Activity Of Lysosomal Hydrolases Research Articles

AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD(+) elevation.

Summary AMPK activation is beneficial for cellular homeostasis and senescence prevention. However, the molecular events involved in AMPK activation are not well defined. In this study, we addressed the mechanism underlying the protective effect of AMPK on oxidative stress‐induced senescence. The results showed that AMPK was inactivated in senescent cells. However, pharmacological activation of AMPK by metformin and berberine significantly prevented the development of senescence and, accordingly, inhibition of AMPK by Compound C was accelerated. Importantly, AMPK activation prevented hydrogen peroxide‐induced impairment of the autophagic flux in senescent cells, evidenced by the decreased p62 degradation, GFP‐RFP‐LC3 cancellation, and activity of lysosomal hydrolases. We also found that AMPK activation restored the NAD + levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD + synthesis. In addition, the mechanistic relationship of autophagic flux and NAD + synthesis and the involvement of mTOR and Sirt1 activities were assessed. In summary, our results suggest that AMPK prevents oxidative stress‐induced senescence by improving autophagic flux and NAD + homeostasis. This study provides a new insight for exploring the mechanisms of aging, autophagy and NAD + homeostasis, and it is also valuable in the development of innovative strategies to combat aging.

The neurological manifestations of Fabry disease. A review

Fabry disease (Anderson-Fabry disease) is an X-linked recessive lysosomal storage disorder resulting from deficient activity of lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A), which leads to progressive accumulation of globotriaosylceramide (Gb3) in various cells, predominantly endothelial and vascular smooth muscle cells, with clinical manifestations affecting major organs including the central nervous system. Manifestations of Fabry disease include progressive renal and cardiac insufficiency, neuropathic pain, stroke and cerebral disease, skin and gastrointestinal symptoms. Clinical onset usually occurs in childhood, but many severe patients are diagnosed in adulthood. Females may be severely affected as males and both may die prematurely due to stroke, heart disease and renal failure. Early recognition of symptoms, enzyme activity levels, concentration of Gb3 levels in the blood, urine and skin biopsies, as well as genetic testing (GLA gene) enable establishment of early diagnosis and therapeutic intervention with enzyme replacement therapy. Enzyme replacement therapy can stabilize or reduce the progression of the disease. Early therapy may prevent complications of the disease.

Quantification of Lysosomal Membrane Permeabilization by Cytosolic Cathepsin and β-N-Acetyl-Glucosaminidase Activity Measurements

Programmed cell death involving lysosomal membrane permeabilization (LMP) is an alternative cell death pathway induced under various cellular conditions and by numerous cytotoxic stimuli. The method presented here to quantify LMP takes advantage of the detergent digitonin, which creates pores in cellular membranes by replacing cholesterol. The difference in cholesterol content between the plasma membrane (high) and lysosomal membrane (low) allows titration of digitonin to a concentration that permeabilizes the plasma membrane but leaves lysosomal membranes intact. The extent of LMP is determined by measuring the cytosolic activity of lysosomal hydrolases (e.g., cysteine cathepsins) and/or β-N-acetyl-glucosaminidase in the digitonin-extracted cytoplasm and comparing it to the total cellular enzyme activity. Digitonin extraction of the cytosol can be combined with precipitation of protein and/or western blot analysis for detection of lysosomal proteins (e.g., cathepsins).

Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages.

