Redox-regulated pathway of tyrosine phosphorylation underlies NF-κB induction by an atypical pathway independent of the 26S proteasome.

Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

Compensatory increase in USP14 activity accompanies impaired proteasomal proteolysis during aging.

The deubiquitinating enzyme, USP14, found in association with the proteasome is essential in mediating ubiquitin trimming and in ensuring ubiquitin-homeostasis. As aging is accompanied by a significant decline in proteasomal proteolysis in primary human T lymphocytes, we evaluated the contributory role of USP14 in this decline. Our studies for the first time demonstrate that enzymatic activity of proteasome-associated USP14 is significantly higher in T cells obtained from elderly donors. Additionally, such an increase in USP14 activity could be mimicked by chemically inhibiting the proteasome, using lactacystin. Thus, USP14 activity appears to be reciprocally regulated by the catalytic function of the 26S proteasome. To determine whether the inhibition of USP14 activity counter regulates proteasomal proteolysis, T cells pretreated with a small molecule inhibitor of USP14, IU1, were activated and assessed for IκBα degradation as a measure of proteasomal proteolysis. While T cells obtained from young donors demonstrated increased degradation of IκBα, those from the elderly remained unaffected by IU1 pretreatment. Taken together, these results demonstrate that the decrease in proteolysis of proteasomal substrates during aging is independent of the increased USP14 activity and that the reciprocal regulation of USP14 and proteasomal catalytic activity may be necessary to maintain cellular ubiquitin homeostasis.

Altered regulation of CXCR4 expression during aging contributes to increased CXCL12-dependent chemotactic migration of CD4(+) T cells.

Chemokine-dependent migration of T lymphocytes assures recirculation of naïve T cells to secondary lymphoid organs and tissue-specific trafficking of memory-effector T cells. Previous studies carried out in rodents have demonstrated age-associated modulation of the expression of chemokine receptors such as CXCR4 and CCR5; however, little is known about the molecular mechanisms that regulate receptor expression and turnover in T cells, during advancing age in humans. Our recent results demonstrating increased chemotactic migration in response to CXCL12 in CD4(+) T cells obtained from the elderly, as compared to those from young donors, led us to hypothesize that increase in surface expression, because of altered endocytic regulation of CXCR4 on T cells during aging, might be directly responsible for increased migration toward CXCL12. Studies presented here demonstrate a significant increase in the surface expression of CXCR4 in CD4(+) T cells from elderly human donors, relative to those from the young. Additionally, CXCL12-mediated endocytosis of CXCR4 was differentially regulated during aging, which could be attributed to alterations in the ubiquitination of CXCR4. Thus, altered ubiquitination of CXCR4 may contribute to the increased surface expression and enhanced T-cell migration to chemotactic stimuli in the elderly.

Impairment of non-muscle myosin IIA in human CD4+ T cells contributes to functional deficits in the elderly.

Physiological aging imposes significant alterations in the repertoire of T cells and all associated functions. Although several studies have reported defects upon antigen-induced activation of T cells during aging, the molecular mechanisms that control T-cell receptor (TCR) downmodulation remain to be fully defined. While previous studies have assessed the role of F-actin in regulating activation-induced TCR internalization, few have delineated the roles of motor proteins, such as non-muscle myosin IIA (NMMIIA). In this study, we describe a series of experiments supporting the hypothesis that effective TCR downmodulation requires not only efficient reorganization of the actin cytoskeleton, but also functional NMMIIA. For the first time, we show that CD4(+) T cells from elderly human donors have dysfunctional NMMIIA that contributes to delaying activation-induced TCR internalization and impairing calcium mobilization. Additionally, our results demonstrate that chemical inhibition of NMMIIA in CD4(+) T cells from young donors also results in complete abrogation of TCR internalization, strongly supporting the fundamental role of NMMIIA in modulating this event. Recent observations that the generation of an efficient T-cell response requires migration prompted us to investigate whether NMMIIA also plays a regulatory role in CD4(+) T-cell migration. We show that chemical inhibition of NMMIIA downmodulates chemotactic migration in CD4(+) T cells from both young and elderly donors. Together, these data demonstrate a significant contribution of dysfunctional NMMIIA to TCR-mediated functional defects during aging.

Aging and immune function: molecular mechanisms to interventions.

