**Atg8-Family Proteins—Structural Features and Molecular Interactions in Autophagy and Beyond**

**Nicole Wesch 1,**†**, Vladimir Kirkin 2,**† **and Vladimir V. Rogov 1,3,4,\***

Received: 8 August 2020; Accepted: 27 August 2020; Published: 1 September 2020

**Abstract:** Autophagy is a common name for a number of catabolic processes, which keep the cellular homeostasis by removing damaged and dysfunctional intracellular components. Impairment or misbalance of autophagy can lead to various diseases, such as neurodegeneration, infection diseases, and cancer. A central axis of autophagy is formed along the interactions of autophagy modifiers (Atg8-family proteins) with a variety of their cellular counter partners. Besides autophagy, Atg8-proteins participate in many other pathways, among which membrane trafficking and neuronal signaling are the most known. Despite the fact that autophagy modifiers are well-studied, as the small globular proteins show similarity to ubiquitin on a structural level, the mechanism of their interactions are still not completely understood. A thorough analysis and classification of all known mechanisms of Atg8-protein interactions could shed light on their functioning and connect the pathways involving Atg8-proteins. In this review, we present our views of the key features of the Atg8-proteins and describe the basic principles of their recognition and binding by interaction partners. We discuss affinity and selectivity of their interactions as well as provide perspectives for discovery of new Atg8-interacting proteins and therapeutic approaches to tackle major human diseases.

**Keywords:** Atg8; LC3; GABARAP; LIR motif; SAR; UBL; autophagy


*2.3. Characterization of the Nanomicelles*

Spermidine loading was evaluated in 50 mg/mL NS dispersions diluted (1:3, *v*/*v*) with an ethanol:water (1:1, *v*/*v*) mixture and analyzed by a fluorimetric method in comparison with the empty nanomicelles. The concentration obtained represented spermidine, both encapsulated in the nanomicelles and free in the aqueous phase. Therefore, to obtain the concentration of free spermidine, the nanomicelle dispersion was centrifuged in a 3.5 mL Ultra 5 KDa filter (Merck Millipore, Burlington, MA, USA) at 4000× *g* for 30 min and the ultrafiltrate was analyzed for spermidine content after dilution 1:3 with an ethanol:water (1:1, *v*/*v*) mixture. The difference between spermidine concentration in the nanomicelle dispersion and in the ultrafiltrate provided the concentration of the encapsulated spermidine. The nanomicelle loading was obtained as the ratio between the concentration (*w*/*v*) of the encapsulated spermidine and the concentration (*w*/*v*) of the nanomicelle dispersion.

The fluorimetric evaluation of spermidine in the nanomicelle dispersion and in the ultrafiltrate was carried out by a polyamine assay kit (Biovision, Milan, Italy) containing enzymes for the generation of hydrogen peroxide from spermidine and subsequent reaction of hydrogen peroxide with a fluorometric probe (Ex/Em = 535/587 nm) to yield a signal proportional to the amount of the polyamine present. The analysis was carried out according to the manufacturer's instructions.

Fenretinide loading was evaluated in 50 mg/mL NF dispersions diluted (1:3 *v*/*v*) with an ethanol:water (1:1, *v*/*v*) mixture and analyzed by UV spectroscopy (Shimadzu UV-1601) at 360 nm in comparison with the empty nanomicelles. The concentration obtained represented fenretinide, both encapsulated in the nanomicelles and free in the aqueous phase. To obtain the concentration of the free drug, the nanomicelle dispersion was centrifuged in a 3.5 mL Ultra 5 KDa filter at 4000× *g* for 30 min, and the ultrafiltrate was spectrophotometrically analyzed for drug content after dilution 1:3 with an ethanol:water (1:1, *v*/*v*) mixture. The difference between the drug concentration in the nanomicelle suspension and in the ultrafiltrate provided the concentration of the encapsulated drug. The nanomicelle loading was obtained as the ratio between the concentration (*w*/*v*) of the encapsulated drug and the concentration (*w*/*v*) of the nanomicelle dispersion.

