Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Receptor-binding domain-based SARS-CoV-2 vaccine adjuvanted with cyclic di-adenosine monophosphate enhances humoral and cellular immunity in mice.

Novel adjuvants are highly desired to improve immune responses of SARS-CoV-2 vaccines. This work reports the potential of the stimulator of interferon genes (STING) agonist adjuvant, the cyclic di-adenosine monophosphate (c-di-AMP), in a SARS-CoV-2 vaccine based on the receptor binding domain (RBD). Here, mice immunized with two doses of monomeric RBD adjuvanted with c-di-AMP intramuscularly were found to exhibit stronger immune responses compared to mice vaccinated with RBD adjuvanted with aluminum hydroxide (Al(OH)3 ) or without adjuvant. After two immunizations, consistent enhancements in the magnitude of RBD-specific immunoglobulin G (IgG) antibody response were observed by RBD + c-di-AMP (mean: 15360) compared to RBD + Al(OH)3 (mean: 3280) and RBD alone (n.d.). Analysis of IgG subtypes indicated a predominantly Th1-biased immune response (IgG2c, mean: 14480; IgG2b, mean: 1040, IgG1, mean: 470) in mice vaccinated with RBD + c-di-AMP compared to a Th2-biased response in those vaccinated with RBD + Al(OH)3 (IgG2c, mean: 60; IgG2b: n.d.; IgG1, mean: 16660). In addition, the RBD + c-di-AMP group showed better neutralizing antibody responses as determined by pseudovirus neutralization assay and by plaque reduction neutralization assay with SARS-CoV-2 wild type. Moreover, the RBD + c-di-AMP vaccine promoted interferon-γ secretion of spleen cell cultures after RBD stimulation. Furthermore, evaluation of IgG-antibody titers in aged mice showed that di-AMP was able to improve RBD-immunogenicity at old age after 3 doses (mean: 4000). These data suggest that c-di-AMP improves immune responses of a SARS-CoV-2 vaccine based on RBD, and would be considered a promising option for future COVID-19 vaccines.

Phosphatidylinositol 3-Phosphate Mediates the Establishment of Infectious Bursal Disease Virus Replication Complexes in Association with Early Endosomes.

Infectious bursal disease virus (IBDV) is the archetypal member of the family Birnaviridae and the etiological agent of Gumboro disease, a highly contagious immunosuppressive infection of concern to the global poultry sector for its adverse health effects in chicks. Unlike most double-stranded RNA (dsRNA) viruses, which enclose their genomes within specialized cores throughout their viral replication cycle, birnaviruses organize their bisegmented dsRNA genome in ribonucleoprotein (RNP) structures. Recently, we demonstrated that IBDV exploits endosomal membranes for replication. The establishment of IBDV replication machinery on the cytosolic leaflet of endosomal compartments is mediated by the viral protein VP3 and its intrinsic ability to target endosomes. In this study, we identified the early endosomal phosphatidylinositol 3-phosphate [PtdIns(3)P] as a key host factor of VP3 association with endosomal membranes and consequent establishment of IBDV replication complexes in early endosomes. Indeed, our data reveal a crucial role for PtdIns(3)P in IBDV replication. Overall, our findings provide new insights into the replicative strategy of birnaviruses and strongly suggest that it resembles those of positive-strand RNA (+ssRNA) viruses, which replicate in association with host membranes. Furthermore, our findings support the role of birnaviruses as evolutionary intermediaries between +ssRNA and dsRNA viruses and, importantly, demonstrate a novel role for PtdIns(3)P in the replication of a dsRNA virus.IMPORTANCEInfectious bursal disease virus (IBDV) infects chicks and is the causative agent of Gumboro disease. During IBDV outbreaks in recent decades, the emergence of very virulent variants and the lack of effective prevention/treatment strategies to fight this disease have had devastating consequences for the poultry industry. IBDV belongs to the peculiar family Birnaviridae Unlike most dsRNA viruses, birnaviruses organize their genomes in ribonucleoprotein complexes and replicate in a core-independent manner. We recently demonstrated that IBDV exploits host cell endosomes as platforms for viral replication, a process that depends on the VP3 viral protein. In this study, we delved deeper into the molecular characterization of IBDV-endosome association and investigated the role of host cell phosphatidylinositide lipids in VP3 protein localization and IBDV infection. Together, our findings demonstrate that PtdIns(3)P serves as a scaffold for the association of VP3 to endosomes and reveal its essential role for IBDV replication.

Molecular definitions of autophagy and related processes.

Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication.

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.

