Effectiveness of ustekinumab in patients with psoriatic arthritis in a real-world, multicenter study.

OBJECTIVE: To assess the effectiveness and survival of ustekinumab (UST) among patients with psoriatic arthritis (PsA) treated under routine clinical care. METHODS: Multicenter study. Epidemiological and clinical data was collected through electronic medical records of all patients with PsA who started UST in 15 hospitals of Spain. RESULTS: Two hundred and one patients were included, 130 (64.7%) with 45 mg and 71 (35.3%) with 90 mg. One hundred and thirty one patients (65.2%) had previously received another biological therapy. The median baseline DAS 28 ESR was 3.99, and Psoriasis Area and Severity Index (PASI) was 3. Overall, there was a significant decrease in DAS66/68 CRP, swollen joint count (SJC), tender joint count (TJC), and PASI in the first month of treatment, with earlier improvement in skin (PASI) than joints outcomes. Survival was numerically lower in patients with UST 45 mg (58.1%) than 90 mg (76.1%), although significant differences were not found (p = 0.147). When comparing naïve and < 1 TNF blocker versus > 2 TNF blocker-experienced patients, a significantly earlier response was seen in the former group regarding SJC (p = 0.029) at 1 month. Fifty-one patients (25.3%) stopped UST due to joint inefficacy and 4 patients due to adverse events (1.9%). Drug survival was significantly better in patients with fewer lines of previous biological agents (p = 0.003 for < 1 TNF blocker versus > 2 TNF blocker users). CONCLUSIONS: UST was effective in PsA patients in a routine clinical care setting. Patients with UST 90 mg and fewer lines of previous biologics achieved better and faster responses. Key Points • Largest cohort of patients with PsA in treatment with UST with specific rheumatological indication. • First cohort of patients with PsA comparing effectiveness of UST according to 45/90 mg dose.

Monitoring spatiotemporal changes in chaperone-mediated autophagy in vivo.

Autophagy malfunctioning occurs in multiple human disorders, making attractive the idea of chemically modulating it with therapeutic purposes. However, for many types of autophagy, a clear understanding of tissue-specific differences in their activity and regulation is missing because of lack of methods to monitor these processes in vivo. Chaperone-mediated autophagy (CMA) is a selective type of autophagy that until now has only been studied in vitro and not in the tissue context at single cell resolution. Here, we develop a transgenic reporter mouse that allows dynamic measurement of CMA activity in vivo using image-based procedures. We identify previously unknown spatial and temporal differences in CMA activity in multiple organs and in response to stress. We illustrate the versatility of this model for monitoring CMA in live animals, organotypic cultures and cell cultures from these mice, and provide practical examples of multiorgan response to drugs that modulate CMA.

Analysis of Chaperone-Mediated Autophagy.

Chaperone-mediated autophagy (CMA) is a selective type of autophagy whereby a specific subset of intracellular proteins is targeted to the lysosome for degradation. These proteins are identified by a chaperone that targets them to lysosomes. There, they are translocated into the organelle lumen through a lysosomal membrane receptor/translocation complex. CMA plays an important role in maintaining cellular proteostasis by eliminating damaged and altered proteins. CMA also participates in the control of the cellular energetic balance through recycling of amino acids resulting from lysosomal proteolysis of the substrate proteins. Lastly, due to the intrinsic protein selectivity of CMA, this type of autophagy exerts regulatory functions by mediating timely degradation of key cellular proteins that participate in processes such as lipid and glucose metabolism, cell cycle, DNA repair, and cellular reprogramming, among others. Dysfunctional CMA occurs with age and has now been described in a growing list of human pathologies such as metabolic disorders, neurodegeneration, cancer, immunodeficiency, and diabetes. In this chapter, we describe current methodologies to quantitatively analyze CMA activity in different experimental models.

Autophagy and regulation of cilia function and assembly.

Motile and primary cilia (PC) are microtubule-based structures located at the cell surface of many cell types. Cilia govern cellular functions ranging from motility to integration of mechanical and chemical signaling from the environment. Recent studies highlight the interplay between cilia and autophagy, a conserved cellular process responsible for intracellular degradation. Signaling from the PC recruits the autophagic machinery to trigger autophagosome formation. Conversely, autophagy regulates ciliogenesis by controlling the levels of ciliary proteins. The cross talk between autophagy and ciliated structures is a novel aspect of cell biology with major implications in development, physiology and human pathologies related to defects in cilium function.

