High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell’s DNA replication and repair machineries to replicate their own genomes. How this host–pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

GAPDH, a heme chaperone, has been previously implicated in the incorporation of heme into iNOS and soluble guanylyl cyclase (sGC). Since sGC is critical for myoglobin (Mb) heme‐maturation, we investigated the role of GAPDH in the maturation of this globin, as well as hemoglobins α, β, and γ. Utilizing cell culture systems, we found that overexpression of wild‐type GAPDH increased, whereas GAPDH mutants H53A and K227A decreased, the heme content of Mb and Hbα and Hbβ. Overexpression of wild‐type GAPDH fully recovered the heme‐maturation inhibition observed with the GAPDH mutants. Partial rescue was observed by overexpression of sGCβ1 but not by overexpression of a sGCΔβ1 deletion mutant, which is unable to bind the sGCα1 subunit required to form the active sGCα1β1 complex. Wild type and mutant GAPDH was found to be associated in a complex with each of the globins and Hsp90. GAPDH at endogenous levels was found to be associated with Mb in differentiating C2C12 myoblasts, and with Hbγ or Hbα in differentiating HiDEP‐1 erythroid progenitor cells. Knockdown of GAPDH in C2C12 cells suppressed Mb heme‐maturation. GAPDH knockdown in K562 erythroleukemia cells suppressed Hbα and Hbγ heme‐maturation as well as Hb dimerization. Globin heme incorporation was not only dependent on elevated sGCα1β1 heterodimer formation, but also influenced by iron provision and magnitude of expression of GAPDH, d‐aminolevulinic acid, and FLVCR1b. Together, our data support an important role for GAPDH in the maturation of myoglobin and γ, β, and α hemoglobins.

Background: In early autoimmune neuroinflammation, interferon (IFN)γ and its upregulation of the immunoproteasome (iP) is pathologic. However, during chronic multiple sclerosis (MS), IFNγ has protective properties. Although dysregulation of the iP has been implicated in neurodegeneration, its function remains to be fully elucidated. Here, we demonstrate that IFNγ signaling in regional astrocytes induces the iP and promotes protection of the CNS during chronic autoimmunity. Methods: In a multiple sclerosis (MS) brain, we evaluated mRNA expression and labeled postmortem MS brainstem and spinal cord for iP subunits and indicators of oxidative stress. Primary regional human astrocytes were analyzed for iP regulation and function by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, OxyBlot, and reactive oxygen species and caspase activity detection assays. Following immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55 , the role of IFNγ signaling and the iP during chronic experimental autoimmune encephalomyelitis (EAE) were assessed using pharmacologic inhibition of the iP and genetic interruption of IFNγ signaling specifically in astrocytes. Central nervous system (CNS) tissues were analyzed by immunohistochemistry (IHC) and immunofluorescence, and cell-specific colocalization was quantified. Results: In MS tissue, iP expression was enhanced in the spinal cord compared to brainstem lesions, which correlated with a decrease in oxidative stress. In vitro, IFNγ stimulation enhanced iP expression, reduced reactive oxygen species burden, and decreased oxidatively damaged and poly-ubiquitinated protein accumulation preferentially in human spinal cord astrocytes, which was abrogated with the use of the iP inhibitor, ONX 0914. During the chronic phase of an MS animal model, EAE, ONX 0914 treatment exacerbated the disease and led to increased oxidative stress and polyubiquitinated protein buildup. Finally, mice with astrocyte-specific loss of the IFNγ receptor exhibited worsened chronic EAE associated with reduced iP expression, enhanced lesion size and oxidative stress, and poly-ubiquitinated protein accumulation in astrocytes. Conclusions: Taken together, our data reveal a protective role for IFNγ in chronic neuroinflammation and identify a novel function of the iP in astrocytes during CNS autoimmunity.

During multiple sclerosis (MS), an inflammatory and neurodegenerative disease of the central nervous system (CNS), symptoms, and outcomes are determined by the location of inflammatory lesions. While we and others have shown that T cell cytokines differentially regulate leukocyte entry into perivascular spaces and regional parenchymal localization in murine models of MS, the molecular mechanisms of this latter process are poorly understood. Here, we demonstrate that astrocytes exhibit region-specific responses to T cell cytokines that promote hindbrain versus spinal cord neuroinflammation. Analysis of cytokine receptor expression in human astrocytes showed region-specific responsiveness to Th1 and Th17 inflammatory cytokines. Consistent with this, human and murine astrocytes treated with these cytokines exhibit differential expression of the T cell localizing molecules VCAM-1 and CXCR7 that is both cytokine and CNS region-specific. Using in vivo models of spinal cord versus brain stem trafficking of myelin-specific T cells and astrocyte-specific deletion strategies, we confirmed that Th1 and Th17 cytokines differentially regulate astrocyte expression of VCAM-1 and CXCR7 in these locations. Finally, stereotaxic injection of individual cytokines into the hindbrain or spinal cord revealed region-and cytokine-specific modulation of localizing cue expression by astrocytes. These findings identify a role for inflammatory cytokines in mediating local astrocyte-dependent mechanisms of immune cell trafficking within the CNS during neuroinflammation.astrocyte, CXCR7, cytokine, neuroinflammation, regional heterogeneity, T cell, VCAM-1 Jessica L. Williams and Sindhu Manivasagam contributed equally to this study.