Methicillin-resistant Staphylococcus aureus (MRSA) causes invasive, drug-resistant skin and soft tissue infections. Reports that S. aureus bacteria survive inside macrophages suggest that the intramacrophage environment may be a niche for persistent infection; however, mechanisms by which the bacteria might evade macrophage phagosomal defenses are unclear. We examined the fate of the S. aureus-containing phagosome in THP-1 macrophages by evaluating bacterial intracellular survival and phagosomal acidification and maturation and by testing the impact of phagosomal conditions on bacterial viability. Multiple strains of S. aureus survived inside macrophages, and in studies using the MRSA USA300 clone, the USA300-containing phagosome acidified rapidly and acquired the late endosome and lysosome protein LAMP1. However, fewer phagosomes containing live USA300 bacteria than those containing dead bacteria associated with the lysosomal hydrolases cathepsin D and β-glucuronidase. Inhibiting lysosomal hydrolase activity had no impact on intracellular survival of USA300 or other S. aureus strains, suggesting that S. aureus perturbs acquisition of lysosomal enzymes. We examined the impact of acidification on S. aureus intramacrophage viability and found that inhibitors of phagosomal acidification significantly impaired USA300 intracellular survival. Inhibition of macrophage phagosomal acidification resulted in a 30-fold reduction in USA300 expression of the staphylococcal virulence regulator agr but had little effect on expression of sarA, saeR, or sigB. Bacterial exposure to acidic pH in vitro increased agr expression. Together, these results suggest that S. aureus survives inside macrophages by perturbing normal phagolysosome formation and that USA300 may sense phagosomal conditions and upregulate expression of a key virulence regulator that enables its intracellular survival.

Increased expression of transcription factor EB (TFEB) is associated with autophagy, migratory phenotype and poor prognosis in non-small cell lung cancer.

We investigated the role of lysosomal biogenesis and hydrolase activity in the clinical behavior and postoperative outcome of lung cancer. Using immunohistochemistry we investigated the expression of the transcription factor EB (TFEB) which orchestrates lysosomal biogenesis, the lysosome membrane protein LAMP2a and of the lysosomal hydrolase cathepsin D in a series of 98 non-small cell lung carcinomas (NSCLC) treated with surgery alone. In vitro experiments with the A549 and H1299 lung cancer cell lines were also performed. Overexpression of TFEB, LAMP2a and Cathepsin D was noted in 47/98 (47.9%), 43/98 (43.9%) and 39/98 (39.8%) cases, respectively, and were significantly correlated with each other and with adenocarcinomas. High LAMP2a was related to high histology grade. Linear regression analysis confirmed significant association of TFEB with BNIP3 (p=0.0003, r=0.35) and LC3A with LAMP2a expression (p=0.0002, r=0.37). An inverse association of Cathepsin D expression with stone-like structures (SLS) was recorded (p=0.02, r=0.22). On univariate analysis all three lyososomal variables were associated with poor prognosis (p=0.05, 0.04 and 0.01, for TFEB, Cathepsin D and LAMP2a, respectively). Multivariate analysis showed that the SLS number (p=0.0001, HR5.37), Cathepsin D expression (p=0.01, HR=2.2) and stage (p=0.01, HR=1.5) were independent prognostic variables. Silencing of TFEB with siRNAs in the A549 and H1299 lung cancer cell lines did not affect proliferation but resulted in reduced migration ability. Lysosomal biogenesis is linked to autophagosomal protein expression in NSCLC and characterizes subgroups of high risk patients after complete surgical lung tumor resection.

Role of saposin C and D in auditory and vestibular function.

Saposins are small proteins derived from a precursor protein, prosaposin. Each of the four saposins (A-D) is necessary for the activity of lysosomal glycosphingolipid hydrolases. Individual saposin mutations lead to lysosomal storage diseases, some of which are associated with hearing loss. Here we evaluate the effects of the loss of saposins C and D on auditory and vestibular function in transgenic mice. Transgenic mice with either loss of saposin C function or a combined loss of saposin C + D function were studied. Light microscopy and immunofluorescence were used to evaluate histologic and morphologic changes in the auditory and vestibular organs. Acoustic brainstem response thresholds and distortion product otoacoustic emissions were used to study the auditory phenotype. A null mutation of saposin C did not result in any identifiable histologic changes or loss of hearing through postnatal day 55. Combined losses of saposins C and D similarly did not result in any changes in organ of Corti histology or loss of hearing. However, inclusions within the vestibular end organs was noted, consistent with afferent and efferent neuronal sprouting, although to a much milder degree than seen in the previously studied prosaposin knockout mouse. Loss of saposin C and D function, although causing mild phenotypic changes in the vestibular end organs, otherwise results in minimal functional impairment and no changes in the auditory system. It is more likely that the auditory and vestibular effects of the loss of prosaposin are mediated through the actions of saposin A and/or B. NA.

Autophagosome-lysosome fusion is independent of V-ATPase-mediated acidification.