Not all vascular smooth muscle cell exosomes calcify equally in chronic kidney disease.

Prevention of medial calcification in patients with chronic kidney disease requires the maintenance of vascular smooth muscle cell fitness. To preserve viability under chronic kidney disease-induced stress, vascular smooth muscle cells increase exosome formation and release, but the result is aggravated pathological calcification. Now Chen et al. report that microvesicles from calcifying vascular smooth muscle cells may propagate procalcifying signals to normal vascular smooth muscle cells. To help design effective strategies to impair procalcifying cell-to-cell communication, this commentary updates the current understanding of the main regulators of microvesicle/exosome biogenesis and secretion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development.

Hemin induces mitophagy in a leukemic erythroblast cell line.

BACKGROUND INFORMATION: In eukaryotic cells, autophagy is considered a lysosomal catabolic process which participates in the degradation of intracellular components in a vacuolar structure termed autolysosome. This pathway plays a significant role in the erythropoiesis process, contributing to the clearance of some organelles (such as mitochondria) that are not necessary in the mature red blood cells. Nevertheless, the role of autophagy in erythrocyte maturation has not been fully established. RESULTS: Here, we have demonstrated that hemin (a physiological erythroid maturation stimulator) is able to induce the expression of critical autophagic genes (i.e., Map1a1b (LC3), Beclin-1 gen, Atg5) in an erythroleukemia cell type. We have also shown that hemin increased the size of autophagic vacuoles which were labelled with LC3 and the degradative lysosomal marker dye quenched-bovine serum albumin. In addition, we have determined by Western blot a rise in the lipidated form of the autophagic protein LC3 (i.e., LC3-II) upon hemin treatment. Moreover, we provide evidence that hemin induces mitochondrial membrane depolarisation and that mitochondria sequestration by autophagy requires the active form of the NIX protein. CONCLUSIONS: We have found that the physiological erythroid maturation stimulator hemin is able to induce mitophagy in K562 cells, and that the autophagy adaptor NIX is necessary for mitophagy progression. K562 cells have been used as a relevant model to determine the possible therapeutic role of new differentiating compounds. SIGNIFICANCE: It has been proposed that autophagy induction is a feasible new therapeutic key in fighting cancer. Our results suggest that hemin is favoring erythroid maturation by inducing an autophagic response in K562 cells, being a possible therapeutic candidate that may help in the chronic myelogenous leukemia (CML) treatment.

Autophagy response: manipulating the mTOR-controlled machinery by amino acids and pathogens.

Macroautophagy is a self-degradative process that normally maintains cellular homeostasis via a lysosomal pathway. It is induced by different stress signals, including nutrients and growth factors' restriction as well as pathogen invasions. These stimuli are modulated by the serine/threonine protein kinase mammalian target of rapamycin (mTOR) which control not only autophagy but also protein translation and gene expression. This review focuses on the important role of mTOR as a master regulator of cell growth and the autophagy pathway. Here, we have discussed the role of intracellular amino acid availability and intracellular pH in the redistribution of autophagic structures, which may contribute to mammalian target of rapamycin complex 1 (mTORC1) activity regulation. We have also discussed that mTORC1 complex and components of the autophagy machinery are localized at the lysosomal surface, representing a fascinating mechanism to control the metabolism, cellular clearance and also to restrain invading intracellular pathogens.

cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response.

Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla) is the S. aureus-secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus-containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell.

Endocytic SNAREs are involved in optimal Coxiella burnetii vacuole development.

Coxiella burnetii is a Gram-negative intracellular bacterium. As previously described, both the endocytic and the autophagic pathways contribute to the maturation of Coxiella replicative vacuoles (CRVs). The large CRVs share the properties of both phagolysosomal and autophagolysosomal compartments. Vamp3, Vamp7 and Vamp8 are v-SNAREs involved in the endocytic pathway which participate mainly in the fusion between endosomes and lysosomes. In the present study we observed that Vamp7 interacts with C. burnetii at different infection times (1 h-48 h p.i.). We have determined that a truncated mutant of Vamp7 (Vamp7 NT) and a siRNA against this SNARE protein affects the optimal development of CRVs, suggesting that Vamp7 mediates fusion events that are required for the biogenesis of CRVs. Indeed, we have observed that overexpression of Vamp7 NT inhibited the heterotypic fusion with lysosomes and the homotypic fusion between individual Coxiella phagosomes and CRVs. Moreover, we have detected in the vacuole membrane, at different infection times, the Vamp7 partners (Vti1a and Vti1b). Interestingly, treatment with chloramphenicol reduced the colocalization between C. burnetii and Vamp7, Vti1a or Vti1b, indicating that the recruitment of these SNAREs proteins is a bacteria-driven process that favours the CRV biogenesis, likely by facilitating the interaction with the endolysosomal compartment.

Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation.

BACKGROUND INFORMATION: Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. RESULTS: Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCßII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCßII but not PKCßI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCßII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. CONCLUSIONS: This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking.

Staphylococcus aureus phagocytosis is affected by senescence.

Senescent cells accumulate in multicellular animals with aging, resulting in organ or tissue dysfunction. These alterations increase the incidence of a variety of illnesses, including infectious diseases, and, in certain instances, its severity. In search of a rationale for this phenomenon, we focused on the endophagocytic pathway in senescent cells. We first described the endocytic vesicle populations at different stages of maturation using confocal microscopy. There was an increase in the number of vacuoles per cell, which was partially explained by an increase in cell size. No changes in vesicle maturation or degradation capacities were determined by microscopy or Western blot assays. Also, we studied the internalization of various endophagocytic cargoes in senescent cells and observed only a decrease in the intracellular recovery of bacteria such as Staphylococcus aureus. Afterwards, we studied the intracellular traffic of S. aureus, and observed no differences in the infection between control and senescent cells. In addition we quantified the recovery of bacteria from control and senescent cells infected in the presence of several inhibitors of endophagosomal maturation, and no changes were observed. These results suggest that bacterial internalization is affected in senescent cells. Indeed, we confirmed this hypothesis by determining minor bacterial adherence and internalization by confocal microscopy. Furthermore, it is important to highlight that we found very similar results with cells from aged animals, specifically BMDMs. This alteration in senescent cells enlightens the diminished bacterial clearance and may be a factor that increases the propensity to suffer severe infectious conditions in the elderly.

Comparative analysis of humoral immune response upon the three first vaccines applied in Argentina: IgG production and neutralizing capacity against SARS-CoV-2.

The population that has not received a SARS-CoV-2 vaccine is at high risk for infection whereas vaccination prevents COVID-19 severe disease, hospitalization, and death. In Argentina, to date, more than 50 million doses of vaccines against SARS-CoV-2 have been administered. The three main vaccines applied are Sputnik V, Oxford-AstraZeneca, and Sinopharm. In this study, we have compared the antibody response of voluntary individuals at day 0 (first dose vaccination day) and at 21-25 days post first and second dose. Our results indicate that at 21-25 days after the administration of the first doses of Sputnik V the large majority of the people vaccinated 80% (n = 15) presented high humoral responses as determined by the measurement of IgG against the Spike protein and the Receptor Binding Domain (RBD). In the case of those vaccinated with AstraZeneca, the percentage was 80% (n = 15) whereas this value was reduced to only 25% (n = 16) in persons that received Sinopharm. However, after the second doses, most of the recipients had significant levels of antibodies. The virus neutralizing capacity of the antibodies generated was evaluated using a pseudotyped VSV-SARS-CoV2 Spike expressing eGFP and the data was analyzed by fluorescence microscopy and flow cytometry. The results indicate that a good correlation exists between the levels of IgG and the neutralizing capacity of the antibodies against the recombinant virus. Our results stand out the importance of applying the second dose of Sinopharm. Thus, the present report provides data that will contribute to decisions making about the vaccine implementation plans of action for, not only our region but our country to support the fight against the COVID-19 global pandemic.

The PhoP/PhoQ system and its role in Serratia marcescens pathogenesis.

Serratia marcescens is able to invade, persist, and multiply inside nonphagocytic cells, residing in nonacidic, nondegradative, autophagosome-like vacuoles. In this work, we have examined the physiological role of the PhoP/PhoQ system and its function in the control of critical virulence phenotypes in S. marcescens. We have demonstrated the involvement of the PhoP/PhoQ system in the adaptation of this bacterium to growth on scarce environmental Mg(2+), at acidic pH, and in the presence of polymyxin B. We have also shown that these environmental conditions constitute signals that activate the PhoP/PhoQ system. We have found that the two S. marcescens mgtE orthologs present a conserved PhoP-binding motif and demonstrated that mgtE1 expression is PhoP dependent, reinforcing the importance of PhoP control in magnesium homeostasis. Finally, we have demonstrated that phoP expression is activated intracellularly and that a phoP mutant strain is defective in survival inside epithelial cells. We have shown that the Serratia PhoP/PhoQ system is involved in prevention of the delivery to degradative/acidic compartments.

Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay.