Abstract The immune system of an organism is an essential component of the defense mechanism aimed at combating pathogenic stress. Age-associated immune dysfunction, also dubbed "immune senescence," manifests as increased susceptibility to infections, increased onset and progression of autoimmune diseases, and onset of neoplasia. Over the years, extensive research has generated consensus in terms of the phenotypic and functional defects within the immune system in various organisms, including humans. Indeed, age-associated alterations such as thymic involution, T cell repertoire skewing, decreased ability to activate naïve T cells and to generate robust memory responses, have been shown to have a causative role in immune decline. Further, understanding the molecular mechanisms underlying the generation of proteotoxic stress, DNA damage response, modulation of ubiquitin proteasome pathway, and regulation of transcription factor NFκB activation, in immune decline, have paved the way to delineating signaling pathways that cross-talk and impact immune senescence. Given the role of the immune system in combating infections, its effectiveness with age may well be a marker of health and a predictor of longevity. It is therefore believed that a better understanding of the mechanisms underlying immune senescence will lead to an effective interventional strategy aimed at improving the health span of individuals. Antioxid. Redox Signal. 14, 1551-1585.

Proteasome inhibition up-regulates inflammatory gene transcription induced by an atypical pathway of NF-kappaB activation.

Proteasome inhibition has become synonymous with inhibition of NF-kappaB activity. However, hyperactive NF-kappaB responses often accompany physiological conditions marked by proteasomal defects, i.e. advancing age, geriatric diseases, and bortezomib resistance. These paradoxical NF-kappaB responses are likely to be impervious to proteasomal defects because they stem from atypical NF-kappaB signaling induced by upstream mechanisms which are proteasome-independent. While this atypical pathway does not require proteasome for NF-kappaB nuclear translocation, a role for proteasome in regulating nuclear NF-kappaB remains unexplored. We now demonstrate that proteasome stringently controls transcription of inflammatory mediators regulated by this atypical NF-kappaB pathway. Proteolytic activity of the proteasome mediates the removal of the NF-kappaB subunit, p65/RelA, from inflammatory genes, thereby terminating atypical NF-kappaB-dependent transcriptional responses. For the first time, we demonstrate that both 19S and 20S components of the 26S proteasome complex are recruited to an inflammatory gene promoter; additionally, the 19S and 20S complexes appear to play distinct roles in the negative regulation of NF-kappaB-dependent transcription. By demonstrating that proteasome regulates the termination of atypical NF-kappaB-dependent transcriptional responses, these studies clearly indicate a novel, regulatory role for proteasome in atypical NF-kappaB signaling. Moreover, these results signal a potential interplay between lowered proteasomal function and increased inflammation and may explain why inflammation accompanies physiological conditions under which proteasomal function is compromised, such as during advancing age or following bortezomib treatment. Given this role for proteasome in inflammation resolution, restoration of proteasome function may constitute a novel mechanism for intervening in chronic inflammatory diseases.

Catalytic activity of the proteasome fine-tunes Brg1-mediated chromatin remodeling to regulate the expression of inflammatory genes.

The induction of key pro-inflammatory genes is regulated by the SWI/SNF class of ATP-dependent remodeling complexes. In particular, the catalytic ATPase subunit, Brg1, is distinctly involved in the chromatin remodeling required for activating pro-inflammatory genes in a temporally, ordered fashion. Despite advances in our understanding of the role for Brg1 in the kinetics of inflammatory responses, little is known about the precise mechanisms which down-regulate Brg1 activity. Biochemical studies implicate a role for the proteasome in the regulation of SWI/SNF assembly and function; however, it is unclear if proteasome-dependent mechanisms modulate its remodeling activity or recruitment to chromatin in order to regulate inflammatory gene transcription. We now demonstrate for the first time that proteasome function represents an important mechanism for limiting inducible association of Brg1 with promoters of SWI/SNF-regulated, inflammatory genes. As a result, catalytic activity of the proteasome fine-tunes the kinetics of inflammatory gene transcription by inhibiting both premature and persistent chromatin remodeling at SWI/SNF-regulated genes. These results provide mechanistic insight into the interplay between nucleosome remodeling, inflammation and proteasome, and underscore the critical role of the proteasome in controlling both extent and duration of inflammatory responses.

Ca2+ homeostasis regulates Xenopus oocyte maturation.

In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte's competence to undergo maturation in preparation for fertilization and embryonic development.

Lower expression of catalytic and structural subunits of the proteasome contributes to decreased proteolysis in peripheral blood T lymphocytes during aging.

Aging, in the immune system, is characterized by a decreased ability to respond to exogenous insults, resulting in increased susceptibility to infections and blunted response to vaccination. While significant age-associated deficits in immune function have been documented, the underlying molecular mechanisms are still being investigated. A consistent decline in the proteolytic activity of the proteasome has been demonstrated with advancing age, implicating an important role for the proteasome in immune senescence, by studies that largely employed proteasome-enriched preparations from cell lysates. With the availability of novel cell permeable active site probes designed specifically for assaying proteasomal activity in live cells, we now confirm our earlier data demonstrating lower catalytic activity of the proteasome in primary human T cells obtained from the elderly when compared to those from young donors. Loss in proteasomal catalytic activity translated into a loss in functional activity, as was observed in a degradation assay employing an ubiquitinated protein substrate, Ub-IkappaBalpha. Unlike fluorogenic peptide substrates, use of ubiquitinated protein substrates not only confer greater stringency in terms of proteasomal hydrolysis, but also involve the participation of the 19S regulatory component. This age-associated loss in proteasomal activity is accompanied by alteration in the levels of catalytic, structural and regulatory subunits, with no change in that of the 11S activator or the inhibitor PAAF1. Oxidative modification, such as carbonylation and lipid-peroxidation, of proteasomal subunits was also detected in T cells from the elderly. Thus, oxidative modification and lower levels of proteasomal subunits contribute to decreased proteolytic activity during immune-senescence.