Autophagic pathways and metabolic stress.

Autophagy is an essential intracellular process that mediates degradation of intracellular proteins and organelles in lysosomes. Autophagy was initially identified for its role as alternative source of energy when nutrients are scarce but, in recent years, a previously unknown role for this degradative pathway in the cellular response to stress has gained considerable attention. In this review, we focus on the novel findings linking autophagic function with metabolic stress resulting either from proteins or lipids. Proper autophagic activity is required in the cellular defense against proteotoxicity arising in the cytosol and also in the endoplasmic reticulum, where a vast amount of proteins are synthesized and folded. In addition, autophagy contributes to mobilization of intracellular lipid stores and may be central to lipid metabolism in certain cellular conditions. In this review, we focus on the interrelation between autophagy and different types of metabolic stress, specifically the stress resulting from the presence of misbehaving proteins within the cytosol or in the endoplasmic reticulum and the stress following a lipogenic challenge. We also comment on the consequences that chronic exposure to these metabolic stressors could have on autophagic function and on how this effect may underlie the basis of some common metabolic disorders.

Chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is the only type of autophagy in mammalian cells able to selectively degrade cytosolic proteins in lysosomes. CMA is maximally activated in response to stressors such as prolonged starvation, exposure to toxic compounds, or oxidative stress. We have found that CMA activity decreases in aging and in some age-related disorders such as Parkinson's disease. Impaired CMA under these conditions may be responsible for the accumulation of damaged proteins inside cells and for their higher vulnerability to stressors. In contrast to other forms of autophagy, where substrates are engulfed or sequestered along with other cytosolic components, CMA substrates are translocated one-by-one across the lysosomal membrane. Changes in the levels/activity of the lysosomal components required for substrate translocation can be used to stimulate CMA activity. However, the most unequivocal method to measure CMA is by directly tracking the translocation of substrate proteins into isolated lysosomes.

Autophagy as a cell-repair mechanism: activation of chaperone-mediated autophagy during oxidative stress.

Proper removal of oxidized proteins is an important determinant of success when evaluating the ability of cells to handle oxidative stress. The ubiquitin/proteasome system has been considered the main responsible mechanism for the removal of oxidized proteins, as it can discriminate between normal and altered proteins, and selectively target the latter ones for degradation. A possible role for lysosomes, the other major intracellular proteolytic system, in the removal of oxidized proteins has been often refused, mostly on the basis of the lack of selectivity of this system. Although most of the degradation of intracellular components in lysosomes (autophagy) takes place through "in bulk" sequestration of complete cytosolic regions, selective targeting of proteins to lysosomes for their degradation is also possible via what is known as chaperone-mediated autophagy (CMA). In this work, we review recent evidence supporting the participation of CMA in the clearance of oxidized proteins in the forefront of the cellular response to oxidative stress. The consequences of an impairment in CMA activity, observed during aging and in some age-related disorders, are also discussed.

Autophagic vacuoles are enriched in amyloid precursor protein-secretase activities: implications for beta-amyloid peptide over-production and localization in Alzheimer's disease.

In Alzheimer's disease (AD), the neuropathologic hallmarks of beta-amyloid deposition and neurofibrillary degeneration are associated with early and progressive pathology of the endosomal-lysosomal system. Abnormalities of autophagy, a major pathway to lysosomes for protein and organelle turnover, include marked accumulations of autophagy-related vesicular compartments (autophagic vacuoles or AVs) in affected neurons. Here, we investigated the possibility that AVs contain the proteases and substrates necessary to cleave the amyloid precursor protein (APP) to A beta peptide that forms beta-amyloid, a key pathogenic factor in AD. AVs were highly purified using a well-established metrizamide gradient procedure from livers of transgenic YAC mice overexpressing wild-type human APP. By Western blot analysis, AVs contained APP, beta CTF - the beta-cleaved carboxyl-terminal domain of APP, and BACE, the protease-mediating beta-cleavage of APP. beta-Secretase activity measured against a fluorogenic peptide was significantly enriched in the AV fraction relative to whole-liver lysate. Compared to other recovered subcellular fractions, AVs exhibited the highest specific activity of gamma-secretase based on a fluorogenic assay and inhibition by a specific inhibitor of gamma-secretase, DAPT. AVs were also the most enriched subcellular fraction in levels of the gamma-secretase components presenilin and nicastrin. Immunoelectron microscopy demonstrated selective immunogold labeling of AVs with antibodies specific for the carboxyl termini of human A beta 40 and A beta 42. These data indicate that AVs are a previously unrecognized and potentially highly active compartment for A beta generation and suggest that the abnormal accumulation of AVs in affected neurons of the AD brain contributes to beta-amyloid deposition.