The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts. In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved. Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes. In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process. Lysosomal-dependent or -independent T. cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells. In this context, we have previously shown that induction of autophagy in host cells before infection favors T. cruzi invasion. Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T. cruzi entry sites and that the newly formed T. cruzi parasitophorous vacuole has characteristics of an autophagolysosome. This review summarizes the current knowledge of the molecular and cellular mechanisms of T. cruzi invasion in nonphagocytic cells. Based on our findings, we propose a new model in which T. cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells.

Mycobacterium marinum induces a marked LC3 recruitment to its containing phagosome that depends on a functional ESX-1 secretion system.

Autophagy has been implicated as part of the innate immune system against different intracellular microorganisms. Mycobacterium marinum is the causative agent of the fish-tank granuloma and has been widely used as an alternative model to study pathogenic mycobacteria. In this report, we show an active interaction of M. marinum with the autophagic protein LC3, an event that requires pathogen viability and bacterial protein synthesis. Interestingly, M. marinum lacking the region of difference 1 (RD1) is unable to recruit LC3, indicating that a functional ESX-1 secretion system is an absolute requirement for this process. In addition, phagocytosis of the bacteria is also a condition for the LC3 rearrangement induced by M. marinum. We present evidence that this pathogen resides temporarily in a LC3-decorated compartment with late endocytic features but mostly devoid of lysosomal enzymes or degradative properties. In addition our results indicate that autophagy induction by rapamycin treatment leads to maturation of the M. marinum-containing compartment.

FKBP8 is a novel molecule that participates in the regulation of the autophagic pathway.

Autophagy is a homeostatic process by which misfolded proteins, organelles and cytoplasmic material are engulfed in autophagosomal vesicles and degraded through a lisosomal pathway. FKBP8 is a member of the FK506-binding proteins family (FKBP) usually found in mitochondria and the endoplasmic reticulum. This protein plays a critical role in cell functions such as protein trafficking and folding. In the present report we demonstrate that the depletion of FKBP8 abrogated autophagy activation induced by starvation, whereas the overexpression of this protein triggered the autophagy cascade. We found that FKBP8 co-localizes with ATG14L and BECN1, both members of the VPS34 lipid kinase complex, which regulates the initial steps in the autophagosome formation process. We have also demonstrated that FKBP8 is necessary for VPS34 activity. Our findings indicate that the regulatory function of FKBP8 in the autophagy process depends of its transmembrane domain. Surprisingly, this protein was not found in autophagosomal vesicles, which reinforces the notion that the FKBP8 only participates in the initial steps of the autophagosome formation process. Taken together, our data provide evidence that FKBP8 modulates the early steps of the autophagosome formation event by interacting with the VPS34 lipid kinase complex. SUMMARY: In this article, the protein FKBP38 is reported to be a novel modulator of the initial steps of the autophagic pathway, specifically in starvation-induced autophagy. FKBP38 interacts with the VPS34 lipid kinase complex, with the transmembrane domain of FKBP38 being critical for its biological function.

The early secretory pathway contributes to the growth of the Coxiella-replicative niche.

Coxiella burnetii is a Gram-negative obligate intracellular bacterium. After internalization, this bacterium replicates in a large parasitophorous vacuole that has features of both phagolysosomes and autophagosomal compartments. We have previously demonstrated that early after internalization Coxiella phagosomes interact with both the endocytic and the autophagic pathways. In this report, we present evidence that the Coxiella-replicative vacuoles (CRVs) also interact with the secretory pathway. Rab1b is a small GTPase responsible for the anterograde transport between the endoplasmic reticulum and the Golgi apparatus. We present evidence that Rab1b is recruited to the CRV at later infection times (i.e., after 6 h of infection). Interestingly, knockdown of Rab1b altered vacuole growth, indicating that this protein was required for the proper biogenesis of the CRV. In addition, overexpression of the active GTPase-defective mutant (GFP-Rab1b Q67L) affected the development of the Coxiella-replicative compartment inhibiting bacterial growth. On the other hand, disruption of the secretory pathway by brefeldin A treatment or by overexpression of Sar1 T39N, a defective dominant-negative mutant of Sar1, affected the typical spaciousness of the CRVs. Taken together, our results show for the first time that the Coxiella-replicative niche also intercepts the early secretory pathway.

Glucose deprivation causes oxidative stress and stimulates aggresome formation and autophagy in cultured cardiac myocytes.

Aggresomes are dynamic structures formed when the ubiquitin-proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6 h. However, at longer times (18 h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with gamma-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.