The ATP-dependent proton pump V-ATPase ensures low intralysosomal pH, which is essential for lysosomal hydrolase activity. Based on studies with the V-ATPase inhibitor BafilomycinA1, lysosomal acidification is also thought to be required for fusion with incoming vesicles from the autophagic and endocytic pathways. Here we show that loss of V-ATPase subunits in the Drosophila fat body causes an accumulation of non-functional lysosomes, leading to a block in autophagic flux. However, V-ATPase-deficient lysosomes remain competent to fuse with autophagosomes and endosomes, resulting in a time-dependent formation of giant autolysosomes. In contrast, BafilomycinA1 prevents autophagosome–lysosome fusion in these cells, and this defect is phenocopied by depletion of the Ca2+ pump SERCA, a secondary target of this drug. Moreover, activation of SERCA promotes fusion in a BafilomycinA1-sensitive manner. Collectively, our results indicate that lysosomal acidification is not a prerequisite for fusion, and that BafilomycinA1 inhibits fusion independent of its effect on lysosomal pH.

The role of proteases of food objects and enteral microbiota in nutritive and thermal adaptations of the digestive system in fishes

The review presents the data on the activity and some thermal characteristics of proteases in the potential food objects of fishes and some enteral microbiota representatives that provide an induced autolysis and symbiotic digestion. It is shown that during the active feeding period the total protease activity in the prey tissues exceeds that in the fish gastric mucosa by 5–10 times. At low temperature, the relative activity of the prey tissue lysosomal hydrolases (20–35%) and enteral microbiota enzymes (up to 45%) may exceed that of proteases synthesized by the hepatopancreas and acting in the consumers’ intestinal mucosa (less than 10% of maximal activity). The data presented indicate the important role of proteases of food objects and enteral microbiota in nutritive adaptations of the fish digestive system.

Disruption of sphingolipid metabolism augments ceramide-induced autophagy in preeclampsia

Bioactive sphingolipids including ceramides are involved in a variety of pathophysiological processes by regulating cell death and survival. The objective of the current study was to examine ceramide metabolism in preeclampsia, a serious disorder of pregnancy characterized by oxidative stress, and increased trophoblast cell death and autophagy. Maternal circulating and placental ceramide levels quantified by tandem mass spectrometry were elevated in pregnancies complicated by preeclampsia. Placental ceramides were elevated due to greater de novo synthesis via high serine palmitoyltransferase activity and reduced lysosomal breakdown via diminished ASAH1 expression caused by TGFB3-induced E2F4 transcriptional repression. SMPD1 activity was reduced; hence, sphingomyelin degradation by SMPD1 did not contribute to elevated ceramide levels in preeclampsia. Oxidative stress triggered similar changes in ceramide levels and acid hydrolase expression in villous explants and trophoblast cells. MALDI-imaging mass spectrometry localized the ceramide increases to the trophophoblast layers and syncytial knots of placentae from pregnancies complicated by preeclampsia. ASAH1 inhibition or ceramide treatment induced autophagy in human trophoblast cells via a shift of the BOK-MCL1 rheostat toward prodeath BOK. Pharmacological inhibition of ASAH1 activity in pregnant mice resulted in increased placental ceramide content, abnormal placentation, reduced fetal growth, and increased autophagy via a similar shift in the BOK-MCL1 system. Our results reveal that oxidative stress-induced reduction of lysosomal hydrolase activities in combination with elevated de novo synthesis leads to ceramide overload, resulting in increased trophoblast cell autophagy, and typifies preeclampsia as a sphingolipid storage disorder.

Effect of U18666a on Beta-Glucosidase, Sphingomyelinase, and Beta-Galactosidase Activities in Astrocytes of Young Rats

