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Activity of heparanase, endoglycosidase that cleaves heparan sulfate side chains in heparan sulfate proteoglycans, is highly implicated in tumor progression and metastasis. Heparanase inhibitors are therefore being evaluated clinically as anti-cancer therapeutics. Heparanase 2 (Hpa2) is a close homolog of heparanase that lacks HS-degrading activity and functions as an endogenous inhibitor of heparanase. As a result, Hpa2 appears to attenuate tumor growth but mechanisms that regulate Hpa2 expression and determine the ratio between heparanase and Hpa2 are largely unknown. We have recently reported that the expression of Hpa2 is induced by endoplasmic reticulum (ER) and proteotoxic stresses, but the mechanism(s) underlying Hpa2 gene regulation was obscure. Here we expand the notion that Hpa2 is regulated by conditions of stress. We report that while ER and hypoxia, each alone, resulted in a 3-7 fold increase in Hpa2 expression, combining ER stress and hypoxia resulted in a noticeable, over 40-fold increase in Hpa2 expression. A prominent induction of Hpa2 expression was also quantified in cells exposed to heat shock, proteotoxic stress, lysosomal stress, and chemotherapy (cisplatin), strongly implying that Hpa2 is regulated by conditions of stress. Furthermore, analyses of the Hpa2 gene promoter led to the identification of activating-transcription-factor 3 (ATF3) as a transcription factor that mediates Hpa2 induction by stress, thus revealing, for the first time, a molecular mechanism that underlies Hpa2 gene regulation. Induction of Hpa2 and ATF3 by conditions of stress that often accompany the rapid expansion of tumors is likely translated to improved survival of cancer patients.

The activating transcription factor 3 (ATF3) and the c-Jun dimerization protein 2 (JDP2) are members of the basic leucine zipper (bZIP) family of transcription factors. These proteins share a high degree of homology and both can activate or repress transcription. Deficiency of either one of them in the non-cancer host cells was shown to reduce metastases. As ATF3 and JDP2 compensate each other’s function, we studied the double deficiency of ATF3 and JDP2 in the stromal tumor microenvironment. Here, we show that mice with ATF3 and JDP2 double deficiency (designated thereafter dKO) developed larger tumors with high vascular perfusion and increased cell proliferation rate compared to wild type (WT) mice. We further identify that the underlying mechanism involves tumor associated fibroblasts which secrete high levels of stromal cell-derived factor 1 (SDF-1) in dKO fibroblasts. SDF-1 depletion in dKO fibroblasts dampened tumor growth and blood vessel perfusion. Furthermore, ATF3 and JDP2 were found to regulate SDF-1 transcription and secretion in fibroblasts, a phenomenon that is potentiated in the presence of cancer cells. Collectively, our results suggest that ATF3 and JDP2 regulate the expression of essential tumor promoting factors expressed by fibroblasts within the tumor microenvironment, and thus restrain tumor growth.

The mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes. The three main MAPK cascades are the extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 kinases. A typical MAPK cascade is composed of MAP3K-MAP2K-MAPK kinases that are held by scaffold proteins. Scaffolds function to assemble the protein tier and contribute to the specificity and efficacy of signal transmission. WD repeat domain 62 (WDR62) is a JNK scaffold protein, interacting with JNK, MKK7, and several MAP3Ks. The loss of WDR62 in human leads to microcephaly and pachygyria. Yet the role of WDR62 in cellular function is not fully studied. We used the CRISPR/Cas9 and short hairpin RNA approaches to establish a human breast cancer cell line MDA-MB-231 with WDR62 loss of function and studied the consequence to JNK signaling. In growing cells, WDR62 is responsible for the basal expression of c-Jun. In stressed cells, WDR62 specifically mediates TNFα−dependent JNK activation through the association with both the adaptor protein, TNF receptor-associated factor 2 (TRAF2), and the MAP3K protein, mixed lineage kinase 3. TNFα-dependent JNK activation is mediated by WDR62 in HCT116 and HeLa cell lines as well. MDA-MB-231 WDR62-knockout cells display increased resistance to TNFα−induced cell death. Collectively, WDR62 coordinates the TNFα receptor signaling pathway to JNK activation through association with multiple kinases and the adaptor protein TRAF2.

Mitogen-activated protein kinases (MAPKs) form a kinase tier module in which MAPK, MAP2K and MAP3K are held by scaffold proteins. The scaffold proteins serve as a protein platform for selective and spatial kinase activation. The precise mechanism by which the scaffold proteins function has not yet been fully explained. WD40-repeat protein 62, WDR62 is a novel scaffold protein of the c-Jun N-terminal kinase (JNK) pathway. WDR62 is a 1523 a.a. long protein with no significant sequence homology to a known gene. Previously WDR62 was shown to associate with JNK and MKK4/7 in a modular fashion. Here, we show that WDR62 is able to associate with multiple members of the MAP3K of the mixed lineage kinase family and we map WDR62-MLK3 interacting domains. We identify two separable interacting domains within WDR62 and MLK3 proteins that can cross associate. MLK3 association with WDR62 is independent of JNK and MKK4/7 domains and activities. CDC42 activation disrupts WDR62-MLK3 association independent of MLK3 kinase activity.

Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-α, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent.

The 26S proteasome is a large cytoplasmic protease that degrades polyubiquitinated proteins to short peptides in a processive manner. The proteasome 19S regulatory subcomplex tethers the target protein via its polyubiquitin adduct and unfolds the target polypeptide, which is then threaded into the proteolytic site-containing 20S subcomplex. Hul5 is a 19S subcomplex-associated ubiquitin ligase that elongates ubiquitin chains on proteasome-bound substrates. We isolated hul5 Delta as a mutation with which fusions of an unstable cyclin to stable reporter proteins accumulate as partially processed products. These products appear transiently in the wild type but are strongly stabilized in 19S ATPase mutants and in the hul5 Delta mutant, supporting a role for the ATPase subunits in the unfolding of proteasome substrates before insertion into the catalytic cavity and suggesting a role for Hul5 in the processive degradation of proteins that are stalled on the proteasome.

Pho85 cyclins (Pcls), activators of the yeast cyclin-dependent kinase (CDK) Pho85, belong together with the p35 activator of mammalian CDK5 to a distinct structural cyclin class. Different Pcls target Pho85 to distinct substrates. Pcl5 targets Pho85 specifically to Gcn4, a yeast transcription factor involved in the response to amino acid starvation, eventually causing the degradation of Gcn4. Pcl5 is itself highly unstable, an instability that was postulated to be important for regulation of Gcn4 degradation. We used hybrids between different Pcls to circumscribe the substrate recognition function to the core cyclin box domain of Pcl5. Furthermore, the cyclin hybrids revealed that Pcl5 degradation is uniquely dependent on two distinct degradation signals: one N-terminal and one C-terminal to the cyclin box domain. Whereas the C-terminal degradation signal is independent of Pho85, the N-terminal degradation signal requires phosphorylation of a specific threonine residue by the Pho85 molecule bound to the cyclin. This latter mode of degradation depends on the SCF ubiquitin ligase. Degradation of Pcl5 after self-catalyzed phosphorylation ensures that activity of the Pho85/ Pcl5 complex is self-limiting in vivo. We demonstrate the importance of this mechanism for the regulation of Gcn4 degradation and for cell growth under conditions of amino acid starvation.