Petro Starokadomskyy

COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A

The COMMD1 protein, implicated in copper homeostasis, is found to regulate endosomal sorting of the copper transporter ATP7A through a novel protein complex containing CCDC22, CCDC93, and C16orf62, which link COMMD1 to the WASH complex.

CCDC22 deficiency in humans blunts activation of proinflammatory NF-kappa B signaling

NF-κB is a master regulator of inflammation and has been implicated in the pathogenesis of immune disorders and cancer. Its regulation involves a variety of steps, including the controlled degradation of inhibitory IκB proteins. In addition, the inactivation of DNA-bound NF-κB is essential for its regulation. This step requires a factor known as copper metabolism Murr1 domain-containing 1 (COMMD1), the prototype member of a conserved gene family. While COMMD proteins have been linked to the ubiquitination pathway, little else is known about other family members. Here we demonstrate that all COMMD proteins bind to CCDC22, a factor recently implicated in X-linked intellectual disability (XLID). We showed that an XLID-associated CCDC22 mutation decreased CCDC22 protein expression and impaired its binding to COMMD proteins. Moreover, some affected individuals displayed ectodermal dysplasia, a congenital condition that can result from developmental NF-κB blockade. Indeed, patient-derived cells demonstrated impaired NF-κB activation due to decreased IκB ubiquitination and degradation. In addition, we found that COMMD8 acted in conjunction with CCDC22 to direct the degradation of IκB proteins. Taken together, our results indicate that CCDC22 participates in NF-κB activation and that its deficiency leads to decreased IκB turnover in humans, highlighting an important regulatory component of this pathway.

DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.

COMMD1 (Copper Metabolism MURR1 Domain-containing Protein 1) Regulates Cullin RING Ligases by Preventing CAND1 (Cullin-associated Nedd8-dissociated Protein 1) Binding

Cullin RING ligases (CRLs), the most prolific class of ubiquitin ligase enzymes, are multimeric complexes that regulate a wide range of cellular processes. CRL activity is regulated by CAND1 (Cullin-associated Nedd8-dissociated protein 1), an inhibitor that promotes the dissociation of substrate receptor components from the CRL. We demonstrate here that COMMD1 (copper metabolism MURR1 domain-containing 1), a factor previously found to promote ubiquitination of various substrates, regulates CRL activation by antagonizing CAND1 binding. We show that COMMD1 interacts with multiple Cullins, that the COMMD1-Cul2 complex cannot bind CAND1, and that, conversely, COMMD1 can actively displace CAND1 from CRLs. These findings highlight a novel mechanism of CRL activation and suggest that CRL regulation may underlie the pleiotropic activities of COMMD1.

A bird’s-eye view of autophagy

Autophagy is a process in which a eukaryotic (but not prokaryotic) cell destroys its own components through the lysosomal machinery. This tightly regulated process is essential for normal cell growth, development, and homeostasis, serving to maintain a balance between synthesis and degradation, resulting in the recycling of cellular products. Here we try to expand the concept of autophagy and define it as a general mechanism of regulation encompassing various levels of the biosphere. Interestingly, one of the consequences of such an approach is that we must presume an existence of the autophagic processes in the prokaryotic domain.

Bimolecular Affinity Purification: A Variation of TAP with Multiple Applications

The identification of true interacting partners of any given bait can be plagued by the nonspecific purification of irrelevant proteins. To avoid this problem, Tandem Affinity Purification (TAP) is a widely used procedure in molecular biology as this reduces the chance of nonspecific proteins being present in the final preparation. In this approach, two different affinity tags are fused to the protein bait. Herein, we review in detail a variation on the TAP procedure that we have previously developed, where the affinity moieties are placed on two different proteins that form a complex in vivo. This variation, which we refer to as Bimolecular Affinity Purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known proteins. We have utilized BAP for characterization of molecular complexes and evaluation of proteins interaction. Another application of BAP is the isolation of ubiquitin-like proteins (UBL)-modified fractions of a given protein and characterization of the lysine-acceptor site and structure of UBL-chains.

Detection of IκB Degradation Dynamics and IκB-α Ubiquitination

The NF-κB signaling pathway is a primary regulator of inflammation that has been implicated in the pathogenesis of immune disorders and cancer. This signaling network is strictly regulated; in a nonactivated state, NF-κB transcription factors are sequestered in the cytoplasm by the IκB family of proteins. Various pro-inflammatory stimuli result in the phosphorylation and subsequent ubiquitination of IκBs. These events lead to rapid degradation of IκB and allow translocation of the transcription factors to the nucleus. Therefore, ubiquitination and degradation of IκBs are critical steps in NF-κB pathway activation and can serve as a quantitative parameter to assess pathway activation. In this article, we present a detailed protocol for the quantification of in vivo ubiquitination and turnover of IκB-α in response to a variety of cellular stimuli.

Methodology to study NF-κB/RelA ubiquitination in vivo.

Nuclear factor-kappa B (NF-κB) is a family of transcription factors that regulate immune responses, cell proliferation, differentiation, and survival. Activity of the NF-κΒ pathway on a cellular level is tightly controlled through various mechanisms, one of which is the ubiquitin-dependent degradation of chromatin-bound NF-κB subunits. In general, the ubiquitination of NF-κB regulates the duration of gene transcription activated in response to inflammatory signals. In this article, we present protocols to examine the in vivo ubiquitination status of RelA, a critical protein of the NF-κB family.