Niemann-Pick type C disease (NPC) is a neurodegenerative genetic disorder caused by accumulation of lipids, especially cholesterol, in the perinuclear space. U18666A is a cholesterol transport-inhibiting agent, being used to mimic NPC, mainly in fibroblasts. The objective of this study was to observe the effect of the drug U18666A, which causes the accumulation of cholesterol in the cytoplasm of astrocytes from newborn rats, on some lysosomal hydrolase activities. Filipin staining and fluorescence microscopy, through CellM software, were used for visualization and quantification of cholesterol. The dose of U18666A that provided the greatest accumulation of cholesterol was that of 0.25 µg/mL in incubation for 48 h. Primary rat astrocytes incubated with the drug (NPC) showed a significantly higher amount of cholesterol than those without U18666A (controls). The measurement of activity of enzymes sphingomyelinase and beta-glucosidase in astrocytes of rats with NPC was significantly lower than that of control astrocytes, which is consistent with the disease in humans. The activity of the enzyme beta-galactosidase showed no significant difference between both groups. We concluded that U18666A appears to be an excellent intracellular cholesterol transport-inhibiting agent affecting some metabolic pathways in astrocytes of young rats, which mimics NPC in these animals. Just like the change in the activity of lysosomal enzymes has been demonstrated, other biochemical parameters of the cell can be tested with this animal model, thus contributing to a better understanding of the disease.

Preventive effect of phytic acid on lysosomal hydrolases in normal and isoproterenol-induced myocardial infarction in Wistar rats

This study was aimed to evaluate the preventive role of phytic acid on lysosomal enzymes in isoproterenol (ISO)-induced myocardial infarction (MI) in male Wistar rats. Rats subcutaneously injected with ISO (85 mg/kg) at an interval of 24 h for two days showed a significant increase in the activities of lysosomal enzymes (glucuronidase, N-acetyl glucosaminidase, galactosidase, cathepsin-B and cathepsin-D) were increased significantly in serum and the heart of ISO-induced rats, but the activities of glucuronidase and cathepsin-D were decreased significantly in the lysosomal fraction of the heart. Pretreatment with phytic acid (25 and 50 mg/kg) daily for a period of 56 d positively altered activities of lysosomal hydrolases in ISO-induced rats. Thus, phytic acid possesses a cardioprotective effect in ISO-induced MI in rats.

X-linked myopathy with excessive autophagy: a failure of self-eating

Autophagic vacuolar myopathies (AVMs) are a group of disorders united by shared histopathological features on muscle biopsy that include the aberrant accumulation of autophagic vacuoles. The classic conditions that compose the AVMs include Pompe Disease, Danon Disease and X-linked myopathy with excessive autophagy (XMEA). Other disorders, including acquired myopathies like chloroquine toxicity, also have features of an autophagic myopathy. This review is focused on XMEA, a myopathy with onset of slowly progressive proximal weakness and elevated serum creatine kinase (2×to 20×normal) typically in the first decade of life. However, both late-adult onset and severe, sometimes lethal, neonatal cases also occur. Skeletal muscle pathology is characterized by numerous cytoplasmic autophagic vacuoles, complex muscle fiber splitting with internalization of capillaries, and complement C5b-9 deposition within vacuoles and along the sarcolemma. The autophagic vacuoles have sarcolemmal features. Mutations in the VMA21 gene at Xq28 cause XMEA by reducing the activity of lysosomal hydrolases. The VMA21 protein regulates the assembly of the V-ATPase required to acidify the lysosome. Increased lysosomal pH and poor degradation of cellular debris may secondarily induce autophagy, the net effect being accumulation of autophagolysosomes. The relationship of XMEA to other lysosomal disorders of muscle and potential therapeutic interventions for XMEA are discussed.

Innate immune responses in the brain of sphingolipid lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are mainly caused by the defective activity of lysosomal hydrolases. A sub-class of LSDs are the sphingolipidoses, in which sphingolipids accumulate intra-cellularly. We here discuss the role of innate immunity in the sphingolipidoses, and compare the pathways of activation in two classical sphingolipidoses, namely Gaucher disease and Sandhoff disease, and in Niemann-Pick C disease, in which the main storage material is cholesterol but sphingolipids also accumulate. We discuss the mechanisms leading to neuroinflammation, and the different pathways of neuroinflammation in the different diseases, and suggest that intervention in these pathways may be a useful therapeutic approach to address these devastating human diseases.

