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

Correction: Evolution of the Twist Subfamily Vertebrate Proteins: Discovery of a Signature Motif and Origin of the Twist1 Glycine-Rich Motifs in the Amino-Terminus Disordered Domain.

[This corrects the article DOI: 10.1371/journal.pone.0161029.]

Mechanisms of Regulation of the <i>CHRDL1</i> Gene by the TWIST2 and ADD1/SREBP1c Transcription Factors

Setleis syndrome (SS) is a rare focal facial dermal dysplasia caused by recessive mutations in the basic helix-loop-helix (bHLH) transcription factor, TWIST2. Expression microarray analysis showed that the chordin-like 1 (CHRDL1) gene is up-regulated in dermal fibroblasts from three SS patients with the Q119X TWIST2 mutation. METHODS: Putative TWIST binding sites were found in the upstream region of the CHRDL1 gene and examined by electrophoretic mobility shift (EMSA) and reporter gene assays. RESULTS: EMSAs showed specific binding of TWIST1 and TWIST2 homodimers, as well as heterodimers with E12, to the more distal E-boxes. An adjoining E-box was bound by ADD1/SREBP1c. EMSA analysis suggested that TWIST2 and ADD1/SREBP1c could compete for binding. Luciferase (luc) reporter assays revealed that the CHRDL1 gene upstream region drives its expression and ADD1/SREBP1c increased it 2.6 times over basal levels. TWIST2, but not the TWIST2-Q119X mutant, blocked activation by ADD1/SREBP1c, but overexpression of TWIST2-Q119X increased luc gene expression. In addition, EMSA competition assays showed that TWIST2, but not TWIST1, competes with ADD1/SREBP1c for DNA binding to the same site. CONCLUSIONS: Formation of an inactive complex between the TWIST2 Q119X and Q65X mutant proteins and ADD1/SREBP1c may prevent repressor binding and allow the binding of other regulators to activate CHRDL1 gene expression

Amino acid comparison between ancestor and human Twist sequences.

<p>Functional motifs (nuclear localization signal (NLSs), basic Helix-loop-Helix (bHLH), glycine-rich regions, and Twist box domains) are depicted by bars on top of the sequence. Twist2 lacks both glycine regions present only in Twist1. The Twist_BB (closest to ancestor) protein lacks the first glycine region but contains some glycine residues in the second glycine region (black box). The Twist_BB protein contains extra amino acid residues at the start of its N-terminus and in the Twist box domain of the C-terminus not present in either human Twist proteins. The bHLH domains are approximately 90% conserved in all three sequences. Overall, Twist1 amino acid sequence is 54% similar to Twist_BB, while Twist2 has 64% similarity, which suggests that the ancestor of both Twist paralogs was a “Twist2-like” protein. Below the protein sequence: (*) = conserved residues; (:) = conservative mutations; (.) = semi-conservative mutations; () = non-conservative mutations. Twist_BB = <i>Branchiostoma belcheri</i> (Lancelet). Sequence names used represent the common name of the species to which they belong. The alignment was performed with PSI-COFFEE.</p

Secondary structure prediction of TWIST proteins.

<p><b>A) Structural characteristics of TWIST1 protein. B) Structural characteristics of TWIST2 proteins.</b> The analyses show that both TWIST1 and TWIST2 sequences have a disordered amino terminus domain as expected. It also predicts a high degree of structure for the bHLH domain, and the carboxy terminus in a disordered/flexible region. Pink tube = helix; Yellow arrow = strand; Black line = coil; Blue bars = confidence of prediction; Pred = predicted secondary structure; A.A. = target sequence. The FFpred web server was used for structure prediction.</p

Evolutionary Conservation of Intrinsic Protein Disorder.

<p><b>A) Disorder conservation prediction of Twist1 proteins.</b> The disorder feature found in the N-terminus region of Twist1 is evolutionary conserved. <b>B) Disorder conservation prediction of Twist2 proteins.</b> The disorder feature found in the N-terminus region of Twist2 is evolutionary conserved. Disorder classification: Structured (white, S) if there is zero disorder. Constrained (green, C) if both the sequence and disorder conservation scores are 5 or greater. Non-conserved (red, N) if the disorder conservation score is below 5. (-) means gap. DisCons tool was used to asses for evolutionary conservation of intrinsic protein disorder.</p