Particle size, polydispersity, and zeta potential were measured at 37 ◦C (Malvern Nano-ZS Spectrometer, Malvern, UK) on the nanomicelle suspensions prepared in PBS and on the nanomicelle suspensions progressively diluted up to 1:100 (*v*/*v*) starting from 50 mg/mL concentration. Dilutions were made with PBS containing 10% (*v*/*v*) Human Serum (HS) to simulate the in-vivo dilution of the nanomicelles injected into the bloodstream. A minimum of 12 measurements per sample were made. Results were the combination of 3 10-min runs for a total accumulation correlation function time of 30 min. The results were volume-weighted.

Leakage of spermidine and fenretinide from NS and NF, respectively, was measured by dialysis at 37 ◦C, as previously described [21,22], with some minor changes. Briefly, the nanomicelles suspensions, prepared in NaCl 0.9% (*w*/*v*) at 50 mg/mL, were diluted to 10 mg/mL with pH 7.4 PBS containing 10% HS, and 1 mL of the diluted suspension was placed in a releasing chamber separated from a receiving compartment by a dialysis membrane (Mw cutoff 5KD, Fisher Scientific). The receiving compartment was filled with 10 mL pH 7.4 PBS containing 10% HS.

Leakage from the nanomicelles was determined by evaluating spermidine or fenretinide concentrations in the receiving phase at increasing time intervals. Spermidine concentration was obtained by the polyamine assay kit previously described, and fenretinide was evaluated spectrophotometrically by its maximum absorption wavelength (360 nm). Sink conditions were monitored throughout the experiment.


Psychosocial Effect of Maxillofacial Deformities

*Maxillofacial Surgery and Craniofacial Deformity - Practices and Updates*

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10.1682/JRRD.2013.10.0218

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**120**

**123**

**Chapter 9**

*Morio Tonogi*

also improve sleep quality.

respiratory disturbance

**1. Introduction**

**Abstract**

The Effects of Maxillomandibular

Maxillomandibular advancement (MMA) using a standardized surgical procedure consisting of a LeFort I osteotomy and bilateral sagittal split ramus osteotomy and genioglossus advancement (GA) using a genioplasty improve airway volume, oxygen desaturation, and the AHI in patients with OSA. However, there are few reports on changes in sleep quality following MMA and GA. We assessed the effects of MMA and GA on sleep quality by comparing oxygen desaturation, AHI, and sleep architecture before and after surgery. Methods: Eight patients underwent polysomnography (PSG) and CT scan before and after surgery. Conclusions: Our study finds that %TST and %REM were both increased, while %S1 and NA both decreased. Based on these results, it appears that both the quality and quantity of sleep were improved. MMA and GA improve sleep respiratory disturbance and can

**Keywords:** maxillomandibular advancement, sleep quality, genioplasty,

maxillary retrusion can all cause upper airway obstruction [1–8].

Obstructive sleep apnea (OSA) is a disorder characterized by intermittent and recurrent episodes of partial or complete upper airway obstruction during sleep. Obesity, a narrow nasopharynx and oropharynx, large soft palate, large tonsils, large tongue, tongue retroposition, micrognathia, mandibular retrognathia, and

Maxillomandibular advancement (MMA) and genioglossus advancement (GA)

improve airway volume, oxygen desaturation, and the apnea-hypopnea index (AHI) in patients with OSA. However, there are few reports on changes in sleep quality and architecture following MMA and GA [9]. Therefore, we assessed the

Advancement and Genioglossus

Advancement on Sleep Quality

*Takako Sato, Ryota Nakamura, Akio Himejima,*

*Hiroaki Azaki, Junya Aoki, Keiichi Yanagawa,*

*Akemi Kusano, Serim Kang, Saori Ohtani,*

*Kentarou Yamada, Kanako Yamagata,*

*Keiji Shinozuka, Takeya Yamada and*

**Chapter 9**


**3. Classification**

Cerebral Palsy causes functional limitations that lead to disorders of motor and postural development ascribed to non-progressive disorders that happen in fetal development or the child's brain [12]. It has been classified mainly into three categories viz. Physiological, Topographical and Functional classification system, includes Gross Motor Function Classification System (GMFCS), the Manual Ability Classification System (MACS), the Communication Function Classification System (CFCS), and the Eating and Drinking Ability Classification System (EDACS) which are complementary to each other.


