Despite much evidence of the involvement of the proteasomeubiquitin signaling system in temperature stress response, the dynamics of the ubiquitylome during cold response has not yet been studied. Here, we have compared quantitative ubiquitylomes from a strain deficient in proteasome substrate recruitment and a reference strain during cold response. We have observed that a large group of proteins showing increased ubiquitylation in the proteasome mutant at low temperature is comprised by reverses suppressor of Ty-phenotype 5 (Rsp5)-regulated plasma membrane proteins. Analysis of internalization and degradation of plasma membrane proteins at low temperature showed that the proteasome becomes determinant for this process, whereas, at 30°C, the proteasome is dispensable. Moreover, our observations indicate that proteasomes have increased capacity to interact with lysine 63-polyubiquitylated proteins during low temperature in vivo. These unanticipated observations indicate that, during cold response, there is a proteolytic cellular reprogramming in which the proteasome acquires a role in the endocytic-vacuolar pathway.Proteasomal and endocytic-vacuolar pathways are responsible for the degradation of a large portion of proteins in eukaryotic cells, and their activities are fundamental for protein homeostasis. The endocytic-vacuolar pathway degrades internalized plasma membrane (PM) 3 proteins, proteins trafficking from Golgi to the endosome, and proteins from autophagic processes (1). The proteasome is the fate of cytosolic and nuclear proteins, co-translationally incomplete or misfolded proteins, partially aggregated proteins, and transmembrane proteins from the endoplasmic reticulum (2, 3). Despite the functional divergence between endocytic-vacuolar and proteasomal pathways, they share a key regulatory aspect: client proteins are modified by ubiquitin, and as a consequence ubiquitin-conjugating and ubiquitin-recognizing proteins act as receptors and regulators of both processes. The involvement of distinct ubiquitin-protein ligases, deubiquitylating factors, different polyubiquitin topologies, and specific recognition of ubiquitylated substrates appear to be key factors to ensure the independence of both pathways (1, 4, 5).In the initial steps of the endocytic pathway, yeast PM cargo proteins are mainly ubiquitylated by the Nedd4-type ubiquitin ligase Rsp5 (4, 6, 7). Ubiquitylated cargo proteins undergo endocytosis, a process orchestrated by epsins and epsin-like proteins, which possess two types of ubiquitin binding domains: ubiquitin-interacting motifs and ubiquitin-associated domains (8). Internalized proteins may exhibit monoubiquitylation or Lys-63-dependent polyubiquitylation (4, 7). In the endosome, ubiquitylated cargo is recruited to the ESCRT-0 complex constituted by Vps27 (Hrs) and Hse1 (STAM1/2). Both Vps27 and Hse1 proteins contain ubiquitin binding domains that mediate interaction with ubiquitylated cargo (9). During multivesicular body biogenesis, ubiquitin modification of cargo proteins is preserved...

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Background and Hypothesis:Ultraviolet B (UVB) irradiation from the sun can disrupt normal epidermal keratinocyte function, leading to the development of skin cancer. The integrated stress response (ISR) is a conserved cellular mechanism against environmental stressors, such as UV irradiation, through the phosphorylation of the alpha subunit of the initiation factor eIF2 (eIF2a-P). Previously, we demonstrated that the ISR kinase GCN2 plays a role in the response of keratinocytes exposed to UV irradiation. However, the role of other components of the ISR in UV-induced stress has not been well elucidated. We hypothesize that in addition to GCN2, other components of ISR such as PERK, GCN1, and ZAK1 are involved in apoptotic pathways induced by prolonged UVB exposure. Methods:Gene-specific knockouts (KO) were generated using CRISPR/Cas9 and validated through immunoblotting. Wild-type (WT) and KO NTERT keratinocytes were irradiated with UVB at 0, 100, 200, and 400J/m2. Six hours post-UVB, cell photomicrographs were taken at 0 and 400J/m2. Measurements for apoptosis were then performed by measuring enzymatic levels of caspase-3. Results:Measurements of caspase-3 following UVB exposure on WT NTERT, PERK-KO, and GCN2-KO showed no difference between the groups. GCN1-KO had significantly greater caspase-3 activity at 400J/m2, whereas ZAK1-KO had significantly reduced caspase-3 activity at the same concentration. GCN2/PERK double-KO had significantly increased caspase-3 activity at 400 J/m2 compared to the individual KO cells. Conclusion and Potential Impact:Collectively, our data suggests that the ISR may be involved in the apoptotic responses that ensue in keratinocytes exposed to UVB. How GCN2 and PERK could co-interact with GCN1 or ZAK1 to facilitate apoptotic fate in keratinocytes still warrants further research. Nevertheless, elucidating the role of the ISR in normal keratinocyte response to UV irradiation will allow us to define novel therapeutic targets for the treatment or prevention of UV-related skin malignancies.

Background and Hypothesis:  Chronic cutaneous wounds are a serious health concern afflicting millions of people. One of the primary factors preventing the closure of chronic wounds is the inability of keratinocytes to migrate across the wound bed. Epidermal keratinocytes migrate in a cohesive manner known as the keratinocyte collective cell migration (KCCM). Our lab has demonstrated that the integrated stress response (ISR) plays a key role in the KCCM. The ISR is initiated by stress-sensitive kinases, such as GCN2, and results in decreased global protein synthesis while preferentially increasing the translation of mRNAs encoding cytoprotective proteins. Wound repair also relies on the actin cytoskeleton, but the crosstalk between actin and the ISR is not well established. We hypothesize that the interaction between the ISR and the actin cytoskeleton is critical for KCCM during wound healing.     Methods:  Cutaneous wound healing was approximated in vitro using the KCCM-dependent scratch assay. Wild-type (WT) and GCN2-deleted (KO) keratinocytes were grown on coverslips, differentiated, scratched, and harvested at different times post-wounding. F-actin and vimentin (VIM) expression was monitored over time using fluorescent phalloidin-488 and immunofluorescence. In addition, WT keratinocytes were treated with actin-depolymerizing drugs and induction of ISR was measured using immunoblots.     Results:  Depolymerization of F-actin was observed along the leading edge of both wounded WT and GCN2-KO keratinocytes immediately following wounding. WT keratinocytes upregulated VIM expression at the leading edge whereas VIM expression remained unchanged in the wounded GCN2-KO keratinocytes. Treatment with latrunculin B and cytochalasin D, which both result in actin depolymerization, induced GCN2 phosphorylation in the differentiated WT keratinocytes.    Conclusion and Potential Impact:  F-actin depolymerization elicits a GCN2-mediated induction of the ISR. GCN2 and the ISR are critical components of the cutaneous wound repair process and their crosstalk with the actin cytoskeleton may serve as a novel therapeutic target in the treatment of chronic wounds.