Unique properties of lamp2a compared to other lamp2 isoforms.

Lamp2a acts as a receptor in the lysosomal membrane for substrate proteins of chaperone-mediated autophagy. Using antibodies specific for the cytosolic tail of lamp2a and others recognizing all lamp2 isoforms, we found that in rat liver lamp2a represents 25% of lamp2s in the lysosome. We show that lamp2a levels in the lysosomal membrane in rat liver and fibroblasts in culture directly correlate with rates of chaperone-mediated autophagy in a variety of physiological and pathological conditions. The concentration of other lamp2s in the lysosomal membrane show no correlation under the same conditions. Furthermore, substrate proteins bind to lamp2a but not to other lamp2s. Four positively-charged amino acids uniquely present in the cytosolic tail of lamp2a are required for the binding of substrate proteins. Lamp2a also distributes to an unique subpopulation of perinuclear lysosomes in cultured fibroblasts in response to serum withdrawal, and lamp2a, more than other lamp2s, tends to multimerize. These characteristics may be important for lamp2a to act as a receptor for chaperone-mediated autophagy.

Selective degradation of annexins by chaperone-mediated autophagy.

Annexins are a family of proteins that bind phospholipids in a calcium-dependent manner. Analysis of the sequences of the different members of the annexin family revealed the presence of a pentapeptide biochemically related to KFERQ in some annexins but not in others. Such sequences have been proposed to be a targeting sequence for chaperone-mediated autophagy, a lysosomal pathway of protein degradation that is activated in confluent cells in response to removal of serum growth factors. We demonstrate that annexins II and VI, which contain KFERQ-like sequences, are degraded more rapidly in response to serum withdrawal, while annexins V and XI, without such sequences, are degraded at the same rate in the presence and absence of serum. Using isolated lysosomes, only the annexins containing KFERQ-like sequences are degraded by chaperone mediated-autophagy. Annexins V and XI could associate with lysosomes but did not enter the lysosomes and were not proteolytic substrates. Furthermore, four annexins containing KFERQ-like sequences, annexins I, II, IV, and VI, are enriched in lysosomes with high chaperone-mediated autophagy activity as expected for substrate proteins. These results provide striking evidence for the importance of KFERQ motifs in substrates of chaperone-mediated autophagy.

Age-related decline in chaperone-mediated autophagy.

Intracellular protein degradation rates decrease with age in many tissues and organs. In cultured cells, chaperone-mediated autophagy, which is responsible for the selective degradation of cytosolic proteins in lysosomes, decreases with age. In this work we use lysosomes isolated from rat liver to analyze age-related changes in the levels and activities of the main components of chaperone-mediated autophagy. Lysosomes from "old" (22-month-old) rats show lower rates of chaperone-mediated autophagy, and both substrate binding to the lysosomal membrane and transport into lysosomes decline with age. A progressive age-related decrease in the levels of the lysosome-associated membrane protein type 2a that acts as a receptor for chaperone-mediated autophagy was responsible for decreased substrate binding in lysosomes from old rats as well as from late passage human fibroblasts. The cytosolic levels and activity of the 73-kDa heat-shock cognate protein required for substrate targeting to lysosomes were unchanged with age. The levels of lysosome-associated hsc73 were increased only in the oldest rats. This increase may be an attempt to compensate for reduced activity of the pathway with age.

When lysosomes get old.

Changes in the lysosomes of senescent tissues and organisms are common and have been used as biomarkers of aging. Lysosomes are responsible for the degradation of many macromolecules, including proteins. At least five different pathways for the delivery of substrate proteins to lysosomes are known. Three of these pathways decline with age, and the molecular explanations for these deficiencies are currently being studied. Other aspects of lysosomal proteolysis increase or do not change with age in spite of marked changes in lysosomal morphology and biochemistry. Age-related changes in certain lysosomal pathways of proteolysis remain to be studied. This area of research is important because abnormalities in lysosomal protein degradation pathways may contribute to several characteristics and pathologies associated with aging.

Regulation of lamp2a levels in the lysosomal membrane.