**3. Olive oil phenolic compounds**

Historically, the health promoting properties of EVOO were attributed to the high concentration of monounsaturated fatty acids (MUFAs), in particular oleic acid, contained in EVOO. However other seed oils (i.e. sunflower, soybean, and rapeseed), which also contain high concentrations of oleic acid, do not exhibit the same health benefits as EVOO (LópezMiranda et al., 2010, Harper et al., 2006, Aguilera et al., 2004). In addition to oleic acid, EVOO contains a minor, yet significant phenolic fraction that other seed oils lack and this fraction has generated much interest regarding its health promoting properties.

Currently, 36 phenolic compounds have been identified in EVOO and studies (human, animal, *in vivo* and *in vitro*) have demonstrated that olive oil phenolics have positive effects on certain physiological parameters such as plasma lipoproteins, oxidative damage, inflammatory markers, platelet and cellular function, antimicrobial activity, and bone health (for review see (Cicerale et al., 2010)), possibly reducing the risk of chronic disease development.


**5. Antimicrobial susceptibility and management of patients**

Choosing an initial antibiotic for suspected VAP is a difficult task. A scheme of empiric antibiotic therapy must take into account that *S. aureus*, *P. aeruginosa*, *Acinetobacter* spp., and Enterobacteriaceae members together represent more than 80% of VAP cases worldwide and several strains are defined as MDR pathogens [26]. To provide suitable antibiotic exposure regarding the possibility of infection by MDR pathogens, the empiric therapy should contain multiple agents with broader spectrum of activity [25].

However, antibiotic choices should be based on local prevalence and the antimicrobial susceptibility profile of the usual pathogens, since data from guidelines or other hospitals can be ineffective [61]. For empiric MRSA coverage, vancomycin or linezolid are strongly recommended. On the other hand, if it is indicated as MSSA coverage, the following antibiotics should be used: piperacillin-tazobactam, cefepime, levofloxacin, imipenem, or meropenem. Suspected etiology for MRSA or MSSA should be based on the presence of risk factors [61].

It has been reported that more than 50% of MRSA are also resistant to macrolides, lincosamides, fluoroquinolones, and aminoglycosides. This high level of resistance not only impedes successful therapy but also allows the microorganism to persist in the hospital, expanding its reservoir. So, vancomycin is the first-line treatment to VAP patients caused by MRSA. Nevertheless, some studies have described *S. aureus* strains with decreased susceptibility to vancomycin (vancomycin intermediate-resistant *S. aureus*, VISA). The acquired-resistance of MRSA to vancomycin is related to acquire mutations that appear in MRSA during vancomycin therapy [62, 63]. More recently, studies describing MRSA strains with high-level vancomycin resistance (vancomycin-resistant *S. aureus*, VRSA) were described. The mechanism of resistance is associated to the presence of transposon Tn1546, acquired from vancomycinresistant *Enterococcus faecalis*, which is known to alter cell wall structure and metabolism, but the resistance mechanisms in VISA and VRSA isolates are less well defined [62].

have been considered the agents of choice for infections caused by susceptible pathogens, but the rapid increase in carbapenem resistance rates has complicated this issue. Other mechanisms include: changes in OMPs, penicillin-binding proteins, and efflux pumps; resistance to aminoglycosides, mediated by aminoglycoside phosphotransferases, acetyltransferases, and adenyltransferases; resistance to quinolones, polymyxins, tetracyclines, among others [28].

Microbiology of Ventilator-Associated Pneumonia http://dx.doi.org/10.5772/intechopen.69430 115

A recent cohort study of bacteremia associated with pneumonia found that inappropriate initial antibiotic treatment seems to be the most important independent determinant of mortality and is the only identified mortality predictor amenable to intervention [67]. These Gramnegative bacteria are responsible for increasing numbers of infections encountered in hospitals, particularly among immunocompromised patients, and community-acquired infections are also increasing in prevalence. Furthermore, the impact of *P. aeruginosa* and *A. baumannii*

The diagnosis of VAP is usually based on clinical, radiographic, and microbiological criteria. Microbiological diagnosis is important in the management of VAP, since early diagnosis can influence clinical outcomes. The usual methods for microbiological diagnosis are based on quantitative or semiquantitative culture, but the results can take 48 h or more to be available. The Gram stain method has been used as screening of infection and to guide initial antibiotic therapy. However, utility of microscopy examination of respiratory secretions is still

Molecular methods can also be used to obtain results more quickly and initiate rational antibiotic therapy of patients with VAP. Many method formats are available for the detection of target genes for microbial identification and also for the detection of antimicrobial resistance