Screening and identification of small molecule inhibitors of ErbB2-induced invasion

ERBB2 amplification and overexpression are strongly associated with invasive cancer with high recurrence and poor prognosis. Enhanced ErbB2 signaling induces cysteine cathepsin B and L expression leading to their higher proteolytic activity (zFRase activity), which is crucial for the invasion of ErbB2-positive breast cancer cells in vitro. Here we introduce a simple screening system based on zFRase activity as a primary readout and a following robust invasion assay and lysosomal distribution analysis for the identification of compounds that can inhibit ErbB2-induced invasion. With an unbiased kinase inhibitor screen, we identified Bohemine/Roscovitine, Gö6979 and JAK3 inhibitor VI as compounds that can efficiently decrease cysteine cathepsin activity. Using the well-established and clinically relevant ErbB1 and ErbB2 inhibitor lapatinib as a positive control, we studied their ability to inhibit ErbB2-induced invasion in 3-dimensional Matrigel cultures. We found one of them, JAK3 inhibitor VI, capable of inhibiting invasion of highly invasive ErbB2-positive ovarian cancer cells as efficiently as lapatinib, whereas Gö6979 and Roscovitine displayed more modest inhibition. All compounds reversed the malignant, ErbB2-induced and invasion-supporting peripheral distribution of lysosomes. This effect was most evident for lapatinib and JAK3 inhibitor VI and milder for Gö6979 and Roscovitine. Our results further showed that JAK3 inhibitor VI function was independent of JAK kinases but involved downregulation of cathepsin L. We postulate that the screening method and the verification experiments that are based on oncogene-induced changes in lysosomal hydrolase activity and lysosomal distribution could be used for identification of novel inhibitors of ErbB2-induced invasiveness. Additionally, we introduce a novel function for lapatinib in controlling malignant lysosomal distribution, that may also be involved in its capability to inhibit ErbB2-induced invasion in vivo.

Fluorescence-based visualization of autophagic activity predicts mouse embryo viability

Embryo quality is a critical parameter in assisted reproductive technologies. Although embryo quality can be evaluated morphologically, embryo morphology does not correlate perfectly with embryo viability. To improve this, it is important to understand which molecular mechanisms are involved in embryo quality control. Autophagy is an evolutionarily conserved catabolic process in which cytoplasmic materials sequestered by autophagosomes are degraded in lysosomes. We previously demonstrated that autophagy is highly activated after fertilization and is essential for further embryonic development. Here, we developed a simple fluorescence-based method for visualizing autophagic activity in live mouse embryos. Our method is based on imaging of the fluorescence intensity of GFP-LC3, a versatile marker for autophagy, which is microinjected into the embryos. Using this method, we show that embryonic autophagic activity declines with advancing maternal age, probably due to a decline in the activity of lysosomal hydrolases. We also demonstrate that embryonic autophagic activity is associated with the developmental viability of the embryo. Our results suggest that embryonic autophagic activity can be utilized as a novel indicator of embryo quality.

Determination of the lysosomal hydrolase activity in blood collected on filter paper, an alternative to screen high risk populations

This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease.

Cribriform pattern in brain MRI: A diagnostic clue for mucopolysaccharidoses

Mucopolysaccharidoses (MPS) represents a heterogeneous group of inherited lysosomal storage disorders characterised by defective degradation of long-chain complex carbohydrates called glycosoaminoglycans (GAGs). To date, 11 distinct types of MPS have been described, each as a result of deficient enzymatic activity of specific lysosomal hydrolase. The most common types are Hurler and Hunter syndromes. We report a case of a child presenting with macrocephaly, clinically suspected to be due to hydrocephalus. An MRI (3 Tesla) brain study demonstrated the cribriform pattern in the brain caused by dilated perivascular spaces, which is a diagnostic clue for the presence of MPS.

Cribriform pattern in brain MRI: A diagnostic clue for mucopolysaccharidoses

Mucopolysaccharidoses (MPS) represents a heterogeneous group of inherited lysosomal storage disorders characterised by defective degradation of long-chain complex carbohydrates called glycosoaminoglycans (GAGs). To date, 11 distinct types of MPS have been described, each as a result of deficient enzymatic activity of specific lysosomal hydrolase. The most common types are Hurler and Hunter syndromes. We report a case of a child presenting with macrocephaly, clinically suspected to be due to hydrocephalus. An MRI (3 Tesla) brain study demonstrated the cribriform pattern in the brain caused by dilated perivascular spaces, which is a diagnostic clue for the presence of MPS.