The selective degradation of cytosolic proteins in lysosomes by chaperone-mediated autophagy depends, at least in part, on the levels of a substrate receptor at the lysosomal membrane. We have previously identified this receptor as the lysosome-associated membrane protein type 2a (lamp2a) and showed that levels of lamp2a at the lysosomal membrane directly correlate with the activity of the proteolytic pathway. Here we show that levels of lamp2a at the lysosomal membrane are mainly controlled by changes in its half-life and its distribution between the lysosomal membrane and the matrix. The lysosomal degradation of lamp2a requires the combined action of at least two different proteolytic activities at the lysosomal membrane. Lamp2a is released from the membrane by the action of these proteases, and then the truncated lamp2a is rapidly degraded within the lysosomal matrix. Membrane degradation of lamp2a is a regulated process that is inhibited in the presence of substrates for chaperone-mediated autophagy and under conditions that activate that type of autophagy. Uptake of substrate proteins also results in transport of some intact lamp2a from the lysosomal membrane into the matrix. This fraction of lamp2a can be reinserted back into the lysosomal membrane. The traffic of lamp2a through the lysosomal matrix is not mediated by vesicles, and lamp2a reinsertion requires the lysosomal membrane potential and protein components of the lysosomal membrane. The distribution of lamp2a between the lysosomal membrane and matrix is a dynamic process that contributes to the regulation of lysosomal membrane levels of lamp2a and consequently to the activity of the chaperone-mediated autophagic pathway.

Direct lysosomal uptake of alpha 2-microglobulin contributes to chemically induced nephropathy.

BACKGROUND: An abnormal accumulation of alpha 2-microglobulin (alpha 2 mu) in kidney lysosomes of male rats has been described in the nephropathy resulting from exposure to a variety of chemicals. The increment in lysosomal levels of alpha 2 mu cannot be explained by a decrease in its proteolytic susceptibility. Because a portion of alpha 2 mu resides in the cytosol of kidney cells, we decided to analyze whether this cytosolic form also contributes to the abnormal lysosomal accumulation of alpha 2 mu after exposure to chemicals. METHODS: Intact kidney lysosomes were isolated from untreated or 2,2,4-trimethylpentane (TMP) treated rats, and their ability to take up alpha 2 mu was compared. RESULTS: alpha 2 mu can be directly transported into isolated lysosomes in the presence of the heat shock cognate protein of 73 kDa (hsc73). alpha 2 mu specifically binds to a lysosomal membrane glycoprotein of 96 kDa, previously identified as the receptor for the hsc73-mediated lysosomal pathway of protein degradation. In rats exposed to TMP, the specific lysosomal transport of alpha 2 mu increases, as well as the ability of lysosomes to directly transport other substrates for this pathway. The increased lysosomal transport is mainly due to an increase in the levels of the receptor protein in the lysosomal membrane. CONCLUSIONS: The hsc73-mediated lysosomal pathway contributes to the normal degradation of alpha 2 mu in rat kidney and liver, and the activity of this pathway is increased after exposure to TMP. Our results suggest that the chemically induced accumulation of cytosolic alpha 2 mu in lysosomes is mediated by an increased rate of direct uptake into lysosomes.

IkappaB is a substrate for a selective pathway of lysosomal proteolysis.

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor kappa B (IkappaB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IkappaB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IkappaB is directly transported into isolated lysosomes in a process that requires binding of IkappaB to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit IkappaB uptake by lysosomes. Ubiquitination and phosphorylation of IkappaB are not required for its targeting to lysosomes. The lysosomal degradation of IkappaB is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of IkappaB is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in IkappaB degradation can be completely blocked by lysosomal inhibitors. In Chinese hamster ovary cells exhibiting an increased activity of the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2, the human form of lgp96, the degradation of IkappaB is increased. There are both short- and long-lived pools of IkappaB, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of IkappaB is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating IkappaB degradation in lysosomes. This selective pathway of lysosomal degradation of IkappaB is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor kappa B. In addition, the response of nuclear factor kappa B to several stimuli increases when this lysosomal pathway of proteolysis is activated.

Lysosomes, a meeting point of proteins, chaperones, and proteases.