Semiquantitative culture of endotracheal aspirates (ETA) is the recommended microbiological procedure to diagnose VAP, since it is more sensitive and can be done more rapidly. Other biological specimens have been used, including the ones obtained by invasive sampling, such as: bronchoalveolar lavage (BAL), blind bronchial sampling (mini-BAL), and protected specimen brush (PSB). Blood cultures should also be performed for all patients with suspected VAP. In all cases, samples should be obtained before the patients initiate antibiotic

The main problem with the semiquantitative culture of ETA is that its high sensitivity promotes the unnecessary prescription of antibiotics to some patients. In the case of quantitative cultures of lower respiratory tract secretions, the following threshold cut-offs are usually

–106

may lead to false-negative results and worse clinical outcomes in some patients [61].

, BAL 104

, and PSB 103

CFU/mL. This strategy

resistance on health systems is a major concern in hospitals worldwide.

**6. Laboratory diagnosis**

controversial.

genes.

**6.1. Culture**

therapy [61].

applied to diagnosis true infection: ETA 105

Antibiotic options for Gram-negative coverage are more varied and must contain two antipseudomonal antibiotics from different classes in the presence of risk factors for MDR pathogens for the initial treatment of suspected VAP. If the patient does not present risk factors for MDR pathogens, only one anti-pseudomonas drug should be prescribed [61].

The frequency of infections caused by *P. aeruginosa* has increased in combination with the morbidity and mortality among hospitalized patients, all of which are exacerbated by antimicrobial resistance. Studies have demonstrated that resistance to carbapenems, aminoglycosides, and fluoroquinolones has increased gradually over the past few years, as well as episodes caused by MDR strains. Many *P. aeruginosa* isolates display an intrinsic reduced susceptibility to several antibacterial agents, as well as a tendency to develop resistance during therapy, especially in carbapenem-resistant strains. The most common mechanism of imipenem resistance in *P. aeruginosa* is a combination of chromosomal AmpC production and porin alterations. It also produces extended-spectrum β-lactamases (ESBLs) and can harbor other antibiotic resistance enzymes such as *K. pneumoniae* carbapenemases (KPC) and imipenem metallo-β-lactamases. β-Lactamase production, especially ESBLs, remains the main factor to acquired β-lactam resistance [52, 64, 65].

*K. pneumoniae* may present two major types of antibiotic resistance: (1) expression of ESBLs, which make them resistant to cephalosporins and monobactams and (2) the expression of carbapenemases that make *K. pneumoniae* resistant to almost all available β-lactams, including carbapenems. The first reported of carbapenemase by *K. pneumoniae* was in the USA, in 1996, which was designated KPC. Currently four classes of carbapenemases (classes A–D) have already been described and KPCs are classified into class A. To date, 16 KPC class A variants have already been identified. In addition to KPCs, *K. pneumoniae* strains may carry other forms of carbapenemases, such as class B metallo-β-lactamases (such as New Delhi's metalloβ-lactamase NDM-1 enzymes) and OXA class. In addition to β-lactamases, mutations in outer membrane proteins (OMPs) may also make the bacterium more resistant to β-lactams, particularly if it was in combination with the expression of a carbapenemase [66].

*A. baumannii* is also considered an emerging cause of nosocomial outbreaks, especially by MDR strains in ICUs. The most significant mechanism of carbapenem resistance in *A. baumannii* is the production of carbapenemases, which can be either intrinsic or acquired. Carbapenems have been considered the agents of choice for infections caused by susceptible pathogens, but the rapid increase in carbapenem resistance rates has complicated this issue. Other mechanisms include: changes in OMPs, penicillin-binding proteins, and efflux pumps; resistance to aminoglycosides, mediated by aminoglycoside phosphotransferases, acetyltransferases, and adenyltransferases; resistance to quinolones, polymyxins, tetracyclines, among others [28].

A recent cohort study of bacteremia associated with pneumonia found that inappropriate initial antibiotic treatment seems to be the most important independent determinant of mortality and is the only identified mortality predictor amenable to intervention [67]. These Gramnegative bacteria are responsible for increasing numbers of infections encountered in hospitals, particularly among immunocompromised patients, and community-acquired infections are also increasing in prevalence. Furthermore, the impact of *P. aeruginosa* and *A. baumannii* resistance on health systems is a major concern in hospitals worldwide.