Redox regulation of the proteasome in T lymphocytes during aging.

Proteasome is a major cellular organelle responsible for the regulated turnover of both normal and misfolded proteins. Recent reports from our laboratory have implicated lowered proteasomal chymotryptic activity to be responsible for decreased induction of the transcription factor NFkappaB in T lymphocytes during aging. In this study, we have further analyzed the basis for this decline in proteasomal function, by focusing on the role of oxidative stress. On exposure to the prooxidant BSO, both ATP-stimulatable 26S and ATP-independent 20S proteasomal catalytic activity could be down-regulated in T cells from young donors, mimicking the decline observed in T cells from the elderly. Loss in these catalytic activities, following exposure to prooxidant stimulus, also resulted in a decline in both activation-induced proliferation and degradation of the inhibitor IkappaBalpha, with concomitant increase in the accumulation of carbonylated proteins, mimicking responses seen in T cells from the elderly. Pretreatment with an antioxidant, NAC, could override prooxidant-mediated, but not age-associated, decrease in both 20S and 26S proteasomal activities. These results suggest that the decrease in proteasomal activities observed during aging may be secondary to oxidative stress and underlie immune senescence.

Constitutive degradation of IkappaBalpha in human T lymphocytes is mediated by calpain.

BACKGROUND: Activation-induced induction of transcription factor NFkappaB in T lymphocytes is regulated by its inhibitor IkappaBalpha. NFkappaB activation has been demonstrated to occur either by phosphorylation on serine residues 32 and 36 of the inhibitor, IkappaBalpha, followed by ubiquitination and degradation of the inhibitor by the 26S proteasome, or by a proteasome-independent mechanism involving tyrosine phosphorylation, but not degradation. However, the mechanism underlying constitutive regulation of the levels of the inhibitor, IkappaB, in primary human T lymphocytes, remains to be fully delineated. RESULTS: We demonstrate here, the involvement of a proteasome-independent pathway for constitutive regulation of IkappaBalpha levels in primary human T lymphocytes. Pretreatment with a cell permeable calpain inhibitor, E64D, but not with a proteasome specific inhibitor, lactacystin, blocks stimulus-independent IkappaBalpha degradation in primary human T cells. However, E64D pre-treatment fails to impact on IkappaBalpha levels following stimulation with either TNFalpha or pervanadate. Other isoforms of the inhibitor, IkappaBbeta, and IkappaBgamma, appear not to be subject to a similar ligand-independent regulation. Unlike the previously reported decline in ligand-induced degradation of IkappaBalpha in T cells from the elderly, constitutive degradation does not exhibit an age-associated decline, demonstrating proteasome-independent regulation of the activity. CONCLUSION: Our studies support a role for an E64D sensitive protease in regulating constitutive levels of IkappaBalpha in T cells, independent of the involvement of the 26S proteasome, and suggests a biological role for constitutive degradation of IkappaBalpha in T cells.

Tyrosine phosphorylation-dependent activation of NFkappaB is compromised in T cells from the elderly.

NFkappaB induction and gene regulation are compromised in T lymphocytes during aging. This has been attributed to altered proteasomal function resulting in decreased ubiquitin-mediated degradation of IkappaBalpha. However, little is known about the impact of aging on the mechanisms that lead to the release of active NFkappaB employing pro-oxidant pathways. Oxidant-mediated activation of NFkappaB has been previously shown to involve proteasome independent mechanisms and hence may be an important alternate conduit to the induction of this central transcription factor in aging. Employing H(2)O(2) and pervanadate we not only demonstrate lowered tyrosine phosphorylation of IkappaBalpha, but also compromised induction of nuclear NFkappaB in T cells from the elderly. Lowered tyrosine phosphorylation of IkappaBalpha may be due to a decrease in activity of p56(lck) and ZAP-70, since treatment with piceatannol, an inhibitor of syk and src family kinases, mimics age associated decline in tyrosine phosphorylation of IkappaBalpha in T cells from young donors. Thus, alternate pathways of NFkappaB induction are also impaired in T cells from the elderly and may underlie immune-deficit accompanying aging.