition pattern. Partial inhibition effects displayed by 10 other antibodies define partially overlapping antigenic regions. The binding of these antibodies seems to produce a conformational change in the ₂ molecule, reducing the binding of two other antibodies. The last antibody defines an independent epitope.     

Fibroblasts enhance the in vitro survival of human memory and naïve B cells by maintaining intracellular levels of glutathione

To gain insight into the mechanism of memory B cell survival, we cultured highly purified subpopulations of tonsillar B cells with tonsillar fibroblasts. The fibroblasts greatly enhanced the survival of memory and na?ve B cells but did not delay the rapid apoptosis of germinal center B cells. B cell activation was not observed during the period of culture, as shown by the absence of activation markers and of cycling cells. These findings were reproduced when the B cells were physically separated from the fibroblasts by a semi-permeable transwell-membrane, indicating that the survival factor(s) were diffusible. Several cytokines including IL-6, IL-15, and VEGF were tested for survival activity but none could replace the fibroblasts. However, the addition of reduced glutathione (GSH) to the na?ve and memory B cells significantly enhanced their survival, and depletion of GSH resulted in rapid loss of B cell viability. Furthermore, intracellular glutathione levels were maintained when the B cells were co-cultured with fibroblasts. Our results suggest that glutathione plays an important role in the survival of memory and na?ve B cells in the presence of stromal cells.