Evidence for the existence of a non-catalytic modifier site of peptide hydrolysis by the 20 S proteasome.

The 20 S proteasome is an endoprotease complex that preferentially cleaves peptides C-terminal of hydrophobic, basic, and acidic residues. Recently, we showed that these specific activities, classified as chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide-hydrolyzing (PGPH) activity, are differently affected by Ritonavir, an inhibitor of human immunodeficiency virus-1 protease. Ritonavir competitively inhibited the chymotrypsin-like activity, whereas the trypsin-like activity was enhanced. Here we demonstrate that the Ritonavir-mediated up-regulation of the trypsin-like activity is not affected by specific active site inhibitors of the chymo-trypsin-like and PGPH activity. Moreover, we show that the mutual regulation of chymotrypsin-like and PGPH activities by their substrates as described previously by a "cyclical bite-chew" model is not affected by selective inhibitors of the respective active sites. These data challenge the bite-chew model and suggest that effectors of proteasome activity can act by binding to non-catalytic sites. Accordingly, we propose a kinetic "two-site modifier" model that assumes that the substrate (or effector) may bind to an active site as well as to a second non-catalytic modifier site. This model appears to be valid as it describes the complex kinetic effects of Ritonavir very well. Since Ritonavir partially inhibits major histocompatibility complex class I restricted antigen presentation, the postulated modifier site may be required to coordinate the active centers of the proteasome for the production of class I peptide ligands.

How an inhibitor of the HIV-I protease modulates proteasome activity.

The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.

Heterogeneity in neuroendocrine and immune responses to brief psychological stressors as a function of autonomic cardiac activation.

Human responses to brief psychological stressors are characterized by changes and large individual differences in autonomic, neuroendocrine, and immune function. The authors examined the effects of brief psychological stressors on cardiovascular, neuroendocrine, and cellular immune response in 22 older women to investigate the common effects of stress across systems. They also used interindividual variation in heart rate reactivity, cardiac sympathetic reactivity (as indexed by preejection period reactivity in their reactivity paradigm), and cardiac vagal reactivity (as indexed by respiratory sinus arrhythmia reactivity) to explore the heterogeneity in human responses to brief psychological stressors. The results revealed that brief psychological stressors heightened cardiac activation, elevated plasma catecholamine concentrations, and affected the cellular immune response. It was also found that individuals characterized by high, relative to low, cardiac sympathetic reactivity showed higher stress-related changes in adrenocorticotropic hormone and cortisol plasma levels but comparable changes in epinephrine and norepinephrine concentrations. These data suggest that the effects of psychological stressors on cardiovascular and cellular immune response are governed by coordinated regulatory mechanism(s) and that going beyond the simple notion of heart rate reactivity to examine neural substrates may shed light on the interrelationships among and the regulatory mechanisms for the autonomic, endocrine, and immune responses to stressors.

The effects of an acute psychological stressor on cardiovascular, endocrine, and cellular immune response: a prospective study of individuals high and low in heart rate reactivity.

High and low reactors were preselected on the basis of their heart rate reactivity to a speech stressor in a prescreening session. In the main study, subjects were exposed to a mental arithmetic plus noise stressor. Cardiovascular activity was recorded during baseline and stressor, and blood was drawn prior to and following the stressor for endocrine and immune assays. Results revealed that the stressor decreased the blastogenic response to concanavalin A and increased natural killer cell numbers and cytotoxicity, absolute numbers of CD8+ T-lymphocytes, norepinephrine and epinephrine levels, heart rate, and blood pressure responses. In addition, cortisol and natural killer cell cytotoxicity responses to the stressor differentiated individuals high versus low in heart rate reactivity. These results suggest that the interactions among the autonomic nervous system, endocrine system, and immune system are not only amenable to psychophysiological analysis but that such analyses may play an important role in illuminating underlying mechanisms.