Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human “ubiquitinome” using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport.

Autophagy engulfs cellular components in double-membrane-bound autophagosomes for clearance and recycling after fusion with lysosomes. Thus, autophagy is a key process for maintaining proteostasis and a powerful cell-intrinsic host defense mechanism, protecting cells against pathogens by targeting them through a specific form of selective autophagy known as xenophagy. In this context, ubiquitination acts as a signal of recognition of the cargoes for autophagic receptors, which direct them towards autophagosomes for subsequent breakdown. Nevertheless, autophagy can carry out a dual role since numerous viruses including members of the Orthoherpesviridae family can either inhibit or exploit autophagy for its own benefit and to replicate within host cells. There is growing evidence that Herpes simplex virus type 1 (HSV-1), a highly prevalent human pathogen that infects epidermal keratinocytes and sensitive neurons, is capable of negatively modulating autophagy. Since the effects of HSV-1 infection on autophagic receptors have been poorly explored, this study aims to understand the consequences of HSV-1 productive infection on the levels of the major autophagic receptors involved in xenophagy, key proteins in the recruitment of intracellular pathogens into autophagosomes. We found that productive HSV-1 infection in human neuroglioma cells and keratinocytes causes a reduction in the total levels of Ub conjugates and decreases protein levels of autophagic receptors, including SQSTM1/p62, OPTN1, NBR1, and NDP52, a phenotype that is also accompanied by reduced levels of LC3-I and LC3-II, which interact directly with autophagic receptors. Mechanistically, we show these phenotypes are the result of xenophagy activation in the early stages of productive HSV-1 infection to limit virus replication, thereby reducing progeny HSV-1 yield. Additionally, we found that the removal of the tegument HSV-1 protein US11, a recognized viral factor that counteracts autophagy in host cells, enhances the clearance of autophagic receptors, with a significant reduction in the progeny HSV-1 yield. Moreover, the removal of US11 increases the ubiquitination of SQSTM1/p62, indicating that US11 slows down the autophagy turnover of autophagy receptors. Overall, our findings suggest that xenophagy is a potent host defense against HSV-1 replication and reveals the role of the autophagic receptors in the delivery of HSV-1 to clearance via xenophagy.

Waterborne gastrointestinal infections remain a global health concern, with approximately 1.7 billion diarrhea-related illnesses annually attributable to protozoan parasites. These pathogens are transmitted through contaminated water and exhibit high resistance to chlorination, posing substantial challenges to effective water treatment. This study focused on the most prevalent intestinal parasites in the Los Ríos Region of Chile: Blastocystis sp., Giardia duodenalis, and Entamoeba coli. The objectives were to assess the prevalence of eukaryotic parasites in water samples—covering both drinking and recreational sources—to describe the circulating subtypes of Blastocystis sp. and to identify ecological factors associated with parasite presence. Water samples were analyzed using conventional PCR, next-generation sequencing (NGS) was employed for Blastocystis sp. subtype identification, and the environmental predictors were evaluated using a multivariable logistic regression model. A total of 132 water samples were analyzed, of which 15.2% were positive for Blastocystis sp. and 1.5% for E. coli, while no samples tested positive for G. duodenalis. We identified subtypes ST1–ST4 of Blastocystis sp., along with ST7, ST10, ST14, ST21, and ST23–ST26, the latter being reported for the first time in Chile. Ecological factors significantly associated with Blastocystis sp. presence included higher water temperature and greater rainfall at positive sites. Potable water was associated with significantly lower odds of Blastocystis sp. infection (aOR = 0.04, 95% CI: 0.00–0.87; p = 0.041), while precipitation increased infection odds by 3% per additional millimeter (aOR = 1.03, 95% CI: 1.00–1.06; p = 0.036). Greater distance to the nearest farmhouse was also significantly associated with reduced infection risk, suggesting that proximity to livestock environments may influence Blastocystis sp. transmission. These findings help explain the high prevalence of Blastocystis sp. observed in humans in the Los Ríos Region and highlight the pivotal role of ecological conditions in driving waterborne transmission. To our knowledge, this is the first environmental study in Chile to clearly demonstrate the association between human infection, environmental factors, and the transmission dynamics of Blastocystis sp.

Ubiquitination regulates several biological processes. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1,187 genes of the human “ubiquitinome” using Amyloid Precursor Protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel key regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 caused a robust and rapid inhibition of Golgi-to-ER retrograde transport which leads to a potent blockage of macroautophagy by a mechanism associated with the retention of Atg9A and Rab1A at the Golgi apparatus. Because pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and their K-63-Ub chains, act as a crucial regulator factor for macroautophagy by controlling Golgi-to-ER retrograde transport.