Microbial Sensing by Intestinal Myeloid Cells Controls Carcinogenesis and Epithelial Differentiation

SUMMARY Physiologic microbe-host interactions in the intestine require the maintenance of the microbiota in a luminal compartment through a complex interplay between epithelial and immune cells. However, the roles of mucosal myeloid cells in this process remain incompletely understood. In this study, we identified that decreased myeloid cell phagocytic activity promotes colon tumorigenesis. We show that this is due to bacterial accumulation in the lamina propria and present evidence that the underlying mechanism is bacterial induction of prostaglandin production by myeloid cells. Moreover, we show that similar events in the normal colonic mucosa lead to reductions in Tuft cells, goblet cells, and the mucus barrier of the colonic epithelium. These alterations are again linked to the induction of prostaglandin production in response to bacterial penetration of the mucosa. Altogether, our work highlights immune cell-epithelial cell interactions triggered by the microbiota that control intestinal immunity, epithelial differentiation, and carcinogenesis.

Evolution of the skin manifestations of X-linked pigmentary reticulate disorder

DEAR EDITOR, X-linked reticulate pigmentary disorder (XLPDR, MIM 301220) is a rare syndrome first recognized by Partington. The cardinal manifestations of the disorder are diffuse reticulate hyperpigmentation, hypohidrosis and unique facial features, as well as recurrent pneumonias and sterile inflammation in various organs. The syndrome is exceedingly rare and only 20 patients have been reported worldwide. Recently, we identified that the disorder is associated with a recurrent intronic mutation in POLA1, the gene encoding the catalytic subunit of DNA polymerase alpha. Here, we report a new case of XLPDR and include the first detailed description of the evolution of its dermatological features, as well as confirmation of the same intronic POLA1 mutation. The patient is the fourth child of four and has an unaffected elder sister (Fig. S1a; see Supporting Information). His elder brother has an atrial septal defect, but no other abnormalities. Another brother was born with Potter syndrome and died at 2 h of age. The patient’s mother has restricted hyperpigmentation along Blaschko lines (Fig. S1b; see Supporting Information), which is also observed in his grandmother and aunt, but no other males in the family appear clinically affected. During his first year of life, the patient developed recurrent pneumonia, urethral strictures with occasional bleeding after urination, and bloody diarrhoea (which eventually resolved while on hydrolyzed formula). However, he had no recurrent or unusual nonpulmonary infections and did not have growth delay. At age 7 years, he developed bronchiectasis in the left lower lobe (requiring pulmozyme treatment) and was noted to have decreased visual acuity and photophobia due to bilateral erosive keratitis. At age 13 years, the typical facial features of XLPDR (upswept hair and flared eyebrows) along with reticulate hyperpigmentation were noted (Fig. S1c; see Supporting Information). Laboratory studies (Table S1; see Supporting Information) were otherwise within normal ranges. The diagnosis of XLPDR was confirmed at age 13 years by genetic and immunological tests. The patient was found to be hemizygous for a previously reported POLA1 mutation (NC_000023.10:g.24744696A>G). This noncoding mutation in intron 13 of the POLA1 gene is known to be associated with activated type I interferon responses, and indeed the patient displayed dramatic elevation of interferon-stimulated genes (Fig. S1d; see Supporting Information). While it has been recognized that the pigmentary changes in XLPDR are not present at birth, the evolution of the skin phenotype has not been reported. Retrospective analysis of the new proband indicates no overt changes in skin pigmentation in the first year of life (Fig. S1e; see Supporting Information). The first dermatological manifestations were noted at 14 months of age, when he developed facial telangiectasias (Fig. S1f; see Supporting Information). At the same age, the patient developed hypopigmented spots on the face, extremities and ultimately the trunk (Fig. S1g; see Supporting Information). These skin changes were not associated with pruritus or pain, but the skin was persistently dry. Frank hypohidrosis was noted in infancy, selectively affecting trunk and extremities. In contrast, the head and neck presented profuse sweating since this age. In view of these changes, at age 7 years, a skin biopsy was obtained from his left thigh, which revealed epidermal hypomelanosis with orthokeratosis, focal acanthosis, focal lymphocytosis and mild hypopigmentation (Fig. S1h; see Supporting Information). The dermis contained few melanophages and minimal superficial lymphocytic perivascular infiltrate. Direct immunofluorescence revealed granular deposits of C3 at the dermoepidermal junction, but no deposition of IgG, IgA, IgM or fibrin. Hyperpigmentation was first noted by the parents at 8 years old. At 12 years old, a new skin biopsy revealed orthokeratosis with areas of hyperpigmentation and hypopigmentation of the basal layer of the epidermis. The dermis had mild perivascular inflammatory infiltrates composed of lymphocytes and some melanophages (Fig. S1i; see Supporting Information). Our findings in this case further confirm that XLPDR is associated with constitutive activation of type I interferon responses. Moreover, these data demonstrate lack of genetic heterogeneity in XLPDR, meaning that the identical mutation has been seen in all cases thus far. We speculate that the unique features of the syndrome may be directly related to the specific effects induced by this intronic variant, such as possible variability in mis-splicing among tissues. Unlike Aicardi–Gouti eres syndrome (AGS), the best known interferonopathy, XLPDR does not involve the brain and is associated with a unique set of manifestations including the characteristic facial features and hypohidrosis, which remain unexplained. Among type I interferonopathies, skin findings are seen in activating mutations in TMEM173 (STING), which lead to pulmonary and skin vasculitis, predominantly affecting digits. Additionally, ADAR mutations, which have been linked to AGS, can also result in dyschromatosis symmetrica hereditaria (MIM 127400), characterized by the development in early childhood