Toward the Discovery of Biological Functions Associated with the Mechanosensor Mtl1p of \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e via Integrative Multi-OMICs Analysis

Functional analysis of the Mtl1 protein in Saccharomyces cerevisiae has revealed that this transmembrane sensor endows yeast cells with resistance to oxidative stress through a signaling mechanism called the cell wall integrity pathway (CWI). We observed upregulation of multiple heat shock proteins (HSPs), proteins associated with the formation of stress granules, and the phosphatase subunit of trehalose 6-phosphate synthase which suggests that mtl1Δ strains undergo intrinsic activation of a non-lethal heat stress response. Furthermore, quantitative global proteomic analysis conducted on TMT-labeled proteins combined with metabolome analysis revealed that mtl1Δ strains exhibit decreased levels of metabolites of carboxylic acid metabolism, decreased expression of anabolic enzymes and increased expression of catabolic enzymes involved in the metabolism of amino acids, with enhanced expression of mitochondrial respirasome proteins. These observations support the idea that Mtl1 protein controls the suppression of a non-lethal heat stress response under normal conditions while it plays an important role in metabolic regulatory mechanisms linked to TORC1 signaling that are required to maintain cellular homeostasis and optimal mitochondrial function

Dysregulation of Macrophage-Secreted Cathepsin B Contributes to HIV-1-Linked Neuronal Apoptosis

Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1ADA infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages

The COVID-19 Pandemic: Reflections of Science, Person, and Challenge in Academic Research Settings

In spring of 2021, the Society on NeuroImmune Pharmacology (SNIP) organized a virtual workshop on the coronavirus disease 2019 (COVID-19). The daylong event’s fourth and final symposium, “Well-being and reflections,” offered a glimpse at the pandemic’s impact on the lives of our scientists and educators. This manuscript includes a brief summary of the symposium, a transcription of our incoming president Dr. Santosh Kumar’s lecture, titled “Intervention and improved well-being of basic science researchers during the COVID-19 era: a case study,” and the panel discussion that followed, “Reflection and sharing,” featuring Drs. Jean M. Bidlack, Sylvia Fitting, Santhi Gorantla, Maria Cecilia G. Marcondes, Loyda M. Melendez, and Ilker K. Sariyer. The conclusion of this manuscript includes comments from SNIP’s president Dr. Sulie L. Chang and our Chief Editor, Dr. Howard E. Gendelman. Drs. Sowmya Yelamanchili and Jeymohan Joseph co-chaired the symposium. Graphical abstract: [Figure not available: see fulltext.]

Expression of cathepsin B and cystatin B in the basal ganglia of post-mortem brain tissues.

<p>Basal ganglia tissue from HIV-seronegative (A, F and K) and HIV seropositive (B-E, G-J and L-O) patients were stained with mouse anti-human cathepsin B followed by Alexa 488 conjugate goat anti-mouse (green), or mouse anti-human cystatin B followed by Alexa 488 conjugate goat anti-mouse (green), and rabbit anti-human Iba-1 followed by Alexa 546 goat anti-rabbit (red), and nuclear staining by DAPI (blue). Unstained tissues were used as controls as illustrated in K to O. Magnification: 63×.</p

Post-mortem brain tissues received from the National NeuroAIDS Tissue Consortium.

<p>1)HIVE: HIV encephalitis.</p><p>2)HAD: HIV associated dementia.</p><p>3)MCMD: Minor cognitive motor disorder.</p

Effect of HIV infection on cathepsin B secretion in macrophages.

<p>Cell supernatants (n = 4) from HIV_ADA-infected (solid bars) and uninfected (open bars) macrophage cultures were collected, centrifuged, and tested for cathepsin B, cystatin B and cystatin C expression by antigen capture ELISA. (A) MDM secreted high levels of cathepsin B at all time points assayed. There was an increase in cathepsin B expression in the HIV-infected samples as compared with uninfected controls at 12 dpi (*p<0.05; A). HIV-infected and uninfected macrophages showed no differences in secretion of cystatin C or B (B and C). The ratio of cathepsin B to cystatin B and cathepsin B to cystatin C were calculated over time in culture (D). Cystatin B was present at higher concentrations than cathepsin B at all time points assayed, as indicated by the ratio of cathepsin B to cystatin B lower than 1. However, an increased cathepsin B to cystatin C ratio was observed in both HIV-infected and uninfected macrophages at all time points. At 12 dpi the ratio of cathepsin B to cystatin C in HIV-infected cells was higher in HIV-infected than uninfected cells (*p<0.05, D).</p

Increased cathepsin B mRNA after HIV infection.

<p>MDM from 8 different donors were inoculated with HIV-1_ADA or with serum-free media (uninfected controls) for 12 days and cell pellets collected at 3, 7, and 12 days post-infection. Changes in mRNA levels are shown as <i>Fold change</i>  = 2 ^ΔΔCt  = 2 ^{(Δ Ct control – Δ Ct experimental)} for cathepsin B (white), cystatin B (grey), and cystatin C (black). The mRNA levels of cystatins B and C remained similar after HIV infection. A significant increase in mRNA expression was found for cathepsin B in HIV infected MDM at 12 days compared to 3 (*p = 0.038, B) and 7 dpi (*p = 0.028).</p

Secreted cathepsin B is more active in HIV-infected macrophages than in uninfected controls.

<p>Protein activity was measured by adding a synthetic peptide specific for cathepsin B conjugated to a red fluorogenic compound (RR₂-AFC) and read in a fluorometer at 400 nm excitation and 505 nm emission filters. Culture fluids from HIV-infected macrophages showed a significant increase in the activity of secreted cathepsin B at 3 and 12 days post infection (*p≤0.05). Results for cathepsin B activity are expressed as percentages of control (media only). Increased cathepsin B activity over the days after HIV infection (mean estimate increase/day 9.25 (SE 2.61), p = 0.002). The specificity of the assay is shown by the abrogation of any active cathepsin B after the addition of an inhibitor.</p

Cathepsin B does not interacts with cystatin C in HIV-infected MDM.

<p><i>In situ</i> PLA (Duolink) assay shows interaction between cathepsin B and cystatin C in uninfected cells (A, B and C; top panels) and decreased interactions in HIV infected MDM (D, E and F; bottom panels). Expression of cathepsin B (red) and cystatin C (green) was confirmed by immunofluorescence in uninfected (G and H; right top panels) and HIV-infected MDM (I and J; right bottom panels). This is a representative figure from 3 experiments performed.</p

Cathepsin B is released from lysosomes in HIV-infected MDM.

<p>To analyze the lysosomal localization of cathepsin B, cathepsin B and LAMP2 immunoreactivity were assessed by <i>in situ</i> PLA (Duolink) in uninfected and HIV-infected MDM 3, 6 and 12 dpi. Cathepsin B colocalizes with LAMP2 in uninfected MDM (A, B and C; top panels). However, little colocalization is seen in HIV infected MDM (D, E and F; bottom panels). The presence of individual proteins was determined by immunofluorescence staining (G, H, I, J, K and L). As seen in the right panels both, cathepsin B (red) and LAMP2 (green) are expressed in uninfected (G, H, and I) and HIV-infected (J, K, L) cells. The results presented in this figure are representative of 3 experiments.</p