Shai Cohen

Modification by Single Ubiquitin Moieties Rather Than Polyubiquitination Is Sufficient for Proteasomal Processing of the p105 NF-κB Precursor

Activation of NF-kappaB is regulated via numerous ubiquitin- and proteasome-mediated steps; an important one is processing of the precursor p105 to the p50 active subunit. The mechanisms involved are largely unknown, because this is an exceptional case where the ubiquitin system does not destroy its substrate completely. Here, we demonstrate that proteasomal processing of p105 requires ubiquitin but not generation of polyubiquitin chains. In vitro, ubiquitin species that cannot polymerize mediate processing. In yeasts that express nonpolymerizable ubiquitins, processing proceeds normally, whereas degradation of substrates that are dependent on polyubiquitination is inhibited. Similar results were obtained in mammalian cells. Interestingly, processing requires multiple monoubiquitinations, because progressive elimination of lysines in p105 is accompanied by gradual inhibition of p50 generation. Finally, the proteasome recognizes the multiply monoubiquitinated p105. These findings suggest that a proteolytic signal can be composed of a cluster of single ubiquitins, not necessarily a chain.

Dual Effects of IκB Kinase β-Mediated Phosphorylation on p105 Fate: SCFβ-TrCP-Dependent Degradation and SCFβ-TrCP-Independent Processing

ABSTRACT Processing of the p105 NF-κB precursor to yield the p50 active subunit is a unique and rare case in which the ubiquitin system is involved in limited processing rather than in complete destruction of its target. The mechanisms involved in this process are largely unknown, although a glycine repeat in the middle of p105 has been identified as a processing stop signal. IκB kinase (IKK)β-mediated phosphorylation at the C-terminal domain with subsequent recruitment of the SCFβ-TrCP ubiquitin ligase leads to accelerated processing and degradation of the precursor, yet the roles that the kinase and ligase play in each of these two processes have not been elucidated. Here we demonstrate that IKKβ has two distinct functions: (i) stimulation of degradation and (ii) stimulation of processing. IKKβ-induced degradation is dependent on SCFβ-TrCP, which acts through multiple lysine residues in the IκBγ domain. In contrast, IKKβ-induced processing of p105 is β-transduction repeat-containing protein (β-TrCP) independent, as it is not affected by expression of a dominant-negative β-TrCP or following its silencing by small inhibitory RNA. Furthermore, removal of all 30 lysine residues from IκBγ results in complete inhibition of IKK-dependent degradation but has no effect on IKK-dependent processing. Yet processing still requires the activity of the ubiquitin system, as it is inhibited by dominant-negative UbcH5a. We suggest that IKKβ mediates its two distinct effects by affecting, directly and indirectly, two different E3s.

ABIN-1 negatively regulates NF-κB by inhibiting processing of the p105 precursor

p105 plays dual roles in NF-kappaB signaling: in its precursor form it inhibits NF-kappaB activation, but limited processing by the ubiquitin system generates the p50 active subunit of the transcription factor. Here we show that ABIN-1, an A20-binding protein that is also known to attenuate NF-kappaB activation, inhibits p105 processing. p105 and ABIN-1 physically interact with one another, but the binding is not necessary for inhibition of processing. Rather, it appears to stabilize ABIN-1 and to increase its level, which further augments its inhibitory effect. Deletion of the processing inhibitory domain (PID) of p105 abrogates the inhibition which also requires the ABIN homology domain (AHD)-2 of ABIN-1. Together, the effects of ABIN-1 on p105 processing and of p105 on stabilizing ABIN-1 act to potentiate the NF-kappaB inhibitory activity of ABIN-1.