The cornea in mucopolysaccharidosis IH-S: structural and ultrastructural study

Type I mucopolysaccharidoses (MPS I) include three autosomal recessive disorders (Hurler, MPS IH; Scheie, MPS IS; and Hurler-Scheie, MPS IH-S) caused by the deficient activity of the lysosomal hydrolase α-L-iduronidase with the consequent accumulation of dermatan and heparan sulfate in the lysosomes of several cell types [1]. MPS IH-S is an attenuated disease and the patients show minor facial and skeletal dysmorphism, regular intelligence, mild cardiac and respiratory disease, hepatosplenomegaly, and a normal lifespan. The most common feature is corneal opacification [2], whose morphological basis was not studied in detail. In this work we performed a structural and ultrastructural analysis of the cornea in a patient with MPS IH-S. The patient underwent penetrating keratoplasty and the corneal button was immediately processed for light and electron microscopy. From the micrographs a morphometric analysis was also performed. The corneal epithelium showed superficial cells with few microfolds and evident intercellular spaces. The wing cell layer was formed either by cells with well-evident tonofilaments and small peripheral clear vesicles, or with bilobed nucleus and large paranuclear vesicles filled with granular material. The basal cells showed polygonal shape, with many small vesicles, placed generally in the supranuclear cytoplasm: the intercellular space was enlarged by granular material. The Bowman’s layer was either normal in thickness and structure, or thinner and formed by granular material of variable electron density. The stroma was formed by irregular lamellae of differently oriented collagen, by a large number of keratocytes filled with vesicles, and by intercellular granular material. The corneal endothelium showed degenerative changes. The morphometric analysis of the collagen fibrils diameter provided a mean diameter of 21.71±2.09 nm. Hemidesmosomes were less numerous in the basal cells when compared to the normal cornea. Stromal keratocytes were reduced in their number, particularly in the anterior stroma. Our data showed in MPS IH-S patient pronounced changes of the epithelium, of the Bowman’s layer and of the stroma, consistent with the corneal opacity. As the etiology of the disease is a deficiency of α-L-iduronidase and the consequent accumulation of glycosaminoglycans, we are of the opinion that the stromal keratocytes are the first cells to be involved in the pathogenesis of the corneal disease. The accumulation of the aberrant products seems able to induce morphological changes of both the Bowman’s layer and the corneal epithelium.

Inhibition of retromer activity by herpesvirus saimiri tip leads to CD4 downregulation and efficient T cell transformation.

The mammalian retromer is an evolutionally conserved protein complex composed of a vacuolar protein sorting trimer (Vps 26/29/35) that participates in cargo recognition and a sorting nexin (SNX) dimer that binds to endosomal membranes. The retromer plays an important role in efficient retrograde transport for endosome-to-Golgi retrieval of the cation-independent mannose-6-phosphate receptor (CI-MPR), a receptor for lysosomal hydrolases, and other endosomal proteins. This ultimately contributes to the control of cell growth, cell adhesion, and cell migration. The herpesvirus saimiri (HVS) tyrosine kinase-interacting protein (Tip), required for the immortalization of primary T lymphocytes, targets cellular signaling molecules, including Lck tyrosine kinases and the p80 endosomal trafficking protein. Despite the pronounced effects of HVS Tip on T cell signal transduction, the details of its activity on T cell immortalization remain elusive. Here, we report that the amino-terminal conserved, glutamate-rich sequence of Tip specifically interacts with the retromer subunit Vps35 and that this interaction not only causes the redistribution of Vps35 from the early endosome to the lysosome but also drastically inhibits retromer activity, as measured by decreased levels of CI-MPR and lower activities of cellular lysosomal hydrolases. Physiologically, the inhibition of intracellular retromer activity by Tip is ultimately linked to the downregulation of CD4 surface expression and to the efficient in vitro immortalization of primary human T cells to interleukin-2 (IL-2)-independent permanent growth. Therefore, HVS Tip uniquely targets the retromer complex to impair the intracellular trafficking functions of infected cells, ultimately contributing to efficient T cell transformation.