Lysosomes, classically considered as nonspecific systems for protein degradation, have recently also been shown to be able selectively to degrade specific intracellular proteins. Here we review this selective pathway of lysosomal protein degradation that involves cytosolic and intralysosomal chaperones and a receptor in the lysosomal membrane. This pathway is highly selective for cytosolic proteins containing a lysosomal targeting signal. The selective lysosomal degradation pathway is active under conditions of nutrient deprivation and plays an important role in the regulation of intracellular protein levels in stress situations. Several physiological and pathological modifications in the activity of this selective lysosomal pathway of protein degradation are discussed.

How do intracellular proteolytic systems change with age?

One of the common features of cells from senescent tissues is the accumulation of abnormal proteins. Several hypotheses have been proposed to explain the origin of those abnormal proteins. A defect in proteolytic systems usually responsible for the elimination of altered proteins from the cells could clearly contribute to such accumulation. Here we describe the effect of age on the major proteolytic systems within cells: the ubiquitin-proteasome pathway, the calcium-activated calpain pathways, and multiple lysosomal pathways. Our group has contributed to the characterization of a selective pathway of degradation of cytosolic proteins in lysosomes that is activated under conditions of nutrient deprivation. In this lysosomal pathway of proteolysis proteins are transported through the lysosomal membrane assisted by cytosolic and lysosomal molecular chaperones and a receptor protein in the lysosomal membrane. The activity of this pathway significantly decreases with age, and this decrease might account for the cytosolic accumulation of aberrant substrate proteins in senescent cells. The cellular consequences of the decline of this lysosomal pathway together with possible methods to restore the reduced function are also addressed in this review.

A population of rat liver lysosomes responsible for the selective uptake and degradation of cytosolic proteins.

Two populations of rat liver lysosomes can be distinguished on the basis of their density. A major difference between these populations is that one contains the heat shock cognate protein of 73 kDa (hsc73) within the lysosomal lumen. The lysosomal fraction containing hsc73 exhibits much higher efficiencies in the in vitro uptake and degradation of glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A, two well established substrates of the selective lysosomal pathway of intracellular protein degradation. Preloading of the lysosomal population that is devoid of lumenal hsc73 with hsc73 isolated from cytosol activated the selective transport of substrate proteins into these lysosomes. Furthermore, treatment of animals with leupeptin, an inhibitor of lysosomal cathepsins, or 88 h of starvation also increased the amount of hsc73 within their lysosomal lumen, and these in vivo treatments also activated the selective transport of substrate proteins in vitro. Thus, the hsc73 located within lysosomes appears to be required for efficient uptake of cytosolic proteins by these organelles. The difference in hsc73 content between the lysosomal populations appears to be due to differences in their ability to take up hsc73 combined with differences in the intralysosomal degradation rates of hsc73. The increased stability of hsc73 in one population of lysosomes is primarily a consequence of this lysosomal population's more acidic pH.

A receptor for the selective uptake and degradation of proteins by lysosomes.

Multiple pathways of protein degradation operate within cells. A selective protein import pathway exists for the uptake and degradation of particular cytosolic proteins by lysosomes. Here, the lysosomal membrane glycoprotein LGP96 was identified as a receptor for the selective import and degradation of proteins within lysosomes. Specific substrates of this proteolytic pathway bound to the cytosolic tail of a 96-kilodalton lysosomal membrane protein in two different binding assays. Overexpression of human LGP96 in Chinese hamster ovary cells increased the activity of the selective lysosomal proteolytic pathway in vivo and in vitro.

LAPTM5: a novel lysosomal-associated multispanning membrane protein preferentially expressed in hematopoietic cells.

While a large body of knowledge about cell membrane proteins exists, much less is known about the repertoire and function of integral membrane proteins of intracellular organelles. In looking for novel classes of genes that are functionally important to hematopoietic cells, we have cloned the cDNA for a gene preferentially expressed in adult hematopoietic tissues. During embryonic development the gene is expressed in both hematopoietic and nonhematopoietic tissues. In cell lines the gene is expressed specifically in hematopoietic lineages, whereas in normal adult tissues the mRNA is preferentially detected at high levels in lymphoid and myeloid tissues. The predicted protein is a pentaspanner with no homology to known genes and conserved across evolution. Immunocytological and cell fractionation studies with a specific antibody revealed a protein localizing in lysosomes. The gene, provisionally named LAPTM5, maps to chromosome 1p34. The expression pattern of the gene together with preliminary evidence that the protein interacts with ubiquitin indicates that the protein may have a special functional role during embryogenesis and in adult hematopoietic cells.