Effects of anti-TNF-α treatment on lipid profile in rheumatic diseases: an analytical cohort study

BackgroundThe aim was to assess the influence of long-term treatment with tumor necrosis factor alpha (TNF-α) inhibitors on total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and atherogenic index (AI) in rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) patients.MethodsA retrospective cohort study was conducted on RA, PsA, and AS patients treated with TNF-α inhibitors for at least 270 days between 2001 and 2011. Levels of TC, TG, LDL, and HDL and the AI were compared with baseline values at 0–6, 6–12, 12–18, and 18–24 months. Patients were further subdivided into three groups according to their HMG CoA reductase inhibitor (statin) treatment status in order to assess their effect on the results.ResultsThe records of 311 patients (152 RA, 90 PsA, and 69 AS) were reviewed. TC and TG increased following treatment with TNF-α inhibitors, from 180.85 ± 2.12 mg/dl and 116.00 ± 3.55 mg/dl at baseline to 188.12 ± 2.35 mg/dl (p = 0.02) and 132.02 ± 4.63 mg/dl at 0–6 months (p < 0.01), respectively, and to 184.88 ± 2.09 mg/dl (p = 0.02) and 129.36 ± 4.32 mg/dl at 18–24 months (p < 0.01), respectively. AI increased following treatment with TNF-α inhibitors, from –0.032 ± 0.017 at baseline to 0.004 ± 0.019 at 18–24 months (p < 0.01). LDL decreased significantly in patients who were treated with statins before and during the entire study period, from 119.97 ± 2.86 mg/dl at baseline to 104.02 ± 3.57 mg/dl at 18–24 months (p < 0.01), in contrast to an increase in LDL values in patients who did not receive statins during the study.ConclusionsTNF-α inhibitor treatment was associated with a significant increase in TC and TG levels and the AI. Adding statins to the treatment was associated with a significant decrease in LDL levels.

A20 inhibits both the degradation and limited processing of the NF-κB p105 precursor: A novel additional layer to its regulator role

p105 plays dual roles in NF-κB signaling: it generates the active subunit p50 and in its precursor form inhibits NF-κB activation. p105 processing occurs under basal conditions and increases following signaling. IκB kinase β (IKKβ) mediates phosphorylation at the C-terminal domain of p105, leads to accelerated processing and degradation of the precursor. A20 is a dual function deubiquitinating and ubiquitin ligating enzyme, involving in turning-off the NF-κB signaling pathway. Here we show that A20 suppresses TNFα-induced proteolysis of p105. We demonstrate that A20 inhibits both the signal induced processing and the degradation of p105 upstream to IKK stimulation. In addition, A20 represses the constitutive processing of the precursor protein in IKK independent manner. Silencing of A20 in cells by siRNA, restores p105 processing and the generation of p50. Functional analysis of A20, shows that the ubiquitin ligase activity mediated by its zinc finger domain (ZF), is required for the basal processing inhibitory effect. Its N-terminal ovarian tumor (OTU) domain, however, is not obligatory. We show that A20 inhibits p50 generation in cells by reducing the ubiquitination of its precursor, p105. Co-immunoprecipitation experiments show that A20 is immunoprecipitated by p105 only in the presence of its recently identified ligase, KPC1. Our data propose an additional novel mechanism to explain the known NF-κB inhibitory effects of A20: by affecting p105 ubiquitination and subsequently its degradation and limited processing.

Significance of extremely elevated ferritin level in medical inpatients

The diagnostic and prognostic utility of extremely elevated ferritin values in hospitalized medical patients is lacking. We aimed to determine the clinical significance of ferritin levels ≥ 1000 ng/mL in adults hospitalized in the general medical service. We scanned the hospital laboratory database for ferritin values ≥ 1000 ng/mL, and evaluated the medical history, diagnoses, and survival of patients hospitalized in the general medical service. We compared the characteristics and outcomes of patients with values up-to versus above 2,999 ng/mL. Ferritin samples ranging from 1,003 to 12,170 ng/mL from 422 patients in the lower and 94 in the higher ferritin groups were included. Malignancy, repeat blood transfusions and recent chemotherapy were more prevalent in the higher ferritin group (p=0.003, p=0.002, and p<0.001, respectively). Infection (58.7%), chronic kidney disease (22.0%), and solid or hematological malignancies (21.6% and 17.1%, respectively) were the leading conditions associated with elevated ferritin. One-year survival was low, and significantly lower in patients in the higher ferritin group (10.8% vs. 16.9%, p=0.004). In conclusion, extremely elevated ferritin values in patients admitted to the general medical service are associated with multiplicity of clinical conditions and poor outcome.